An evolutionarily distinct Hmgn2 variant influences shape recognition in Medaka Fish.
Shuntaro Inoue, Yume Masaki, Shinichi Nakagawa, Saori Yokoi
Communications biology, 7, 1, 973, 973, 2024年08月23日, ［国際誌］
英語, 研究論文（学術雑誌）, Protein sequence diversification significantly impacts physiological traits. In this study, using medaka fish (Oryzias latipes), we identify a novel protein variant affecting shape preference behavior. Re-analysis of sequencing data reveals that LOC101156433 encodes a unique Hmgn2 variant with unusual subnuclear localization, clustered separately from the Hmgn2 clades of other species. Medaka mutants with this variant showed reduce telencephalic regions and altered shape preference, suggesting a link between protein sequence variation and behavioral changes. Additionally, this Hmgn2 variant is common in Acanthopterygii fishes, which are adapted to a variety of environments, indicating its potential evolutionary significance. Our findings highlight the relationship between amino acid sequence variation and the development of new molecular and behavioral adaptations, providing insights into the visual shape perception system in fish.

The Essential Role of Architectural Noncoding RNA Neat1 in Cold-Induced Beige Adipocyte Differentiation in Mice.
Hikaru Toya, Yuko Okamatsu-Ogura, Saori Yokoi, Misuzu Kurihara, Mari Mito, Shintaro Iwasaki, Tetsuro Hirose, Shinichi Nakagawa
RNA (New York, N.Y.), 2024年05月01日, ［国際誌］
英語, 研究論文（学術雑誌）, Neat1 is an architectural RNA that provides the structural basis for nuclear bodies known as paraspeckles. Although the assembly processes by which Neat1 organizes paraspeckle components are well-documented, the physiological functions of Neat1 remain less defined. This is partly because Neat1 knockout (KO) mice, lacking paraspeckles, do not exhibit overt phenotypes under normal laboratory conditions. During our search for conditions that elicit clear phenotypes in Neat1 KO mice, we discovered that the differentiation of beige adipocytes-inducible thermogenic cells that emerge upon cold exposure-is severely impaired in these mutant mice. Neat1_2, the architectural isoform of Neat1, is transiently upregulated during the early stages of beige adipocyte differentiation, coinciding with increased paraspeckle formation. Genes with altered expression during beige adipocyte differentiation typically cluster at specific chromosomal locations, some of which move closer to paraspeckles upon cold exposure. These observations suggest that paraspeckles might coordinate the regulation of these gene clusters by controlling the activity of certain transcriptional condensates that co-regulate multiple genes. We propose that our findings highlight a potential role for Neat1 and paraspeckles in modulating chromosomal organization and gene expression, potentially crucial processes for the differentiation of beige adipocytes.

The early macrophage response to pathogens requires dynamic regulation of the nuclear paraspeckle
Sikandar Azam, Kaitlyn S. Armijo, Chi G. Weindel, Morgan J. Chapman, Alice Devigne, Shinichi Nakagawa, Tetsuro Hirose, Susan Carpenter, Robert O. Watson, Kristin L. Patrick
Proceedings of the National Academy of Sciences, 121, 9, Proceedings of the National Academy of Sciences, 2024年02月21日
研究論文（学術雑誌）, To ensure a robust immune response to pathogens without risking immunopathology, the kinetics and amplitude of inflammatory gene expression in macrophages need to be exquisitely well controlled. There is a growing appreciation for stress-responsive membraneless organelles (MLOs) regulating various steps of eukaryotic gene expression in response to extrinsic cues. Here, we implicate the nuclear paraspeckle, a highly ordered biomolecular condensate that nucleates on the Neat1 lncRNA, in tuning innate immune gene expression in murine macrophages. In response to a variety of innate agonists, macrophage paraspeckles rapidly aggregate (0.5 h poststimulation) and disaggregate (2 h poststimulation). Paraspeckle maintenance and aggregation require active transcription and MAPK signaling, whereas paraspeckle disaggregation requires degradation of Neat1 via the nuclear RNA exosome. In response to lipopolysaccharide treatment, Neat1 KO macrophages fail to properly express a large cohort of proinflammatory cytokines, chemokines, and antimicrobial mediators. Consequently, Neat1 KO macrophages cannot control replication of Salmonella enterica serovar Typhimurium or vesicular stomatitis virus. These findings highlight a prominent role for MLOs in orchestrating the macrophage response to pathogens and support a model whereby dynamic assembly and disassembly of paraspeckles reorganizes the nuclear landscape to enable inflammatory gene expression following innate stimuli.

4.5SH RNA counteracts deleterious exonization of SINE B1 in mice.
Rei Yoshimoto, Yuta Nakayama, Ikuko Nomura, Ikuko Yamamoto, Yumeka Nakagawa, Shigeyuki Tanaka, Misuzu Kurihara, Yu Suzuki, Takehiko Kobayashi, Hiroko Kozuka-Hata, Masaaki Oyama, Mari Mito, Shintaro Iwasaki, Tomohiro Yamazaki, Tetsuro Hirose, Kimi Araki, Shinichi Nakagawa
Molecular cell, 83, 24, 4479, 4493, 2023年12月21日, ［国際誌］
英語, 研究論文（学術雑誌）, 4.5SH RNA is a highly abundant, small rodent-specific noncoding RNA that localizes to nuclear speckles enriched in pre-mRNA-splicing regulators. To investigate the physiological functions of 4.5SH RNA, we have created mutant mice that lack the expression of 4.5SH RNA. The mutant mice exhibited embryonic lethality, suggesting that 4.5SH RNA is an essential species-specific noncoding RNA in mice. RNA-sequencing analyses revealed that 4.5SH RNA protects the transcriptome from abnormal exonizations of the antisense insertions of the retrotransposon SINE B1 (asB1), which would otherwise introduce deleterious premature stop codons or frameshift mutations. Mechanistically, 4.5SH RNA base pairs with complementary asB1-containing exons via the target recognition region and recruits effector proteins including Hnrnpm via its 5' stem loop region. The modular organization of 4.5SH RNA allows us to engineer a programmable splicing regulator to induce the skipping of target exons of interest. Our results also suggest the general existence of splicing regulatory noncoding RNAs.

Shell protein composition specified by the lncRNA NEAT1 domains dictates the formation of paraspeckles as distinct membraneless organelles.
Hiro Takakuwa, Tomohiro Yamazaki, Sylvie Souquere, Shungo Adachi, Hyura Yoshino, Naoko Fujiwara, Tetsuya Yamamoto, Tohru Natsume, Shinichi Nakagawa, Gerard Pierron, Tetsuro Hirose
Nature cell biology, 25, 11, 1664, 1675, Cold Spring Harbor Laboratory, 2023年11月, ［国際誌］
英語, 研究論文（学術雑誌）, Abstract

Many membraneless organelles (MLOs) formed through phase separation play crucial roles in various cellular processes. Although these MLOs co-exist in cells, how they maintain their independence without coalescence or engulfment remains largely unknown. Here, we investigated the molecular mechanism by which paraspeckles with core-shell architecture scaffolded by NEAT1_2 lncRNAs exist as distinct MLOs. We identified NEAT1 deletion mutants that assemble paraspeckles that are incorporated into nuclear speckles. Several paraspeckle proteins, including SFPQ, HNRNPF, and BRG1, prevent this incorporation and thus contribute to the segregation of paraspeckles from nuclear speckles. Shell localization of these proteins in the paraspeckles, which is determined by NEAT1_2 lncRNA domains, is required for this segregation process. Conversely, U2-related spliceosomal proteins are involved in internalizing the paraspeckles into nuclear speckles. This study shows that the paraspeckle shell composition dictates the independence of MLOs in the nucleus, providing insights into the importance of the shell in defining features and functions of MLOs.

SINE-derived short noncoding RNAs: their evolutionary origins, molecular mechanisms, and physiological significance
Rei Yoshimoto, Shinichi Nakagawa
Frontiers in RNA Research, 1, Frontiers Media SA, 2023年08月10日
研究論文（学術雑誌）, Short Interspersed Elements (SINEs) comprise a significant portion of the genomes of higher eukaryotes, including humans and mice. This review focuses on SINE-derived noncoding RNAs (ncRNAs), particularly BC1, BC200, and 4.5SH RNA, which are expressed abundantly and in a species-specific manner. These ncRNAs seem to have independently evolved their functions during evolutionary processes: BC1 and BC200 have become cytoplasmic translation inhibitors, while 4.5SH RNA has developed into a nuclear ncRNA that regulates splicing. This review delves into the unique roles of these ncRNAs, with a special emphasis on the recently discovered splicing regulation function of 4.5SH RNA. Furthermore, we discuss their evolutionary trajectories and potential implications for understanding the complexities of gene regulation.

Nondomain biopolymers: Flexible molecular strategies to acquire biological functions.
Kazuharu Arakawa, Tetsuro Hirose, Toshifumi Inada, Takuhiro Ito, Toshie Kai, Masaaki Oyama, Yukihide Tomari, Takao Yoda, Shinichi Nakagawa
Genes to cells : devoted to molecular & cellular mechanisms, 2023年05月30日, ［国際誌］
英語, 研究論文（学術雑誌）, A long-standing assumption in molecular biology posits that the conservation of protein and nucleic acid sequences emphasizes the functional significance of biomolecules. These conserved sequences fold into distinct secondary and tertiary structures, enable highly specific molecular interactions, and regulate complex yet organized molecular processes within living cells. However, recent evidence suggests that biomolecules can also function through primary sequence regions that lack conservation across species or gene families. These regions typically do not form rigid structures, and their inherent flexibility is critical for their functional roles. This review examines the emerging roles and molecular mechanisms of "nondomain biomolecules," whose functions are not easily predicted due to the absence of conserved functional domains. We propose the hypothesis that both domain- and nondomain-type molecules work together to enable flexible and efficient molecular processes within the highly crowded intracellular environment.

Long noncoding RNA Malat1 inhibits Tead3-Nfatc1-mediated osteoclastogenesis to suppress osteoporosis and bone metastasis.
Yang Zhao, Hongqi Teng, Yalan Deng, Marisela Sheldon, Consuelo Martinez, Jie Zhang, Annie Tian, Yutong Sun, Shinichi Nakagawa, Fan Yao, Hai Wang, Li Ma
Research square, 2023年03月20日, ［国際誌］
英語, MALAT1, one of the few highly conserved nuclear long noncoding RNAs (IncRNAs), is abundantly expressed in normal tissues. Previously, targeted inactivation and genetic rescue experiments identified MALAT1 as a suppressor of breast cancer lung metastasis. On the other hand, Malat1-knockout mice are viable and develop normally. On a quest to discover new roles of MALAT1 in physiological and pathological processes, we found that this lncRNA is downregulated during osteoclastogenesis in humans and mice. Notably, Malat1 deficiency in mice promotes osteoporosis and bone metastasis, which can be rescued by genetic add-back of Malat1. Mechanistically, Malat1 binds to Tead3 protein, a macrophage-osteoclast-specific Tead family member, blocking Tead3 from binding and activating Nfatc1, a master regulator of osteoclastogenesis, which results in the inhibition of Nfatc1-mediated gene transcription and osteoclast differentiation. Altogether, these findings identify Malat1 as a lncRNA that suppresses osteoporosis and bone metastasis.

ILF3 prion-like domain regulates gene expression and fear memory under chronic stress.
Akira Yamashita, Yuichi Shichino, Kazuki Fujii, Yumie Koshidaka, Mayumi Adachi, Eri Sasagawa, Mari Mito, Shinichi Nakagawa, Shintaro Iwasaki, Keizo Takao, Nobuyuki Shiina
iScience, 26, 3, 106229, 106229, 2023年03月17日, ［国際誌］
英語, 研究論文（学術雑誌）, The prion-like domain (PrLD) is a class of intrinsically disordered regions. Although its propensity to form condensates has been studied in the context of neurodegenerative diseases, the physiological role of PrLD remains unclear. Here, we investigated the role of PrLD in the RNA-binding protein NFAR2, generated by a splicing variant of the Ilf3 gene. Removal of the PrLD in mice did not impair the function of NFAR2 required for survival, but did affect the responses to chronic water immersion and restraint stress (WIRS). The PrLD was required for WIRS-sensitive nuclear localization of NFAR2 and WIRS-induced changes in mRNA expression and translation in the amygdala, a fear-related brain region. Consistently, the PrLD conferred resistance to WIRS in fear-associated memory formation. Our study provides insights into the PrLD-dependent role of NFAR2 for chronic stress adaptation in the brain.

Long non-coding RNAs: definitions, functions, challenges and recommendations
John S. Mattick, Paulo P. Amaral, Piero Carninci, Susan Carpenter, Howard Y. Chang, Ling-Ling Chen, Runsheng Chen, Caroline Dean, Marcel E. Dinger, Katherine A. Fitzgerald, Thomas R. Gingeras, Mitchell Guttman, Tetsuro Hirose, Maite Huarte, Rory Johnson, Chandrasekhar Kanduri, Philipp Kapranov, Jeanne B. Lawrence, Jeannie T. Lee, Joshua T. Mendell, Timothy R. Mercer, Kathryn J. Moore, Shinichi Nakagawa, John L. Rinn, David L. Spector, Igor Ulitsky, Yue Wan, Jeremy E. Wilusz, Mian Wu
Nature Reviews Molecular Cell Biology, Springer Science and Business Media LLC, 2023年01月03日
研究論文（学術雑誌）

BAY61-3606 Alters snRNP Composition and Enhances Usage of Suboptimal Splice Acceptor Site.
Kenji Tomita, Shinichi Nakagawa, Hiroyoshi Ariga, Hiroshi Maita
Biological & pharmaceutical bulletin, 46, 2, 147, 157, 2023年, ［国内誌］
英語, 研究論文（学術雑誌）, Intron recognition by the spliceosome mainly depends on conserved intronic sequences such as 5' splice sites, 3' splice sites, and branch sites. Therefore, even substitution of just a single nucleotide in a 5' or 3' splice site abolishes the splicing at the mutated site and leads to cryptic splice site usage. A number of disease-causative mutations have been found in 5' and 3' splice sites, but the genes with these mutations still maintain the correct protein-coding sequence, so recovery of splicing at the mutated splice site may produce a normal protein. Mutations in the spliceosome components have been shown to change the balance between the conformational transition and disassembly of the spliceosome, which affects the decision about whether the reaction of the incorporated substrate will proceed. In addition, the lower disassembly rate caused by such mutations induces splicing of the mutated splice site. We hypothesized that small compounds targeting the spliceosome may include a compound mimicking the effect of those mutations. Thus, we screened a small-compound library and identified a compound, BAY61-3606, that changed the cellular small nuclear ribonucleoprotein composition and also showed activity of enhancing splicing at the mutated 3' splice site of the reporter gene, as well as splicing at the suboptimal 3' splice site of endogenous cassette exons. These results indicate that further analysis of the mechanism of action of BAY61-3606 could enable modulation of the fidelity of splicing.

tRNA-like Transcripts from the NEAT1-MALAT1 Genomic Region Critically Influence Human Innate Immunity and Macrophage Functions
Martina Gast, Vanasa Nageswaran, Andreas W. Kuss, Ana Tzvetkova, Xiaomin Wang, Liliana H. Mochmann, Pegah Ramezani Rad, Stefan Weiss, Stefan Simm, Tanja Zeller, Henry Voelzke, Wolfgang Hoffmann, Uwe Völker, Stefan B. Felix, Marcus Dörr, Antje Beling, Carsten Skurk, David-Manuel Leistner, Bernhard H. Rauch, Tetsuro Hirose, Bettina Heidecker, Karin Klingel, Shinichi Nakagawa, Wolfram C. Poller, Filip K. Swirski, Arash Haghikia, Wolfgang Poller
Cells, 11, 24, 3970, 3970, MDPI AG, 2022年12月08日
研究論文（学術雑誌）, The evolutionary conserved NEAT1-MALAT1 gene cluster generates large noncoding transcripts remaining nuclear, while tRNA-like transcripts (mascRNA, menRNA) enzymatically generated from these precursors translocate to the cytosol. Whereas functions have been assigned to the nuclear transcripts, data on biological functions of the small cytosolic transcripts are sparse. We previously found NEAT1−/− and MALAT1−/− mice to display massive atherosclerosis and vascular inflammation. Here, employing selective targeted disruption of menRNA or mascRNA, we investigate the tRNA-like molecules as critical components of innate immunity. CRISPR-generated human ΔmascRNA and ΔmenRNA monocytes/macrophages display defective innate immune sensing, loss of cytokine control, imbalance of growth/angiogenic factor expression impacting upon angiogenesis, and altered cell–cell interaction systems. Antiviral response, foam cell formation/oxLDL uptake, and M1/M2 polarization are defective in ΔmascRNA/ΔmenRNA macrophages, defining first biological functions of menRNA and describing new functions of mascRNA. menRNA and mascRNA represent novel components of innate immunity arising from the noncoding genome. They appear as prototypes of a new class of noncoding RNAs distinct from others (miRNAs, siRNAs) by biosynthetic pathway and intracellular kinetics. Their NEAT1-MALAT1 region of origin appears as archetype of a functionally highly integrated RNA processing system.

A guide to membraneless organelles and their various roles in gene regulation.
Tetsuro Hirose, Kensuke Ninomiya, Shinichi Nakagawa, Tomohiro Yamazaki
Nature reviews. Molecular cell biology, 2022年11月23日, ［国際誌］
英語, 研究論文（学術雑誌）, Membraneless organelles (MLOs) are detected in cells as dots of mesoscopic size. By undergoing phase separation into a liquid-like or gel-like phase, MLOs contribute to intracellular compartmentalization of specific biological functions. In eukaryotes, dozens of MLOs have been identified, including the nucleolus, Cajal bodies, nuclear speckles, paraspeckles, promyelocytic leukaemia protein (PML) nuclear bodies, nuclear stress bodies, processing bodies (P bodies) and stress granules. MLOs contain specific proteins, of which many possess intrinsically disordered regions (IDRs), and nucleic acids, mainly RNA. Many MLOs contribute to gene regulation by different mechanisms. Through sequestration of specific factors, MLOs promote biochemical reactions by simultaneously concentrating substrates and enzymes, and/or suppressing the activity of the sequestered factors elsewhere in the cell. Other MLOs construct inter-chromosomal hubs by associating with multiple loci, thereby contributing to the biogenesis of macromolecular machineries essential for gene expression, such as ribosomes and spliceosomes. The organization of many MLOs includes layers, which might have different biophysical properties and functions. MLOs are functionally interconnected and are involved in various diseases, prompting the emergence of therapeutics targeting them. In this Review, we introduce MLOs that are relevant to gene regulation and discuss their assembly, internal structure, gene-regulatory roles in transcription, RNA processing and translation, particularly in stress conditions, and their disease relevance.

Transcriptional coregulator Ess2 controls survival of post-thymic CD4+ T cells through the Myc and IL-7 signaling pathways.
Ichiro Takada, Shinya Hidano, Sayuri Takahashi, Kaori Yanaka, Hidesato Ogawa, Megumi Tsuchiya, Atsushi Yokoyama, Shingo Sato, Hiroki Ochi, Tohru Nakagawa, Takashi Kobayashi, Shinichi Nakagawa, Makoto Makishima
The Journal of biological chemistry, 298, 9, 102342, 102342, 2022年09月, ［国際誌］
英語, 研究論文（学術雑誌）, Ess2, also known as Dgcr14, is a transcriptional co-regulator of CD4+ T cells. Ess2 is located in a chromosomal region, the loss of which has been associated with 22q11.2 deletion syndrome (22q11DS), which causes heart defects, skeletal abnormalities, and immunodeficiency. However, the specific association of Ess2 with 22q11DS remains unclear. To elucidate the role of Ess2 in T-cell development, we generated Ess2 floxed (Ess2fl/fl) and CD4+ T cell-specific Ess2 KO (Ess2ΔCD4/ΔCD4) mice using the Cre/loxP system. Interestingly, Ess2ΔCD4/ΔCD4 mice exhibited reduced naïve T-cell numbers in the spleen, while the number of thymocytes (CD4-CD8-, CD4+CD8+, CD4+CD8-, and CD4-CD8+) in the thymus remained unchanged. Furthermore, Ess2ΔCD4/ΔCD4 mice had decreased NKT cells and increased γδT cells in the thymus and spleen. A genome-wide expression analysis using RNA-seq revealed that Ess2 deletion alters the expression of many genes in CD4 single-positive thymocytes, including genes related to the immune system and Myc target genes. In addition, Ess2 enhanced the transcriptional activity of c-Myc. Some genes identified as Ess2 targets in mice show expressional correlation with ESS2 in human immune cells. Moreover, Ess2ΔCD4/ΔCD4 naïve CD4+ T cells did not maintain survival in response to IL-7. Our results suggest that Ess2 plays a critical role in post-thymic T-cell survival through the Myc and IL-7 signaling pathways.

Species-specific formation of paraspeckles in intestinal epithelium revealed by characterization of NEAT1 in naked mole-rat.
Akihiro Yamada, Hikaru Toya, Mayuko Tanahashi, Misuzu Kurihara, Mari Mito, Shintaro Iwasaki, Satoshi Kurosaka, Toru Takumi, Archa Fox, Yoshimi Kawamura, Kyoko Miura, Shinichi Nakagawa
RNA (New York, N.Y.), 28, 8, 1128, 1143, 2022年08月, ［査読有り］, ［最終著者, 責任著者］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2 The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2 that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits a different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.

lncRNA Neat1 regulates neuronal dysfunction post-sepsis via stabilization of hemoglobin subunit beta.
Yan Wu, Pengfei Li, Liu Liu, Andrew J Goodwin, Perry V Halushka, Tetsuro Hirose, Shinichi Nakagawa, Jiliang Zhou, Meng Liu, Hongkuan Fan
Molecular therapy : the journal of the American Society of Gene Therapy, 30, 7, 2618, 2632, 2022年07月06日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, Sepsis-associated encephalopathy (SAE) is characterized by acute and diffuse brain dysfunction and correlates with long-term cognitive impairments with no targeted therapy. We used a mouse model of sepsis-related cognitive impairment to examine the role of lncRNA nuclear enriched abundant transcript 1 (Neat1) in SAE. We observed that Neat1 expression was increased in neuronal cells from septic mice and that it directly interacts with hemoglobin subunit beta (Hbb), preventing its degradation. The Neat1/Hbb axis suppressed postsynaptic density protein 95 (PSD-95) levels and decreased dendritic spine density. Neat1 knockout mice exhibited decreased Hbb levels, which resulted in increased PSD-95 levels, increased neuronal dendritic spine density, and decreased anxiety and memory impairment. Neat1 silencing via the antisense oligonucleotide GapmeR ameliorated anxiety-like behavior and cognitive impairment post-sepsis. In conclusion, we uncovered a previously unknown mechanism of the Neat1/Hbb axis in regulating neuronal dysfunction, which may lead to a novel treatment strategy for SAE.

MiR-150 Attenuates Maladaptive Cardiac Remodeling Mediated by Long Noncoding RNA MIAT and Directly Represses Profibrotic Hoxa4.
Tatsuya Aonuma, Bruno Moukette, Satoshi Kawaguchi, Nipuni P Barupala, Marisa N Sepúlveda, Kyle Frick, Yaoliang Tang, Maya Guglin, Subha V Raman, Chenleng Cai, Suthat Liangpunsakul, Shinichi Nakagawa, Il-Man Kim
Circulation. Heart failure, 15, 4, CIRCHEARTFAILURE121008686, 2022年01月10日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: MicroRNA-150 (miR-150) plays a protective role in heart failure (HF). Long noncoding RNA, myocardial infarction-associated transcript (MIAT) regulates miR-150 function in vitro by direct interaction. Concurrent with miR-150 downregulation, MIAT is upregulated in failing hearts, and gain-of-function single-nucleotide polymorphisms in MIAT are associated with increased risk of myocardial infarction (MI) in humans. Despite the correlative relationship between MIAT and miR-150 in HF, their in vivo functional relationship has never been established, and molecular mechanisms by which these 2 noncoding RNAs regulate cardiac protection remain elusive. METHODS: We use MIAT KO (knockout), Hoxa4 (homeobox a4) KO, MIAT TG (transgenic), and miR-150 TG mice. We also develop DTG (double TG) mice overexpressing MIAT and miR-150. We then use a mouse model of MI followed by cardiac functional, structural, and mechanistic studies by echocardiography, immunohistochemistry, transcriptome profiling, Western blotting, and quantitative real-time reverse transcription-polymerase chain reaction. Moreover, we perform expression analyses in hearts from patients with HF. Lastly, we investigate cardiac fibroblast activation using primary adult human cardiac fibroblasts and in vitro assays to define the conserved MIAT/miR-150/HOXA4 axis. RESULTS: Using novel mouse models, we demonstrate that genetic overexpression of MIAT worsens cardiac remodeling, while genetic deletion of MIAT protects hearts against MI. Importantly, miR-150 overexpression attenuates the detrimental post-MI effects caused by MIAT. Genome-wide transcriptomic analysis of MIAT null mouse hearts identifies Hoxa4 as a novel downstream target of the MIAT/miR-150 axis. Hoxa4 is upregulated in cardiac fibroblasts isolated from ischemic myocardium and subjected to hypoxia/reoxygenation. HOXA4 is also upregulated in patients with HF. Moreover, Hoxa4 deficiency in mice protects the heart from MI. Lastly, protective actions of cardiac fibroblast miR-150 are partially attributed to the direct and functional repression of profibrotic Hoxa4. CONCLUSIONS: Our findings delineate a pivotal functional interaction among MIAT, miR-150, and Hoxa4 as a novel regulatory mechanism pertinent to ischemic HF.

UPA-Seq-Based Search Method for Functional lncRNA Candidates.
Saori Yokoi, Shinichi Nakagawa
Methods in molecular biology (Clifton, N.J.), 2509, 269, 278, 2022年, ［査読有り］, ［招待有り］, ［最終著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Long noncoding RNAs (lncRNAs) constitute a large fraction of the transcriptome in mammals, and recent studies have revealed important functions of lncRNAs in a variety of biological processes. However, the fraction of lncRNAs that have been functionally validated is small, and only sequence and expression information are available for most lncRNAs. Here, we describe the procedures for UV-phenol aqueous-phase RNA sequencing (UPA-seq), a method for searching for functional lncRNA candidates among whole genomes based on the assumption that functional lncRNAs exert their functions through associations with proteins.

The cell type-specific ER membrane protein UGS148 is not essential in mice.
Osamu Takahashi, Mayuko Tanahashi, Saori Yokoi, Mari Kaneko, Kaori Yanaka, Shinichi Nakagawa, Hiroshi Maita
Genes to cells : devoted to molecular & cellular mechanisms, 27, 1, 43, 60, 2022年01月, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Genomes of higher eukaryotes encode many uncharacterized proteins, and the functions of these proteins cannot be predicted from the primary sequences due to a lack of conserved functional domains. In this study, we focused on a poorly characterized protein UGS148 that is highly expressed in a specialized cell type called tanycytes that line the ventral wall of the third ventricle in the hypothalamus. Immunostaining of UGS148 revealed the fine morphology of tanycytes with highly branched apical ER membranes. Immunoprecipitation revealed that UGS148 associated with mitochondrial ATPase at least in vitro, and ER and mitochondrial signals occasionally overlapped in tanycytes. Mutant mice lacking UGS148 did not exhibit overt phenotypes, suggesting that UGS148 was not essential in mice reared under normal laboratory conditions. We also found that RNA probes that were predicted to uniquely detect UGS148 mRNA cross-reacted with uncharacterized RNAs, highlighting the importance of experimental validation of the specificity of probes during the hybridization-based study of RNA localization.

NEAT1 is essential for metabolic changes that promote breast cancer growth and metastasis.
Mi Kyung Park, Li Zhang, Kyung-Won Min, Jung-Hyun Cho, Chih-Chen Yeh, Hyesu Moon, Daniel Hormaechea-Agulla, Hyejin Mun, Seungbeom Ko, Ji Won Lee, Sonali Jathar, Aubrey S Smith, Yixin Yao, Nguyen Thu Giang, Hong Ha Vu, Victoria C Yan, Mary C Bridges, Antonis Kourtidis, Florian Muller, Jeong Ho Chang, Su Jung Song, Shinichi Nakagawa, Tetsuro Hirose, Je-Hyun Yoon, Min Sup Song
Cell metabolism, 33, 12, 2380, 2397, 2021年12月07日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, Accelerated glycolysis is the main metabolic change observed in cancer, but the underlying molecular mechanisms and their role in cancer progression remain poorly understood. Here, we show that the deletion of the long noncoding RNA (lncRNA) Neat1 in MMTV-PyVT mice profoundly impairs tumor initiation, growth, and metastasis, specifically switching off the penultimate step of glycolysis. Mechanistically, NEAT1 directly binds and forms a scaffold bridge for the assembly of PGK1/PGAM1/ENO1 complexes and thereby promotes substrate channeling for high and efficient glycolysis. Notably, NEAT1 is upregulated in cancer patients and correlates with high levels of these complexes, and genetic and pharmacological blockade of penultimate glycolysis ablates NEAT1-dependent tumorigenesis. Finally, we demonstrate that Pinin mediates glucose-stimulated nuclear export of NEAT1, through which it exerts isoform-specific and paraspeckle-independent functions. These findings establish a direct role for NEAT1 in regulating tumor metabolism, provide new insights into the Warburg effect, and identify potential targets for therapy.

NEAT1 lncRNA and amyotrophic lateral sclerosis.
Yoshinori Nishimoto, Shinichi Nakagawa, Hideyuki Okano
Neurochemistry international, 150, 105175, 105175, 2021年09月02日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Amyotrophic lateral sclerosis (ALS) is a representative neurological disease that is known to devastate entire motor neurons within a period of just a few years. Discoveries of the specific pathologies of relevant RNA-binding proteins, including TAR DNA-binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), and the causative genes of both familial and sporadic ALS have provided crucial information that could lead to a cure. In recent ALS research the GGGGCC-repeat expansion in the C9orf72 gene was identified as one of the most important pathological findings, suggesting the significance of both nuclear dysfunction due to dipeptide repeat proteins (DPRs) and RNA toxicity (such as pathological alterations of non-coding RNAs). In research on model animals carrying ALS-related molecules, the determination of whether a factor is protective or toxic has been controversial. Herein, we review the findings regarding NEAT1 RNA and C9orf72 GGGGCC repeats associated with ALS, from the viewpoint of conversion from the protective stage in the nucleus in early-phase ALS to late-phase induction of cell death. This review will provide insights for the development of RNA effectors as novel ALS treatments.

A histone modifier, ASXL1, interacts with NONO and is involved in paraspeckle formation in hematopoietic cells.
Keita Yamamoto, Susumu Goyama, Shuhei Asada, Takeshi Fujino, Taishi Yonezawa, Naru Sato, Reina Takeda, Akiho Tsuchiya, Tomofusa Fukuyama, Yosuke Tanaka, Akihiko Yokoyama, Hikaru Toya, Ayana Kon, Yasuhito Nannya, Rena Onoguchi-Mizutani, Shinichi Nakagawa, Tetsuro Hirose, Seishi Ogawa, Nobuyoshi Akimitsu, Toshio Kitamura
Cell reports, 36, 8, 109576, 109576, 2021年08月24日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Paraspeckles are membraneless organelles formed through liquid-liquid phase separation and consist of multiple proteins and RNAs, including NONO, SFPQ, and NEAT1. The role of paraspeckles and the component NONO in hematopoiesis remains unknown. In this study, we show histone modifier ASXL1 is involved in paraspeckle formation. ASXL1 forms phase-separated droplets, upregulates NEAT1 expression, and increases NONO-NEAT1 interactions through the C-terminal intrinsically disordered region (IDR). In contrast, a pathogenic ASXL mutant (ASXL1-MT) lacking IDR does not support the interaction of paraspeckle components. Furthermore, paraspeckles are disrupted and Nono localization is abnormal in the cytoplasm of hematopoietic stem and progenitor cells (HSPCs) derived from ASXL1-MT knockin mice. Nono depletion and the forced expression of cytoplasmic NONO impair the repopulating potential of HSPCs, as does ASXL1-MT. Our study indicates a link between ASXL1 and paraspeckle components in the maintenance of normal hematopoiesis.

Paraspeckles are constructed as block copolymer micelles.
Tomohiro Yamazaki, Tetsuya Yamamoto, Hyura Yoshino, Sylvie Souquere, Shinichi Nakagawa, Gerard Pierron, Tetsuro Hirose
The EMBO journal, 40, 12, e107270, 2021年06月15日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, Paraspeckles are constructed by NEAT1_2 architectural long noncoding RNAs. Their characteristic cylindrical shapes, with highly ordered internal organization, distinguish them from typical liquid-liquid phase-separated condensates. We experimentally and theoretically investigated how the shape and organization of paraspeckles are determined. We identified the NEAT1_2 RNA domains responsible for shell localization of the NEAT1_2 ends, which determine the characteristic internal organization. Using the soft matter physics, we then applied a theoretical framework to understand the principles that determine NEAT1_2 organization as well as shape, number, and size of paraspeckles. By treating paraspeckles as amphipathic block copolymer micelles, we could explain and predict the experimentally observed behaviors of paraspeckles upon NEAT1_2 domain deletions or transcriptional modulation. Thus, we propose that paraspeckles are block copolymer micelles assembled through a type of microphase separation, micellization. This work provides an experiment-based theoretical framework for the concept that ribonucleoprotein complexes (RNPs) can act as block copolymers to form RNA-scaffolding biomolecular condensates with optimal sizes and structures in cells.

ArcRNAs and the formation of nuclear bodies.
Shinichi Nakagawa, Tomohiro Yamazaki, Taro Mannen, Tetsuro Hirose
Mammalian genome : official journal of the International Mammalian Genome Society, 33, 2, 382, 401, 2021年06月03日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Long noncoding RNAs (lncRNAs) have long been collectively and passively defined as transcripts that do not encode proteins. However, extensive functional studies performed over the last decade have enabled the classification of lncRNAs into multiple categories according to their functions and/or molecular properties. Architectual RNAs (arcRNAs) are a group of lncRNAs that serve as architectural components of submicron-scale cellular bodies or nonmembranous organelles, which are composed of specific sets of proteins and nucleic acids involved in particular molecular processes. In this review, we focus on arcRNAs that function in the nucleus, which provide a structural basis for the formation of nuclear bodies, nonmembranous organelles in the cell nucleus. We will summarize the current list of arcRNAs and proteins associated with classic and more recently discovered nuclear bodies and discuss general rules that govern the formation of nuclear bodies, emphasizing weak multivalent interactions mediated by innately flexible biomolecules.

The lncRNA Caren antagonizes heart failure by inactivating DNA damage response and activating mitochondrial biogenesis.
Michio Sato, Tsuyoshi Kadomatsu, Keishi Miyata, Junco S Warren, Zhe Tian, Shunshun Zhu, Haruki Horiguchi, Aman Makaju, Anna Bakhtina, Jun Morinaga, Taichi Sugizaki, Kaname Hirashima, Kumiko Yoshinobu, Mai Imasaka, Masatake Araki, Yoshihiro Komohara, Tomohiko Wakayama, Shinichi Nakagawa, Sarah Franklin, Koichi Node, Kimi Araki, Yuichi Oike
Nature communications, 12, 1, 2529, 2529, 2021年05月05日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In the past decade, many long noncoding RNAs (lncRNAs) have been identified and their in vitro functions defined, although in some cases their functions in vivo remain less clear. Moreover, unlike nuclear lncRNAs, the roles of cytoplasmic lncRNAs are less defined. Here, using a gene trapping approach in mouse embryonic stem cells, we identify Caren (short for cardiomyocyte-enriched noncoding transcript), a cytoplasmic lncRNA abundantly expressed in cardiomyocytes. Caren maintains cardiac function under pathological stress by inactivating the ataxia telangiectasia mutated (ATM)-DNA damage response (DDR) pathway and activating mitochondrial bioenergetics. The presence of Caren transcripts does not alter expression of nearby (cis) genes but rather decreases translation of an mRNA transcribed from a distant gene encoding histidine triad nucleotide-binding protein 1 (Hint1), which activates the ATM-DDR pathway and reduces mitochondrial respiratory capacity in cardiomyocytes. Therefore, the cytoplasmic lncRNA Caren functions in cardioprotection by regulating translation of a distant gene and maintaining cardiomyocyte homeostasis.

Spliceostatin A interaction with SF3B limits U1 snRNP availability and causes premature cleavage and polyadenylation.
Rei Yoshimoto, Jagat K Chhipi-Shrestha, Tilman Schneider-Poetsch, Masaaki Furuno, A Maxwell Burroughs, Shohei Noma, Harukazu Suzuki, Yoshihide Hayashizaki, Akila Mayeda, Shinichi Nakagawa, Daisuke Kaida, Shintaro Iwasaki, Minoru Yoshida
Cell chemical biology, 28, 9, 1356, 1365, 2021年03月23日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, RNA splicing, a highly conserved process in eukaryotic gene expression, is seen as a promising target for anticancer agents. Splicing is associated with other RNA processing steps, such as transcription and nuclear export; however, our understanding of the interaction between splicing and other RNA regulatory mechanisms remains incomplete. Moreover, the impact of chemical splicing inhibition on long non-coding RNAs (lncRNAs) has been poorly understood. Here, we demonstrate that spliceostatin A (SSA), a chemical splicing modulator that binds to the SF3B subcomplex of the U2 small nuclear ribonucleoprotein particle (snRNP), limits U1 snRNP availability in splicing, resulting in premature cleavage and polyadenylation of MALAT1, a nuclear lncRNA, as well as protein-coding mRNAs. Therefore, truncated transcripts are exported into the cytoplasm and translated, resulting in aberrant protein products. Our work demonstrates that active recycling of the splicing machinery maintains homeostasis of RNA processing beyond intron excision.

Ablation of lncRNA Miat attenuates pathological hypertrophy and heart failure.
Liu Yang, Jianxin Deng, Wenxia Ma, Aijun Qiao, Shiyue Xu, Yang Yu, Chan Boriboun, Xiang Kang, Dunzheng Han, Patrick Ernst, Lufang Zhou, Jiawei Shi, Eric Zhang, Tao-Sheng Li, Hongyu Qiu, Shinichi Nakagawa, Seth Blackshaw, Jianyi Zhang, Gangjian Qin
Theranostics, 11, 16, 7995, 8007, 2021年, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, Rationale: The conserved long non-coding RNA (lncRNA) myocardial infarction associate transcript (Miat) was identified for its multiple single-nucleotide polymorphisms that are strongly associated with susceptibility to MI, but its role in cardiovascular biology remains elusive. Here we investigated whether Miat regulates cardiac response to pathological hypertrophic stimuli. Methods: Both an angiotensin II (Ang II) infusion model and a transverse aortic constriction (TAC) model were used in adult WT and Miat-null knockout (Miat-KO) mice to induce pathological cardiac hypertrophy. Heart structure and function were evaluated by echocardiography and histological assessments. Gene expression in the heart was evaluated by RNA sequencing (RNA-seq), quantitative real-time RT-PCR (qRT-PCR), and Western blotting. Primary WT and Miat-KO mouse cardiomyocytes were isolated and used in Ca2+ transient and contractility measurements. Results: Continuous Ang II infusion for 4 weeks induced concentric hypertrophy in WT mice, but to a lesser extent in Miat-KO mice. Surgical TAC for 6 weeks resulted in decreased systolic function and heart failure in WT mice but not in Miat-KO mice. In both models, Miat-KO mice displayed reduced heart-weight to tibia-length ratio, cardiomyocyte cross-sectional area, cardiomyocyte apoptosis, and cardiac interstitial fibrosis and a better-preserved capillary density, as compared to WT mice. In addition, Ang II treatment led to significantly reduced mRNA and protein expression of the Ca2+ cycling genes Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and ryanodine receptor 2 (RyR2) and a dramatic increase in global RNA splicing events in the left ventricle (LV) of WT mice, and these changes were largely blunted in Miat-KO mice. Consistently, cardiomyocytes isolated from Miat-KO mice demonstrated more efficient Ca2+ cycling and greater contractility. Conclusions: Ablation of Miat attenuates pathological hypertrophy and heart failure, in part, by enhancing cardiomyocyte contractility.

Inhibition of the long non-coding RNA NEAT1 protects cardiomyocytes from hypoxia in vitro via decreased pri-miRNA processing.
Olof Gidlöf, Kerstin Bader, Selvi Celik, Mario Grossi, Shinichi Nakagawa, Tetsuro Hirose, Bernhard Metzler, Björn Olde, David Erlinge
Cell death & disease, 11, 8, 677, 677, 2020年08月13日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, While restoration of coronary blood flow to the ischemic heart is the most effective strategy for reducing infarct size, reperfusion injury represents a significant limiting factor on clinical outcomes in myocardial infarction patients. Ischemic preconditioning (IPC) has been shown to inhibit reperfusion injury and represents an attractive model for studying cardioprotective signal transduction pathways. Long non-coding RNAs (lncRNAs) are a structurally and functionally heterogenous class of RNA transcripts with unknown roles in IPC-induced cardioprotection. Through microarray-based expression profiling of 31,423 lncRNAs in cardiac tissue from IPC mice, we identified the nuclear transcript Neat1 to be rapidly and robustly decreased in response to IPC. siRNA-mediated knock down of Neat1 reduced apoptosis and necrosis in murine cardiomyocytes (CM) and human iPS-derived CMs in response to prolonged hypoxia and hypoxia-reoxygenation, assessed with Annexin V/propidium iodide-staining, a Caspase 3/7 activity assay, LDH release, and western blot for cleaved Caspase 3. Mechanistically, Neat1 was shown to regulate processing of pro-apoptotic microRNA-22 (miR-22) in murine and human CM nuclei using a luciferase reporter assay. Hypoxia-induced downregulation of Neat1 was shown to result in accumulation of unprocessed pri-miRNA and decreased availability of biologically active miRNA, including miR-22. Addition of exogenous synthetic miR-22 reversed the protective effect of Neat1 knock down in human iPS-CM. In conclusion, we have identified the nuclear lncRNA Neat1 as part of a conserved oxygen-sensitive feedback mechanism by regulation of miRNA processing and a potential target in cardioprotection.

Long non-coding RNA Neat1 regulates adaptive behavioural response to stress in mice.
Michail S Kukharsky, Natalia N Ninkina, Haiyan An, Vsevolod Telezhkin, Wenbin Wei, Camille Rabesahala de Meritens, Johnathan Cooper-Knock, Shinichi Nakagawa, Tetsuro Hirose, Vladimir L Buchman, Tatyana A Shelkovnikova
Translational psychiatry, 10, 1, 171, 171, 2020年05月28日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, NEAT1 is a highly and ubiquitously expressed long non-coding RNA (lncRNA) which serves as an important regulator of cellular stress response. However, the physiological role of NEAT1 in the central nervous system (CNS) is still poorly understood. In the current study, we addressed this by characterising the CNS function of the Neat1 knockout mouse model (Neat1-/- mice), using a combination of behavioural phenotyping, electrophysiology and expression analysis. RNAscope® in situ hybridisation revealed that in wild-type mice, Neat1 is expressed across the CNS regions, with high expression in glial cells and low expression in neurons. Loss of Neat1 in mice results in an inadequate reaction to physiological stress manifested as hyperlocomotion and panic escape response. In addition, Neat1-/- mice display deficits in social interaction and rhythmic patterns of activity but retain normal motor function and memory. Neat1-/- mice do not present with neuronal loss, overt neuroinflammation or gross synaptic dysfunction in the brain. However, cultured Neat1-/- neurons are characterised by hyperexcitability and dysregulated calcium homoeostasis, and stress-induced neuronal activity is also augmented in Neat1-/- mice in vivo. Gene expression analysis showed that Neat1 may act as a weak positive regulator of multiple genes in the brain. Furthermore, loss of Neat1 affects alternative splicing of genes important for the CNS function and implicated in neurological diseases. Overall, our data suggest that Neat1 is involved in stress signalling in the brain and fine-tunes the CNS functions to enable adaptive behaviour in response to physiological stress.

Sexually dimorphic role of oxytocin in medaka mate choice.
Saori Yokoi, Kiyoshi Naruse, Yasuhiro Kamei, Satoshi Ansai, Masato Kinoshita, Mari Mito, Shintaro Iwasaki, Shuntaro Inoue, Teruhiro Okuyama, Shinichi Nakagawa, Larry J Young, Hideaki Takeuchi
Proceedings of the National Academy of Sciences of the United States of America, 117, 9, 4802, 4808, 2020年03月03日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, Oxytocin is a central neuromodulator required for facilitating mate preferences for familiar individuals in a monogamous rodent (prairie vole), irrespective of sex. While the role of oxytocin in mate choice is only understood in a few monogamous species, its function in nonmonogamous species, comprising the vast majority of vertebrate species, remains unclear. To address this issue, we evaluated the involvement of an oxytocin homolog (isotocin, referred herein as oxt) in mate choice in medaka fish (Oryzias latipes). Female medaka prefer to choose familiar mates, whereas male medaka court indiscriminately, irrespective of familiarity. We generated mutants of the oxt ligand (oxt) and receptor genes (oxtr1 and oxtr2) and revealed that the oxt-oxtr1 signaling pathway was essential for eliciting female mate preference for familiar males. This pathway was also required for unrestricted and indiscriminate mating strategy in males. That is, either oxt or oxtr1 mutation in males decreased the number of courtship displays toward novel females, but not toward familiar females. Further, males with these mutations exhibited enhanced mate-guarding behaviors toward familiar females, but not toward novel females. In addition, RNA-sequencing (seq) analysis revealed that the transcription of genes involved in gamma-amino butyric acid metabolism as well as those encoding ion-transport ATPase are up-regulated in both oxt and oxtr1 mutants only in female medaka, potentially explaining the sex difference of the mutant phenotype. Our findings provide genetic evidence that oxt-oxtr1 signaling plays a role in the mate choice for familiar individuals in a sex-specific manner in medaka fish.

Forced isoform switching of Neat1_1 to Neat1_2 leads to the loss of Neat1_1 and the hyperformation of paraspeckles but does not affect the development and growth of mice.
Isobe M, Toya H, Mito M, Chiba T, Asahara H, Hirose T, Nakagawa S
RNA (New York, N.Y.), 26, 3, 251, 264, 2020年03月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Neat1 is a long noncoding RNA (lncRNA) that serves as an architectural component of the nuclear bodies known as paraspeckles. Two isoforms of Neat1, the short isoform Neat1_1 and the long isoform Neat1_2, are generated from the same gene locus by alternative 3' processing. Neat1_1 is the most abundant and the best conserved isoform expressed in various cell types, whereas Neat1_2 is expressed in a small population of particular cell types, including the tip cells of the intestinal epithelium. To investigate the physiological significance of isoform switching, we created mutant mice that solely expressed Neat1_2 by deleting the upstream polyadenylation (poly-A) signal (PAS) required for the production of Neat1_1. We observed the loss of Neat1_1 and strong up-regulation of Neat1_2 in various tissues and cells and the subsequent hyperformation of paraspeckles, especially in cells that normally express Neat1_2. However, the mutant mice were born at the expected Mendelian ratios and did not exhibit obvious external and histological abnormalities. These observations suggested that the hyperformation of paraspeckles does not interfere with the development and growth of these animals under normal laboratory conditions.

Architectural RNAs for Membraneless Nuclear Body Formation.
Tomohiro Yamazaki, Shinichi Nakagawa, Tetsuro Hirose
Cold Spring Harbor symposia on quantitative biology, 84, 227, 237, 2020年02月04日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Long noncoding RNAs (lncRNAs) are fundamental regulators of various cellular processes. A subset of lncRNAs, termed architectural RNAs (arcRNAs), function in the formation and maintenance of phase-separated membraneless organelles in multiple eukaryotic species. These membraneless organelles represent an important type of compartmentalization in the crowded cellular environment and have several distinct features. The NEAT1_2 lncRNA is a well-characterized arcRNA that functions as an essential scaffold of paraspeckle nuclear bodies. Here, we describe the biogenesis of paraspeckles on arcRNAs through phase separation, focusing on the specific functions of multiple NEAT1_2 RNA domains and their partner RNA-binding proteins. Finally, we present an updated model of paraspeckle formation and discuss future perspectives of research into arcRNA-instructed architectures of phase-separated nuclear bodies.

What is the switch for coupling transcription and splicing? RNA Polymerase II C-terminal domain phosphorylation, phase separation and beyond.
Maita H, Nakagawa S
Wiley interdisciplinary reviews. RNA, 11, 1, e1574, 2020年01月, ［査読有り］, ［最終著者］, ［国際誌］
英語, Phosphorylation of the RNA polymerase II C-terminal domain (Pol II CTD) has important roles in the kinetic coupling of splicing with transcription, which is essential for many genes to maintain correct splicing patterns. However, because of the extensively repeated low complexity sequences of Pol II CTD, it was unclear how phosphorylation-dependent molecular interactions were able to provide sufficient specificity to spatiotemporally partition various cotranscriptional events. Here we try to view the molecular mechanisms governing cotranscriptional splicing from the role of phase separation based on recent studies showing the ability of Pol II CTD to form droplets. This article is categorized under:   RNA Processing > Splicing Regulation/Alternative Splicing   RNA Processing > Splicing Mechanisms   RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

The long noncoding RNA NEAT1_1 is seemingly dispensable for normal tissue homeostasis and cancer cell growth.
Adriaens C, Rambow F, Bervoets G, Silla T, Mito M, Chiba T, Asahara H, Hirose T, Nakagawa S, Jensen TH, Marine JC
RNA (New York, N.Y.), 25, 12, 1681, 1695, 2019年12月, ［査読有り］, ［国際共著］
研究論文（学術雑誌）

Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD.
Gurumurthy CB, Sato M, Nakamura A, Inui M, Kawano N, Islam MA, Ogiwara S, Takabayashi S, Matsuyama M, Nakagawa S, Miura H, Ohtsuka M
Nature protocols, 14, 8, 2452, 2482, 2019年08月, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes ∼6 weeks to generate the founder mice.

Mammalian NSUN2 introduces 5-methylcytidines into mitochondrial tRNAs.
Shinoda S, Kitagawa S, Nakagawa S, Wei FY, Tomizawa K, Araki K, Araki M, Suzuki T, Suzuki T
Nucleic acids research, 47, 16, 8734, 8745, 2019年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Post-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles in mitochondrial protein synthesis, which produces respiratory chain complexes. In this study, we took advantage of mass spectrometric analysis to map 5-methylcytidine (m5C) at positions 48-50 in eight mouse and six human mt-tRNAs. We also confirmed the absence of m5C in mt-tRNAs isolated from Nsun2 knockout (KO) mice, as well as from NSUN2 KO human culture cells. In addition, we successfully reconstituted m5C at positions 48-50 of mt-tRNA in vitro with NSUN2 protein in the presence of S-adenosylmethionine. Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy clearly revealed NSUN2 foci inside mitochondria. These observations provide novel insights into the role of NSUN2 in the physiology and pathology of mitochondrial functions.

Molecular anatomy of the architectural NEAT1 noncoding RNA: The domains, interactors, and biogenesis pathway required to build phase-separated nuclear paraspeckles.
Hirose T, Yamazaki T, Nakagawa S
Wiley interdisciplinary reviews. RNA, 10, 6, e1545, 2019年05月, ［査読有り］, ［最終著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Long noncoding RNAs (lncRNAs) are extremely diverse and have various significant physiological functions. lncRNAs generally associate with specific sets of RNA-binding proteins (RBPs) to form functional ribonucleoprotein (RNP) complexes. NEAT1 is a highly abundant lncRNA in the mammalian cell nucleus that associates with specific RBPs to form NEAT1 RNPs. Intriguingly, cellular NEAT1 RNPs are extraordinarily large and can be detected using an optical microscope. These gigantic RNPs, so-called paraspeckles, are a type of membraneless nuclear body. Paraspeckles contain approximately 50 NEAT1 RNA molecules together with characteristic RBPs possessing aggregation-prone prion-like domains. Paraspeckle formation proceeds on the nascent NEAT1 transcript in conjunction with NEAT1 biogenesis, which exhibits various features that differ from those exhibited by mRNA biogenesis, including a lack of introns, noncanonical 3' end formation, and nuclear retention. These unique features may be required for the mechanism of paraspeckle formation. NEAT1 possesses three distinct RNA domains (A, B, and C), which function in stabilization (A), isoform switching (B), and paraspeckle assembly (C). In particular, the central C domain contains smaller subdomains that are high-affinity binding sites for the essential paraspeckle proteins (NONO and SFPQ) that subsequently polymerize along NEAT1. Subsequent recruitment of additional essential PSPs (FUS and RBM14) induces liquid-liquid phase separation to build a massive paraspeckle structure. Thus, the molecular anatomy of the NEAT1 arcRNA provides an ideal model to understand how lncRNAs form the functional RNP machinery. This article is characterized under: RNA Export and Localization > Nuclear Export/Import RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Long noncoding RNA NEAT1 modulates immune cell functions and is suppressed in early onset myocardial infarction patients.
Gast M, Rauch B, Haghikia A, Nakagawa S, Haas J, Stroux A, Schmidt D, Schumann P, Weiss S, Jensen L, Kratzer A, Kraenkel N, Müller C, Börnigen D, Hirose T, Blankenberg S, Escher F, Kühl A, Kuss A, Meder B, Landmesser U, Zeller T, Poller W
Cardiovascular research, 2019年03月, ［査読有り］, ［国際共著］

TChIP-Seq: Cell-Type-Specific Epigenome Profiling.
Mari Mito, Mitsutaka Kadota, Shinichi Nakagawa, Shintaro Iwasaki
Journal of visualized experiments : JoVE, 143, 2019年01月23日, ［査読有り］, ［国際誌］
英語, Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

The expression of long noncoding RNA NEAT1 is reduced in schizophrenia and modulates oligodendrocytes transcription.
Katsel P, Roussos P, Fam P, Khan S, Tan W, Hirose T, Nakagawa S, Pletnikov MV, Haroutunian V
NPJ schizophrenia, 5, 1, 3, 2019年01月, ［査読有り］, ［国際共著］

UPA-seq: prediction of functional lncRNAs using differential sensitivity to UV crosslinking.
Taiwa Komatsu, Saori Yokoi, Koichi Fujii, Mari Mito, Yusuke Kimura, Shintaro Iwasaki, Shinichi Nakagawa
RNA (New York, N.Y.), 24, 12, 1785, 1802, 2018年12月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, While a large number of long noncoding RNAs (lncRNAs) are transcribed from the genome of higher eukaryotes, systematic prediction of their functionality has been challenging due to the lack of conserved sequence motifs or structures. Assuming that some lncRNAs function as large ribonucleoprotein complexes and thus are easily crosslinked to proteins upon UV irradiation, we performed RNA-seq analyses of RNAs recovered from the aqueous phase after UV irradiation and phenol-chloroform extraction (UPA-seq). As expected, the numbers of UPA-seq reads mapped to known functional lncRNAs were remarkably reduced upon UV irradiation. Comparison with ENCODE eCLIP data revealed that lncRNAs that exhibited greater decreases upon UV irradiation preferentially associated with proteins containing prion-like domains (PrLDs). Fluorescent in situ hybridization (FISH) analyses revealed the nuclear localization of novel functional lncRNA candidates, including one that accumulated at the site of transcription. We propose that UPA-seq provides a useful tool for the selection of lncRNA candidates to be analyzed in depth in subsequent functional studies.

Long noncoding RNA MALAT1 suppresses breast cancer metastasis.
Kim J, Piao HL, Kim BJ, Yao F, Han Z, Wang Y, Xiao Z, Siverly AN, Lawhon SE, Ton BN, Lee H, Zhou Z, Gan B, Nakagawa S, Ellis MJ, Liang H, Hung MC, You MJ, Sun Y, Ma L
Nature genetics, 50, 12, 1705, 1715, 2018年12月, ［査読有り］, ［国際共著］

Molecular dissection of nuclear paraspeckles: towards understanding the emerging world of the RNP milieu.
Nakagawa S, Yamazaki T, Hirose T
Open biology, 8, 10, 2018年10月, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Paraspeckles are nuclear bodies built on an architectural long noncoding RNA, NEAT1, and a series of studies have revealed their molecular components, fine internal structures and cellular and physiological functions. Emerging lines of evidence suggest that paraspeckle formation is elicited by phase separation of associating RNA-binding proteins containing intrinsically disordered regions, which induce ordered arrangement of paraspeckle components along NEAT1. In this review, we will summarize the history of paraspeckle research over the last couple of decades, especially focusing on the function and structure of the nuclear bodies. We also discuss the future directions of research on long noncoding RNAs that form 'RNP milieux', large and flexible phase-separated ribonucleoprotein complexes.

Long noncoding RNA NEAT1 (nuclear paraspeckle assembly transcript 1) is critical for phenotypic switching of vascular smooth muscle cells.
Ahmed ASI, Dong K, Liu J, Wen T, Yu L, Xu F, Kang X, Osman I, Hu G, Bunting KM, Crethers D, Gao H, Zhang W, Liu Y, Wen K, Agarwal G, Hirose T, Nakagawa S, Vazdarjanova A, Zhou J
Proceedings of the National Academy of Sciences of the United States of America, 115, 37, E8660, E8667, 2018年09月, ［査読有り］, ［国際共著］

Immune system-mediated atherosclerosis caused by deficiency of long noncoding RNA MALAT1 in ApoE-/- mice.
Gast M, Rauch BH, Nakagawa S, Haghikia A, Jasina A, Haas J, Nath N, Jensen L, Stroux A, Böhm A, Friebel J, Rauch U, Skurk C, Blankenberg S, Zeller T, Prasanth KV, Meder B, Kuss A, Landmesser U, Poller W
Cardiovascular research, 2018年08月, ［査読有り］, ［国際共著］

Functional Domains of NEAT1 Architectural lncRNA Induce Paraspeckle Assembly through Phase Separation
Tomohiro Yamazaki, Sylvie Souquere, Takeshi Chujo, Simon Kobelke, Yee Seng Chong, Archa H. Fox, Charles S. Bond, Shinichi Nakagawa, Gerard Pierron, Tetsuro Hirose
Molecular Cell, 70, 6, 1038, 1053.e7, Cell Press, 2018年06月21日, ［査読有り］, ［国際共著］
英語, 研究論文（学術雑誌）, A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction
however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation. The NEAT1 architectural noncoding RNA has a modular domain structure with distinct functions required for paraspeckle formation. Among the functional domains, the middle domain is composed of three distinct, functionally redundant, subdomains that recruit NONO dimers to initiate assembly of the phase-separated paraspeckle structure.

CO2-sensitive tRNA modification associated with human mitochondrial disease.
Huan Lin, Kenjyo Miyauchi, Tai Harada, Ryo Okita, Eri Takeshita, Hirofumi Komaki, Kaoru Fujioka, Hideki Yagasaki, Yu-Ichi Goto, Kaori Yanaka, Shinichi Nakagawa, Yuriko Sakaguchi, Tsutomu Suzuki
Nature communications, 9, 1, 1875, 1875, 2018年05月14日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, It has been generally thought that tRNA modifications are stable and static, and their frequencies are rarely regulated. N6-threonylcarbamoyladenosine (t6A) occurs at position 37 of five mitochondrial (mt-)tRNA species. We show that YRDC and OSGEPL1 are responsible for t6A37 formation, utilizing L-threonine, ATP, and CO2/bicarbonate as substrates. OSGEPL1-knockout cells exhibit respiratory defects and reduced mitochondrial translation. We find low level of t6A37 in mutant mt-tRNA isolated from the MERRF-like patient's cells, indicating that lack of t6A37 results in pathological consequences. Kinetic measurements of t6A37 formation reveal that the Km value of CO2/bicarbonate is extremely high (31 mM), suggesting that CO2/bicarbonate is a rate-limiting factor for t6A37 formation. Consistent with this, we observe a low frequency of t6A37 in mt-tRNAs isolated from human cells cultured without bicarbonate. These findings indicate that t6A37 is regulated by sensing intracellular CO2/bicarbonate concentration, implying that mitochondrial translation is modulated in a codon-specific manner under physiological conditions.

Paraspeckles: Where Long Noncoding RNA Meets Phase Separation
Archa H. Fox, Shinichi Nakagawa, Tetsuro Hirose, Charles S. Bond
Trends in Biochemical Sciences, 43, 2, 124, 135, Elsevier Ltd, 2018年02月01日, ［査読有り］, ［国際共著］
英語, Long noncoding RNA (lncRNA) molecules are some of the newest and least understood players in gene regulation. Hence, we need good model systems with well-defined RNA and protein components. One such system is paraspeckles – protein-rich nuclear organelles built around a specific lncRNA scaffold. New discoveries show how paraspeckles are formed through multiple RNA–protein and protein–protein interactions, some of which involve extensive polymerization, and others with multivalent interactions driving phase separation. Once formed, paraspeckles influence gene regulation through sequestration of component proteins and RNAs, with subsequent depletion in other compartments. Here we focus on the dual aspects of paraspeckle structure and function, revealing an emerging role for these dynamic bodies in a multitude of cellular settings.

Cell Type-Specific Survey of Epigenetic Modifications by Tandem Chromatin Immunoprecipitation Sequencing.
Mari Mito, Mitsutaka Kadota, Kaori Tanaka, Yasuhide Furuta, Kuniya Abe, Shintaro Iwasaki, Shinichi Nakagawa
Scientific reports, 8, 1, 1143, 1143, 2018年01月18日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, The nervous system of higher eukaryotes is composed of numerous types of neurons and glia that together orchestrate complex neuronal responses. However, this complex pool of cells typically poses analytical challenges in investigating gene expression profiles and their epigenetic basis for specific cell types. Here, we developed a novel method that enables cell type-specific analyses of epigenetic modifications using tandem chromatin immunoprecipitation sequencing (tChIP-Seq). FLAG-tagged histone H2B, a constitutive chromatin component, was first expressed in Camk2a-positive pyramidal cortical neurons and used to purify chromatin in a cell type-specific manner. Subsequent chromatin immunoprecipitation using antibodies against H3K4me3-a chromatin modification mainly associated with active promoters-allowed us to survey the histone modifications in Camk2a-positive neurons. Indeed, tChIP-Seq identified hundreds of H3K4me3 modifications in promoter regions located upstream of genes associated with neuronal functions and genes with unknown functions in cortical neurons. tChIP-Seq provides a versatile approach to investigating the epigenetic modifications of particular cell types in vivo.

Neat1 is a p53-inducible lincRNA essential for transformation suppression
Stephano S. Mello, Carolyn Sinow, Nitin Raj, Pawel K. Mazur, Kathryn Bieging-Rolett, Daniela Kenzelmann Broz, Jamie F. Conklin Imam, Hannes Vogel, Laura D. Wood, Julien Sage, Tetsuro Hirose, Shinichi Nakagawa, John Rinn, Laura D. Attardi
GENES & DEVELOPMENT, 31, 11, 1095, 1108, COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2017年06月, ［査読有り］
英語, 研究論文（学術雑誌）, The p53 gene is mutated in over half of all cancers, reflecting its critical role as a tumor suppressor. Although p53 is a transcriptional activator that induces myriad target genes, those p53-inducible genes most critical for tumor suppression remain elusive. Here, we leveraged p53 ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) and RNA-seq (RNA sequencing) data sets to identify new p53 target genes, focusing on the noncoding genome. We identify Neat1, a noncoding RNA (ncRNA) constituent of paraspeckles, as a p53 target gene broadly induced by mouse and human p53 in different cell types and by diverse stress signals. Using fibroblasts derived from Neat1(-/-) mice, we examined the functional role of Neat1 in the p53 pathway. We found that Neat1 is dispensable for cell cycle arrest and apoptosis in response to genotoxic stress. In sharp contrast, Neat1 plays a crucial role in suppressing transformation in response to oncogenic signals. Neat1 deficiency enhances transformation in oncogene-expressing fibroblasts and promotes the development of premalignant pancreatic intraepithelial neoplasias (PanINs) and cystic lesions in Kras(G12D)-expressing mice. Neat1 loss provokes global changes in gene expression, suggesting a mechanism by which its deficiency promotes neoplasia. Collectively, these findings identify Neat1 as a p53-regulated large intergenic ncRNA (lincRNA) with a key role in suppressing transformation and cancer initiation, providing fundamental new insight into p53-mediated tumor suppression.

Unusual semi-extractability as a hallmark of nuclear body-associated architectural noncoding RNAs
Takeshi Chujo, Tomohiro Yamazaki, Tetsuya Kawaguchi, Satoshi Kurosaka, Toru Takumi, Shinichi Nakagawa, Tetsuro Hirose
EMBO JOURNAL, 36, 10, 1447, 1462, WILEY, 2017年05月, ［査読有り］
英語, 研究論文（学術雑誌）, NEAT1_2 long noncoding RNA (lncRNA) is the molecular scaffold of paraspeckle nuclear bodies. Here, we report an improved RNA extraction method: extensive needle shearing or heating of cell lysate in RNA extraction reagent improved NEAT1_2 extraction by 20-fold (a property we term "semi-extractability"), whereas using a conventional method NEAT1_2 was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50 NEAT1_2 molecules are present in a single paraspeckle. Another architectural lncRNA, IGS16, also exhibited similar semi-extractability. A comparison of RNA-seq data from needle-sheared and control samples revealed the existence of multiple semi-extractable RNAs, many of which were localized in sub-nuclear granule-like structures. The semi-extractability of NEAT1_2 correlated with its association with paraspeckle proteins and required the prion-like domain of the RNA-binding protein FUS. This observation suggests that tenacious RNA-protein and protein-protein interactions, which drive nuclear body formation, are responsible for semi-extractability. Our findings provide a foundation for the discovery of the architectural RNAs that constitute nuclear bodies.

Non-coding RNAs in cardiovascular diseases: diagnostic and therapeutic perspectives.
Poller W, Dimmeler S, Heymans S, Zeller T, Haas J, Karakas M, Leistner DM, Jakob P, Nakagawa S, Blankenberg S, Engelhardt S, Thum T, Weber C, Meder B, Hajjar R, andmesser U
European heart journal, 2017年04月, ［査読有り］

Malat1 regulates myogenic differentiation and muscle regeneration through modulating MyoD transcriptional activity
Xiaona Chen, Liangqiang He, Yu Zhao, Yuying Li, Suyang Zhang, Kun Sun, Karl So, Fengyuan Chen, Liang Zhou, Leina Lu, Lijun Wang, Xihua Zhu, Xichen Bao, Miguel A. Esteban, Shinichi Nakagawa, Kannanganattu V. Prasanth, Zhenguo Wu, Hao Sun, Huating Wang
CELL DISCOVERY, 3, 17002, NATURE PUBLISHING GROUP, 2017年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Malat1 is one of the most abundant long non-coding RNAs in various cell types; its exact cellular function is still a matter of intense investigation. In this study we characterized the function of Malat1 in skeletal muscle cells and muscle regeneration. Utilizing both in vitro and in vivo assays, we demonstrate that Malat1 has a role in regulating gene expression during myogenic differentiation of myoblast cells. Specifically, we found that knockdown of Malat1 accelerates the myogenic differentiation in cultured cells. Consistently, Malat1 knockout mice display enhanced muscle regeneration after injury and deletion of Malat1 in dystrophic mdx mice also improves the muscle regeneration. Mechanistically, in the proliferating myoblasts, Malat1 recruits Suv39h1 to MyoD-binding loci, causing trimethylation of histone 3 lysine 9 (H3K9me3), which suppresses the target gene expression. Upon differentiation, the pro-myogenic miR-181a is increased and targets the nuclear Malat1 transcripts for degradation through Ago2-dependent nuclear RNA-induced silencing complex machinery; the Malat1 decrease subsequently leads to the destabilization of Suv39h1/HP1 beta/HDAC1-repressive complex and displacement by a Set7-containing activating complex, which allows MyoD trans-activation to occur. Together, our findings identify a regulatory axis of miR-181a-Malat1-MyoD/Suv39h1 in myogenesis and uncover a previously unknown molecular mechanism of Malat1 action in gene regulation.

Misregulation of an Activity-Dependent Splicing Network as a Common Mechanism Underlying Autism Spectrum Disorders
Mathieu Quesnel-Vallieres, Zahra Dargaei, Manuel Irimia, Thomas Gonatopoulos-Pournatzis, Joanna Y. Ip, Mingkun Wu, Timothy Sterne-Weiler, Shinichi Nakagawa, Melanie A. Woodin, Benjamin J. Blencowe, Sabine P. Cordes
MOLECULAR CELL, 64, 6, 1023, 1034, CELL PRESS, 2016年12月, ［査読有り］
英語, 研究論文（学術雑誌）, A key challenge in understanding and ultimately treating autism is to identify common molecular mechanisms underlying this genetically heterogeneous disorder. Transcriptomic profiling of autistic brains has revealed correlated misregulation of the neuronal splicing regulator nSR100/SRRM4 and its target microexon splicing program in more than one-third of analyzed individuals. To investigate whether nSR100 misregulation is causally linked to autism, we generated mutant mice with reduced levels of this protein and its target splicing program. Remarkably, these mice display multiple autistic-like features, including altered social behaviors, synaptic density, and signaling. Moreover, increased neuronal activity, which is often associated with autism, results in a rapid decrease in nSR100 and splicing of microexons that significantly overlap those misregulated in autistic brains. Collectively, our results provide evidence that misregulation of an nSR100-dependent splicing network controlled by changes in neuronal activity is causally linked to a substantial fraction of autism cases.

Control of Chromosomal Localization of Xist by hnRNP U Family Molecules
Takehisa Sakaguchi, Yuko Hasegawa, Neil Brockdorff, Ken Tsutsui, Kimiko M. Tsutsui, Takashi Sado, Shinichi Nakagawa
DEVELOPMENTAL CELL, 39, 1, 11, 12, CELL PRESS, 2016年10月, ［査読有り］
英語

Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization
Jason A. West, Mari Mito, Satoshi Kurosaka, Toru Takumi, Chiharu Tanegashima, Takeshi Chujo, Kaori Yanaka, Robert E. Kingston, Tetsuro Hirose, Charles Bond, Archa Fox, Shinichi Nakagawa
JOURNAL OF CELL BIOLOGY, 214, 7, 817, 830, ROCKEFELLER UNIV PRESS, 2016年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Paraspeckles are nuclear bodies built on the long noncoding RNA Neat1, which regulates a variety of physiological processes including cancer progression and corpus luteum formation. To obtain further insight into the molecular basis of the function of paraspeckles, we performed fine structural analyses of these nuclear bodies using structural illumination microscopy. Notably, paraspeckle proteins are found within different layers along the radially arranged bundles of Neat1 transcripts, forming a characteristic core-shell spheroidal structure. In cells lacking the RNA binding protein Fus, paraspeckle spheroids are disassembled into smaller particles containing Neat1, which are diffusely distributed in the nucleoplasm. Sequencing analysis of RNAs purified from paraspeckles revealed that AG-rich transcripts associate with Neat1, which are distributed along the shell of the paraspeckle spheroids. We propose that paraspeckles sequester core components inside the spheroids, whereas the outer surface associates with other components in the nucleoplasm to fulfill their function.

Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA
Aparna Anantharaman, Mahdieh Jadaliha, Vidisha Tripathi, Shinichi Nakagawa, Tetsuro Hirose, Michael F. Jantsch, Supriya G. Prasanth, Kannanganattu V. Prasanth
SCIENTIFIC REPORTS, 6, 34043, NATURE PUBLISHING GROUP, 2016年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Paraspeckles are sub-nuclear domains that are nucleated by long noncoding RNA Neat1. While interaction of protein components of paraspeckles and Neat1 is understood, there is limited information on the interaction of non-structural RNA components with paraspeckles. Here, by varying paraspeckle number and size, we investigate how paraspeckles influence the nuclear organization of their nonstructural RNA component Ctn RNA. Our results show that Ctn RNA remains nuclear-retained in the absence of intact paraspeckles, suggesting that they do not regulate nuclear retention of Ctn RNA. In the absence of Neat1, Ctn RNA continues to interact with paraspeckle protein NonO to form residual nuclear foci. In addition, in the absence of Neat1-nucleated paraspeckles, a subset of Ctn RNA localizes to the perinucleolar regions. Concomitant with increase in number of paraspeckles, transcriptional reactivation resulted in increased number of paraspeckle-localized Ctn RNA foci. Similar to Neat1, proteasome inhibition altered the localization of Ctn RNA, where it formed enlarged paraspeckle-like foci. Super-resolution structured illumination microscopic analyses revealed that in paraspeckles, Ctn RNA partially co-localized with Neat1, and displayed a more heterogeneous intra-paraspeckle localization. Collectively, these results show that while paraspeckles do not influence nuclear retention of Ctn RNA, they modulate its intranuclear compartmentalization.

p53 induces formation of NEAT1 lncRNA-containing paraspeckles that modulate replication stress response and chemosensitivity
Carmen Adriaens, Laura Standaert, Jasmine Barra, MathilDe latil, Annelien Verfaillie, Peter Kalev, Bram Boeckx, Paul W. G. Wijnhoven, Enrico Radaelli, William Vermi, Eleonora Leucci, Gaelle Lapouge, Benjamin Beck, Joost van den Oord, Shinichi Nakagawa, Tetsuro Hirose, Anna A. Sablina, Diether Lambrechts, Stein Aerts, Cedric Blanpain, Jean-Christophe Marine
NATURE MEDICINE, 22, 8, 861, +, NATURE PUBLISHING GROUP, 2016年08月, ［査読有り］
英語, 研究論文（学術雑誌）, In a search for mediators of the p53 tumor suppressor pathway, which induces pleiotropic and often antagonistic cellular responses, we identified the long noncoding RNA (lncRNA) NEAT1. NEAT1 is an essential architectural component of paraspeckle nuclear bodies, whose pathophysiological relevance remains unclear. Activation of p53, pharmacologically or by oncogene-induced replication stress, stimulated the formation of paraspeckles in mouse and human cells. Silencing Neat1 expression in mice, which prevents paraspeckle formation, sensitized preneoplastic cells to DNA-damage-induced cell death and impaired skin tumorigenesis. We provide mechanistic evidence that NEAT1 promotes ATR signaling in response to replication stress and is thereby engaged in a negative feedback loop that attenuates oncogene-dependent activation of p53. NEAT1 targeting in established human cancer cell lines induced synthetic lethality with genotoxic chemotherapeutics, including PARP inhibitors, and nongenotoxic activation of p53. This study establishes a key genetic link between NEAT1 paraspeckles, p53 biology and tumorigenesis and identifies NEAT1 as a promising target to enhance sensitivity of cancer cells to both chemotherapy and p53 reactivation therapy.

Gomafu IncRNA knockout mice exhibit mild hyperactivity with enhanced responsiveness to the psychostimulant methamphetamine
Joanna Y. Ip, Masamitsu Sone, Chieko Nashiki, Qun Pan, Kiyoyuki Kitaichi, Kaori Yanaka, Takaya Abe, Keizo Takao, Tsuyoshi Miyakawa, Benjamin J. Blencowe, Shinichi Nakagawa
SCIENTIFIC REPORTS, 6, 27204, NATURE PUBLISHING GROUP, 2016年06月, ［査読有り］
英語, 研究論文（学術雑誌）, The long noncoding RNA Gomafu/MIAT/Rncr2 is thought to function in retinal cell specification, stem cell differentiation and the control of alternative splicing. To further investigate physiological functions of Gomafu, we created mouse knockout (KO) model that completely lacks the Gomafu gene. The KO mice did not exhibit any developmental deficits. However, behavioral tests revealed that the KO mice are hyperactive. This hyperactive behavior was enhanced when the KO mice were treated with the psychostimulant methamphetamine, which was associated with an increase in dopamine release in the nucleus accumbens. RNA sequencing analyses identified a small number of genes affected by the deficiency of Gomafu, a subset of which are known to have important neurobiological functions. These observations suggest that Gomafu modifies mouse behavior thorough a mild modulation of gene expression and/or alternative splicing of target genes.

Simultaneous multicolor detection of RNA and proteins using super-resolution microscopy
Mari Mito, Tetsuya Kawaguchi, Tetsuro Hirose, Shinichi Nakagawa
METHODS, 98, 158, 165, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2016年04月, ［査読有り］
英語, 研究論文（学術雑誌）, A number of non-membranous cellular bodies have been identified in higher eukaryotes, and these bodies contain a specific set of proteins and RNAs that are used to fulfill their functions. The size of these RNA-containing cellular bodies is usually on a submicron scale, making it difficult to observe fine structures using optical microscopy due to the diffraction limitation of visible light. Recently, microscope companies have released super-resolution microscopes that were developed using different principles, enabling the observation of sub-micron structures not resolvable in conventional fluorescent microscopy. Here, we describe multi-color fluorescent in situ hybridization techniques optimized for the simultaneous detection of RNA and proteins using super-resolution microscopy, namely structured illumination microscopy (SIM). (C) 2015 The Authors. Published by Elsevier Inc.

Long noncoding RNA MALAT1-derived mascRNA is involved in cardiovascular innate immunity
Martina Gast, Blanche Schroen, Antje Voigt, Jan Haas, Uwe Kuehl, Dirk Lassner, Carsten Skurk, Felicitas Escher, Xiaomin Wang, Adelheid Kratzer, Katharina Michalik, Anna Papageorgiou, Tim Peters, Madlen Loebel, Sabrina Wilk, Nadine Althof, Kannanganattu V. Prasanth, Hugo Katus, Benjamin Meder, Shinichi Nakagawa, Carmen Scheibenbogen, Heinz-Peter Schultheiss, Ulf Landmesser, Stefanie Dimmeler, Stephane Heymans, Wolfgang Poller
JOURNAL OF MOLECULAR CELL BIOLOGY, 8, 2, 178, 181, OXFORD UNIV PRESS, 2016年04月, ［査読有り］
英語

Natural antisense RNA promotes 3' end processing and maturation of MALAT1 lncRNA
Xinying Zong, Shinichi Nakagawa, Susan M. Freier, Jingyi Fei, Taekjip Ha, Supriya G. Prasanth, Kannanganattu V. Prasanth
NUCLEIC ACIDS RESEARCH, 44, 6, 2898, 2908, OXFORD UNIV PRESS, 2016年04月, ［査読有り］
英語, 研究論文（学術雑誌）, The RNase P-mediated endonucleolytic cleavage plays a crucial role in the 3' end processing and cellular accumulation of MALAT1, a nuclear-retained long noncoding RNA that promotes malignancy. The regulation of this cleavage event is largely undetermined. Here we characterize a broadly expressed natural antisense transcript at the MALAT1 locus, designated as TALAM1, that positively regulates MALAT1 levels by promoting the 3' end cleavage and maturation of MALAT1 RNA. TALAM1 RNA preferentially localizes at the site of transcription, and also interacts with MALAT1 RNA. Depletion of TALAM1 leads to defects in the 3' end cleavage reaction and compromises cellular accumulation of MALAT1. Conversely, overexpression of TALAM1 facilitates the cleavage reaction in trans. Interestingly, TALAM1 is also positively regulated by MALAT1 at the level of both transcription and RNA stability. Together, our data demonstrate a novel feed-forward positive regulatory loop that is established to maintain the high cellular levels of MALAT1, and also unravel the existence of sense-antisense mediated regulatory mechanism for cellular lncRNAs that display RNase P-mediated 3' end processing.

Long Non-Coding RNA Malat-1 Is Dispensable during Pressure Overload-Induced Cardiac Remodeling and Failure in Mice
Tim Peters, Steffie Hermans-Beijnsberger, Abdelaziz Beqqali, Nicole Bitsch, Shinichi Nakagawa, Kannanganattu V. Prasanth, Leon J. de Windt, Ralph J. van Oort, Stephane Heymans, Blanche Schroen
PLOS ONE, 11, 2, e0150236, PUBLIC LIBRARY SCIENCE, 2016年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Background
Long non-coding RNAs (lncRNAs) are a class of RNA molecules with diverse regulatory functions during embryonic development, normal life, and disease in higher organisms. However, research on the role of lncRNAs in cardiovascular diseases and in particular heart failure is still in its infancy. The exceptionally well conserved nuclear lncRNA Metastasis associated in lung adenocarcinoma transcript 1 (Malat-1) is a regulator of mRNA splicing and highly expressed in the heart. Malat-1 modulates hypoxia-induced vessel growth, activates ERK/MAPK signaling, and scavenges the anti-hypertrophic microRNA-133. We therefore hypothesized that Malat-1 may act as regulator of cardiac hypertrophy and failure during cardiac pressure overload induced by thoracic aortic constriction (TAC) in mice.
Results
Absence of Malat-1 did not affect cardiac hypertrophy upon pressure overload: Heart weight to tibia length ratio significantly increased in WT mice (sham: 5.78 +/- 0.55, TAC 9.79 +/- 1.82 g/mm; p<0.001) but to a similar extend also in Malat-1 knockout (KO) mice (sham: 6.21 +/- 1.12, TAC 8.91 +/- 1.74 g/mm; p<0.01) with no significant difference between genotypes. As expected, TAC significantly reduced left ventricular fractional shortening in WT (sham: 38.81 +/- 6.53%, TAC: 23.14 +/- 11.99%; p<0.01) but to a comparable degree also in KO mice (sham: 37.01 +/- 4.19%, TAC: 25.98 +/- 9.75%; p<0.05). Histological hallmarks of myocardial remodeling, such as cardiomyocyte hypertrophy, increased interstitial fibrosis, reduced capillary density, and immune cell infiltration, did not differ significantly between WT and KO mice after TAC. In line, the absence of Malat-1 did not significantly affect angiotensin II-induced cardiac hypertrophy, dysfunction, and overall remodeling. Above that, pressure overload by TAC significantly induced mRNA levels of the hypertrophy marker genes Nppa, Nppb and Acta1, to a similar extend in both genotypes. Alternative splicing of Ndrg2 after TAC was apparent in WT (isoform ratio; sham: 2.97 +/- 0.26, TAC 1.57 +/- 0.40; p<0.0001) and KO mice (sham: 3.64 +/- 0.37; TAC: 2.24 +/- 0.76; p<0.0001) and interestingly differed between genotypes both at baseline and after pressure overload (p<0.05 each).
Conclusion
These findings confirm a role for the lncRNA Malat-1 in mRNA splicing. However, no critical role for Malat-1 was found in pressure overload-induced heart failure in mice, despite its reported role in vascularization, ERK/MAPK signaling, and regulation of miR-133.

MALAT1 long non-coding RNA in cancer
Rei Yoshimoto, Akila Mayeda, Minoru Yoshida, Shinichi Nakagawa
BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS, 1859, 1, 192, 199, ELSEVIER SCIENCE BV, 2016年01月, ［査読有り］
英語, 研究論文（学術雑誌）, A recent massive parallel sequencing analysis has shown the fact that more than 80% of the human genome is transcribed into RNA. Among many kinds of the non-protein coding RNAs, we focus on the metastasis associated lung adenocarcinoma transcript 1 (MALAT1) that is a long non-coding RNA upregulated in metastatic carcinoma cells. Two molecular functions of MALAT1 have been proposed, one is the control of alternative splicing and the other is the transcriptional regulation. In this review, we document the molecular characteristics and functions of MALAT1 and shed light on the implication in the molecular pathology of various cancers. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. (C) 2015 Elsevier B.V. All rights reserved.

Clues to long noncoding RNA taxonomy.
Hirose T, Nakagawa S
Biochimica et biophysica acta, 1859, 1, 1, 2, 2016年01月, ［査読有り］

Regulation of gene expression via retrotransposon insertions and the noncoding RNA 4.5S RNA(H)
Kentaro Ishida, Kenjyo Miyauchi, Yuko Kimura, Mari Mito, Shunpei Okada, Tsutomu Suzuki, Shinichi Nakagawa
GENES TO CELLS, 20, 11, 887, 901, WILEY-BLACKWELL, 2015年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Short interspersed elements (SINEs) comprise a significant portion of mammalian genomes and regulate gene expression through a variety of mechanisms. Here, we show that Myodonta clade-specific 4.5S RNA(H) (4.5SH), an abundant nuclear noncoding RNA that is highly homologous to the retrotransposon SINE B1, controls the expression of reporter gene that contains the antisense insertion of SINE B1 via nuclear retention. The depletion of endogenous 4.5SH with antisense oligonucleotides neutralizes the nuclear retention and changes the subcellular distribution of the reporter transcripts containing the antisense SINE B1 insertion. Importantly, endogenous transcripts with antisense SINE B1 were increased in the cytoplasm after knockdown of 4.5SH, leading to a decrease in cellular growth. We propose a tentative hypothesis that the amplification of the 4.5SH cluster in specific rodent species might delineate their evolutionary direction via the regulation of genes containing the antisense insertion of SINE B1.

MALAT1 long non-coding RNA in cancer.
Yoshimoto R, Mayeda A, Yoshida M, Nakagawa S
Biochimica et biophysica acta, 1859, 1, 192, 199, 2015年10月, ［査読有り］

Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome
Norishige Yamada, Yuko Hasegawa, Minghui Yue, Tomofumi Hamada, Shinichi Nakagawa, Yuya Ogawa
PLoS Genetics, 11, 8, e1005430, Public Library of Science, 2015年08月01日, ［査読有り］
英語, 研究論文（学術雑誌）, To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

Lessons from reverse-genetic studies of lncRNAs.
Nakagawa S
Biochimica et biophysica acta, 1859, 1, 177, 183, 2015年06月, ［査読有り］

Long noncoding RNA in epigenetic gene regulation
Yuko Hasegawa, Shinichi Nakagawa
Long Noncoding RNAs Structures and Functions, 133, 149, Springer Japan, 2015年01月01日, ［査読有り］
英語, 論文集(書籍)内論文, Recent studies have revealed the functional signifi cance of long noncoding RNA (lncRNA) in various biological processes including epigenetic gene regulations. Genomic imprinting is one of the epigenetic processes related to lncRNA in mammals, which controls parent-of-origin-specific gene expression essential for normal development. To date, over 100 genes have been recognized as imprinted genes, the majority of them form clusters on the genome. Each of these imprinting clusters contains DNA regulatory elements called imprinting control regions (ICRs), which are frequently located near the lncRNA genes. In some cases, genetically modifi ed mice and human patients exhibiting imprinting disorder show aberrant expression of these lncRNAs, suggesting a close relationship between genomic imprinting and lncRNAs. DNA methylation and histone modifi cations are the principal molecular mechanisms of epigenetic gene regulation, and recent progress has uncovered diversities and complexities of the lncRNA actions in these processes. In this chapter, we summarize research on genomic imprinting by focusing on the role of lncRNAs to provide insight into lncRNA-mediated regulation of gene expression.

Super-resolution imaging of nuclear bodies by STED microscopy.
Yasushi Okada, Shinichi Nakagawa
Methods in molecular biology (Clifton, N.J.), 1262, 21, 35, 2015年, ［査読有り］, ［国際誌］
英語, The sizes of nuclear bodies and other nuclear structures are normally no more than a few hundred nanometers. This size is below the resolution limit of light microscopy and thus requires electron microscopy for direct observation. Recent developments in super-resolution microscopy have extended the resolution of light microscopy to beyond 100 nm. Here, we describe a super-resolution technique, gated STED, for the analysis of the structure of nuclear bodies, with emphasis on the sample preparation and other technical tips that are important to obtain high-quality super-resolution images.

SiRNA screening of nuclear proteins.
Hasegawa Y, Nakagawa S
Methods in molecular biology (Clifton, N.J.), 1262, 333, 348, 2015年, ［査読有り］

The long noncoding RNA Neat1 is required for mammary gland development and lactation
Laura Standaert, Carmen Adriaens, Enrico Radaelli, Alexandra Van Keymeulen, Cedric Blanpain, Tetsuro Hirose, Shinichi Nakagawa, Jean-Christophe Marine
RNA, 20, 12, 1844, 1849, COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2014年12月, ［査読有り］
英語, 研究論文（学術雑誌）, The lncRNA Neat1 is an essential architectural component of paraspeckle nuclear bodies. Although cell-based studies identified Neat1-paraspeckles as key regulators of gene expression through retention of hyperdited mRNAs and/or transcription factors, it is unclear under which specific physiological conditions paraspeckles are formed in vivo and whether they have any biological relevance. Herein, we show that paraspeckles are assembled in luminal epithelial cells during mammary gland development. Importantly, genetic ablation of Neat1 results in aberrant mammary gland morphogenesis and lactation defects. We provide evidence that the lactation defect is caused by a decreased ability of Neat1-mutant cells to sustain high rates of proliferation during lobular-alveolar development. This study is the first to assign an important biological function to the lncRNA Neat1 and to link it to the presence of paraspeckles nuclear bodies in vivo.

The lncRNA Neat1 is required for corpus luteum formation and the establishment of pregnancy in a subpopulation of mice
Shinichi Nakagawa, Masayuki Shimada, Kaori Yanaka, Mari Mito, Takashi Arai, Eiki Takahashi, Youko Fujita, Toshihiko Fujimori, Laura Standaert, Jean-Christophe Marine, Tetsuro Hirose
DEVELOPMENT, 141, 23, 4618, 4627, COMPANY OF BIOLOGISTS LTD, 2014年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Neat1 is a non-protein-coding RNA that serves as an architectural component of the nuclear bodies known as paraspeckles. Although cell-based studies indicate that Neat1 is a crucial regulator of gene expression, its physiological relevance remains unclear. Here, we find that Neat1 knockout (KO) mice stochastically fail to become pregnant despite normal ovulation. Unilateral transplantation of wildtype ovaries or the administration of progesterone partially rescued the phenotype, suggesting that corpus luteum dysfunction and concomitant low progesterone were the primary causes of the decreased fertility. In contrast to the faint expression observed in most of the adult tissues, Neat1 was highly expressed in the corpus luteum, and the formation of luteal tissue was severely impaired in nearly half of the Neat1 KO mice. These observations suggest that Neat1 is essential for the formation of the corpus luteum and for the subsequent establishment of pregnancy under a suboptimal condition that has not yet been identified.

The oestrogen receptor alpha-regulated lncRNA NEAT1 is a critical modulator of prostate cancer
Dimple Chakravarty, Andrea Sboner, Sujit S. Nair, Eugenia Giannopoulou, Ruohan Li, Sven Hennig, Juan Miguel Mosquera, Jonathan Pauwels, Kyung Park, Myriam Kossai, Theresa Y. MacDonald, Jacqueline Fontugne, Nicholas Erho, Ismael A. Vergara, Mercedeh Ghadessi, Elai Davicioni, Robert B. Jenkins, Nallasivam Palanisamy, Zhengming Chen, Shinichi Nakagawa, Tetsuro Hirose, Neil H. Bander, Himisha Beltran, Archa H. Fox, Olivier Elemento, Mark A. Rubin
NATURE COMMUNICATIONS, 5, 5, 5383, NATURE PUBLISHING GROUP, 2014年11月, ［査読有り］
英語, 研究論文（学術雑誌）, The androgen receptor (AR) plays a central role in establishing an oncogenic cascade that drives prostate cancer progression. Some prostate cancers escape androgen dependence and are often associated with an aggressive phenotype. The oestrogen receptor alpha (ER alpha) is expressed in prostate cancers, independent of AR status. However, the role of ER alpha remains elusive. Using a combination of chromatin immunoprecipitation (ChIP) and RNA-sequencing data, we identified an ER alpha-specific non-coding transcriptome signature. Among putatively ER alpha-regulated intergenic long non-coding RNAs (lncRNAs), we identified nuclear enriched abundant transcript 1 (NEAT1) as the most significantly overexpressed lncRNA in prostate cancer. Analysis of two large clinical cohorts also revealed that NEAT1 expression is associated with prostate cancer progression. Prostate cancer cells expressing high levels of NEAT1 were recalcitrant to androgen or AR antagonists. Finally, we provide evidence that NEAT1 drives oncogenic growth by altering the epigenetic landscape of target gene promoters to favour transcription.

Analysis of the subcellular distribution of RNA by fluorescence in situ hybridization
Shinichi Nakagawa
Regulatory Non-Coding RNAs: Methods and Protocols, 1206, 107, 122, Springer New York, 2014年09月20日, ［査読有り］
英語, 論文集(書籍)内論文, In situ hybridization is a powerful method of examining the spatial distribution of RNA molecules at the subcellular level and serves as a basic technique in the fields of cell and developmental biology. In this technique, target RNAs are fixed in cells using formaldehyde and then hybridized to complementary probes labeled with modified nucleotides that are subsequently detected by immunohistochemical methods. Here, the procedures that are commonly used for the detection of RNA in mammalian tissues and cells are described, focusing on technical tips that improve the sensitivity, productivity, and reproducibility.

Formation of nuclear bodies by the lncRNA Gomafu-associating proteins Celf3 and SF1
Akira Ishizuka, Yuko Hasegawa, Kentaro Ishida, Kaori Yanaka, Shinichi Nakagawa
GENES TO CELLS, 19, 9, 704, 721, WILEY, 2014年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Gomafu/MIAT/Rncr2 is a long noncoding RNA that has been proposed to control retinal cell specification, stem cell differentiation and alternative splicing of schizophrenia-related genes. However, how Gomafu controls these biological processes at the molecular level has remained largely unknown. In this study, we identified the RNA-binding protein Celf3 as a novel Gomafu-associating protein. Knockdown of Celf3 led to the down-regulation of Gomafu, and cross-link RNA precipitation analysis confirmed specific binding between Celf3 and Gomafu. In the neuroblastoma cell line Neuro2A, Celf3 formed novel nuclear bodies (named CS bodies) that colocalized with SF1, another Gomafu-binding protein. Gomafu, however, was not enriched in the CS bodies; instead, it formed distinct nuclear bodies in separate regions in the nucleus. These observations suggest that Gomafu indirectly modulates the function of the splicing factors SF1 and Celf3 by sequestering these proteins into separate nuclear bodies.

Yb Integrates piRNA Intermediates and Processing Factors into Perinuclear Bodies to Enhance piRISC Assembly
Yukiko Murota, Hirotsugu Ishizu, Shinichi Nakagawa, Yuka W. Iwasaki, Shinsuke Shibata, Miharu K. Kamatani, Kuniaki Saito, Hideyuki Okano, Haruhiko Siomi, Mikiko C. Siomi
CELL REPORTS, 8, 1, 103, 113, CELL PRESS, 2014年07月, ［査読有り］
英語, 研究論文（学術雑誌）, PIWI-interacting RNAs (piRNAs) direct Piwi to repress transposons and maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. Here, we show that piRNA intermediates arising from the piRNA cluster flamenco (flam) localize to perinuclear foci adjacent to Yb bodies, termed Flam bodies. RNAi-based screening of piRNA factors revealed that Flam body formation depends on Yb, the core component of Yb bodies, while Piwi and another Yb body component, Armitage, are dispensable for formation. Abolishing the RNA-binding activity of Yb disrupts both Flam bodies and Yb bodies. Yb directly binds flam, but not transcripts from neighboring protein-coding genes. Thus, Yb integrates piRNA intermediates and piRNA processing factors selectively into Flam bodies and Yb bodies, respectively. We suggest that Yb is a key upstream factor in the cytoplasmic phase of the piRNA pathway in ovarian somatic cells.

Nuclear lncRNAs as epigenetic regulators-beyond skepticism.
Nakagawa S, Kageyama Y
Biochimica et biophysica acta, 1839, 3, 215, 222, 2014年03月, ［査読有り］

Gathering around Firre
Shinichi Nakagawa, Tatsuya Hirano
NATURE STRUCTURAL & MOLECULAR BIOLOGY, 21, 3, 207, 208, NATURE PUBLISHING GROUP, 2014年03月, ［査読有り］
英語, Long noncoding RNAs (IncRNAs) have divergent roles in the nuclei of higher eukaryotes, including chromatin modification and regulation of nuclear bodies. A new study adds a new IncRNA function to the current list: serving as a platform for trans-chromosomal associations. At least three gene loci located on different chromosomes are brought together around the transcription site of a IncRNA termed functional intergenic repeating RNA element (Firre).

The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing
G. Barry, J. A. Briggs, D. P. Vanichkina, E. M. Poth, N. J. Beveridge, V. S. Ratnu, S. P. Nayler, K. Nones, J. Hu, T. W. Bredy, S. Nakagawa, F. Rigo, R. J. Taft, M. J. Cairns, S. Blackshaw, E. J. Wolvetang, J. S. Mattick
Molecular Psychiatry, 19, 4, 486, 494, Nature Publishing Group, 2014年, ［査読有り］
英語, 研究論文（学術雑誌）, Schizophrenia (SZ) is a complex disease characterized by impaired neuronal functioning. Although defective alternative splicing has been linked to SZ, the molecular mechanisms responsible are unknown. Additionally, there is limited understanding of the early transcriptomic responses to neuronal activation. Here, we profile these transcriptomic responses and show that long non-coding RNAs (lncRNAs) are dynamically regulated by neuronal activation, including acute downregulation of the lncRNA Gomafu, previously implicated in brain and retinal development. Moreover, we demonstrate that Gomafu binds directly to the splicing factors QKI and SRSF1 (serine/arginine-rich splicing factor 1) and dysregulation of Gomafu leads to alternative splicing patterns that resemble those observed in SZ for the archetypal SZ-associated genes DISC1 and ERBB4. Finally, we show that Gomafu is downregulated in post-mortem cortical gray matter from the superior temporal gyrus in SZ. These results functionally link activity-regulated lncRNAs and alternative splicing in neuronal function and suggest that their dysregulation may contribute to neurological disorders. © 2014 Macmillan Publishers Limited.

NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies
Tetsuro Hirose, Giorgio Virnicchi, Akie Tanigawa, Takao Naganuma, Ruohan Li, Hiroshi Kimura, Takahide Yokoi, Shinichi Nakagawa, Marianne Benard, Archa H. Fox, Gerard Pierron
MOLECULAR BIOLOGY OF THE CELL, 25, 1, 169, 183, AMER SOC CELL BIOLOGY, 2014年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Paraspeckles are subnuclear structures formed around nuclear paraspeckle assembly transcript 1 (NEAT1)/MEN epsilon/beta long noncoding RNA (IncRNA). Here we show that paraspeckles become dramatically enlarged after proteasome inhibition. This enlargement is mainly caused by NEAT1 transcriptional up-regulation rather than accumulation of undegraded paraspeckle proteins. Of interest, however, using immuno-electron microscopy, we find that key paraspeckle proteins become effectively depleted from the nucleoplasm by 50% when paraspeckle assembly is enhanced, suggesting a sequestration mechanism. We also perform microarrays from NEAT1-knockdown cells and find that NEAT1 represses transcription of several genes, including the RNA-specific adenosine deaminase B2 (ADARB2) gene. In contrast, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for ADARB2 transcription. This leads us to hypothesize that ADARB2 expression is controlled by NEAT1-dependent sequestration of SFPQ. Accordingly, we find that ADARB2 expression is strongly reduced upon enhanced SFPQ sequestration by proteasome inhibition, with concomitant reduction in SFPQ binding to the ADARB2 promoter. Finally, NEAT1(-/-) fibroblasts are more sensitive to proteasome inhibition, which triggers cell death, suggesting that paraspeckles/NEAT1 attenuates the cell death pathway. These data further confirm that paraspeckles are stress-responsive nuclear bodies and provide a model in which induced NEAT1 controls target gene transcription by protein sequestration into paraspeckles.

The long non-coding RNA nuclear-enriched abundant transcript 1_2 induces paraspeckle formation in the motor neuron during the early phase of amyotrophic lateral sclerosis
Nishimoto Y, Nakagawa S, Hirose T, Okano HJ, Takao M, Shibata S, Suyama S, Kuwako K, Imai T, Murayama S, Suzuki N, Okano H
EMBO J, 31, 4020, 4034, 2013年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: A long non-coding RNA (lncRNA), nuclear-enriched abundant transcript 1_2 (NEAT1_2), constitutes nuclear bodies known as "paraspeckles". Mutations of RNA binding proteins, including TAR DNA-binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), have been described in amyotrophic lateral sclerosis (ALS). ALS is a devastating motor neuron disease, which progresses rapidly to a total loss of upper and lower motor neurons, with consciousness sustained. The aim of this study was to clarify the interaction of paraspeckles with ALS-associated RNA-binding proteins, and to identify increased occurrence of paraspeckles in the nucleus of ALS spinal motor neurons. RESULTS: In situ hybridization (ISH) and ultraviolet cross-linking and immunoprecipitation were carried out to investigate interactions of NEAT1_2 lncRNA with ALS-associated RNA-binding proteins, and to test if paraspeckles form in ALS spinal motor neurons. As the results, TDP-43 and FUS/TLS were enriched in paraspeckles and bound to NEAT1_2 lncRNA directly. The paraspeckles were localized apart from the Cajal bodies, which were also known to be related to RNA metabolism. Analyses of 633 human spinal motor neurons in six ALS cases showed NEAT1_2 lncRNA was upregulated during the early stage of ALS pathogenesis. In addition, localization of NEAT1_2 lncRNA was identified in detail by electron microscopic analysis combined with ISH for NEAT1_2 lncRNA. The observation indicating specific assembly of NEAT1_2 lncRNA around the interchromatin granule-associated zone in the nucleus of ALS spinal motor neurons verified characteristic paraspeckle formation. CONCLUSIONS: NEAT1_2 lncRNA may act as a scaffold of RNAs and RNA binding proteins in the nuclei of ALS motor neurons, thereby modulating the functions of ALS-associated RNA-binding proteins during the early phase of ALS. These findings provide the first evidence of a direct association between paraspeckle formation and a neurodegenerative disease, and may shed light on the development of novel therapeutic targets for the treatment of ALS.

The long non-coding RNA nuclear-enriched abundant transcript 1-2 induces paraspeckle formation in the motor neuron during the early phase of amyotrophic lateral sclerosis
Yoshinori Nishimoto, Shinichi Nakagawa, Tetsuro Hirose, Hirotaka James Okano, Masaki Takao, Shinsuke Shibata, Satoshi Suyama, Ken-Ichiro Kuwako, Takao Imai, Shigeo Murayama, Norihiro Suzuki, Hideyuki Okano
Molecular Brain, 6, 1, 31, 2013年, ［査読有り］
英語, 研究論文（学術雑誌）, Background: A long non-coding RNA (lncRNA), nuclear-enriched abundant transcript 1-2 (NEAT1-2), constitutes nuclear bodies known as "paraspeckles". Mutations of RNA binding proteins, including TAR DNA-binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), have been described in amyotrophic lateral sclerosis (ALS). ALS is a devastating motor neuron disease, which progresses rapidly to a total loss of upper and lower motor neurons, with consciousness sustained. The aim of this study was to clarify the interaction of paraspeckles with ALS-associated RNA-binding proteins, and to identify increased occurrence of paraspeckles in the nucleus of ALS spinal motor neurons. Results: In situ hybridization (ISH) and ultraviolet cross-linking and immunoprecipitation were carried out to investigate interactions of NEAT1-2 lncRNA with ALS-associated RNA-binding proteins, and to test if paraspeckles form in ALS spinal motor neurons. As the results, TDP-43 and FUS/TLS were enriched in paraspeckles and bound to NEAT1-2 lncRNA directly. The paraspeckles were localized apart from the Cajal bodies, which were also known to be related to RNA metabolism. Analyses of 633 human spinal motor neurons in six ALS cases showed NEAT1-2 lncRNA was upregulated during the early stage of ALS pathogenesis. In addition, localization of NEAT1-2 lncRNA was identified in detail by electron microscopic analysis combined with ISH for NEAT1-2 lncRNA. The observation indicating specific assembly of NEAT1-2 lncRNA around the interchromatin granule-associated zone in the nucleus of ALS spinal motor neurons verified characteristic paraspeckle formation. Conclusions: NEAT1-2 lncRNA may act as a scaffold of RNAs and RNA binding proteins in the nuclei of ALS motor neurons, thereby modulating the functions of ALS-associated RNA-binding proteins during the early phase of ALS. These findings provide the first evidence of a direct association between paraspeckle formation and a neurodegenerative disease, and may shed light on the development of novel therapeutic targets for the treatment of ALS. © 2013 Nishimoto et al.
licensee BioMed Central Ltd.

Alternative 3 '-end processing of long noncoding RNA initiates construction of nuclear paraspeckles
Takao Naganuma, Shinichi Nakagawa, Akie Tanigawa, Yasnory F. Sasaki, Naoki Goshima, Tetsuro Hirose
EMBO JOURNAL, 31, 20, 4020, 4034, NATURE PUBLISHING GROUP, 2012年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Paraspeckles are unique subnuclear structures built around a specific long noncoding RNA, NEAT1, which is comprised of two isoforms produced by alternative 3'-end processing (NEAT1_1 and NEAT1_2). To address the precise molecular processes that lead to paraspeckle formation, we identified 35 paraspeckle proteins (PSPs), mainly by colocalization screening with a fluorescent protein-tagged full-length cDNA library. Most of the newly identified PSPs possessed various putative RNA-binding domains. Subsequent RNAi analyses identified seven essential PSPs for paraspeckle formation. One of the essential PSPs, HNRNPK, appeared to affect the production of the essential NEAT1_2 isoform by negatively regulating the 3'-end polyadenylation of the NEAT1_1 isoform. An in vitro 3'-end processing assay revealed that HNRNPK arrested binding of the CPSF6-NUDT21 (CFIm) complex in the vicinity of the alternative polyadenylation site of NEAT1_1. In vitro binding assays showed that HNRNPK competed with CPSF6 for binding to NUDT21, which was the underlying mechanism to arrest CFIm binding by HNRNPK. This HNRNPK function led to the preferential accumulation of NEAT1_2 and initiated paraspeckle construction with multiple PSPs. The EMBO Journal (2012) 31, 4020-4034. doi:10.1038/emboj.2012.251; Published online 7 September 2012

Paraspeckles: Possible nuclear hubs by the RNA for the RNA
Tetsuro Hirose, Shinichi Nakagawa
Biomolecular Concepts, 3, 5, 415, 428, De Gruyter Mouton, 2012年10月01日, ［査読有り］
英語, The mammalian cell nucleus is a highly compartmentalized system in which multiple subnuclear structures, called nuclear bodies, exist in the nucleoplasmic spaces. Some of the nuclear bodies contain specific long non-coding RNAs (ncRNAs) as their components, and may serve as sites for long ncRNA functions that remain largely enigmatic. A paraspeckle is a nuclear body that is almost ubiquitously observed in mammalian cultured cells but is cell population-specific in adult mouse tissue. The paraspeckle structure is RNase-sensitive. Long ncRNAs, termed MENε/β ncRNAs (also referred to as NEAT1 ncRNAs), have been identified as the RNA components of the paraspeckles. Specific elimination has revealed that MENε/β ncRNAs are essential components for the formation of the intact paraspeckle structure. Paraspeckle formation requires the continual MENε/β ncRNA biogenesis process, including ongoing transcription, alternative 3′-end processing, and stabilization. Some paraspeckle-localized RNA-binding proteins (p54/nrb and PSF) direct paraspeckle formation through the selective stabilization of MENβ ncRNA. Both MENε/β ncRNA expression and their subsequent interactions with paraspeckle proteins can be regulated under environmental and developmental conditions, which are reflected in the size and number of the paraspeckles. However, how paraspeckles function remains largely unsolved. Paraspeckles appear to serve as the site of nuclear retention of specific mRNAs that are selectively transported to the cytoplasm upon certain signals. Alternatively, MENε/β ncRNAs may sequester paraspeckle proteins that function outside the paraspeckles. This review focuses on known aspects of paraspeckles and provides a model of their structure and function.

Paraspeckle nuclear bodies-useful uselessness?
Shinichi Nakagawa, Tetsuro Hirose
CELLULAR AND MOLECULAR LIFE SCIENCES, 69, 18, 3027, 3036, SPRINGER BASEL AG, 2012年09月, ［査読有り］
英語, The nucleus of higher eukaryotes, such as humans and mice, is compartmentalized into multiple nuclear bodies, an organization that allows for the regulation of complex gene expression pathways that are characteristic of these organisms. Paraspeckles are recently discovered, mammalian-specific nuclear bodies built on a long, non-protein-coding RNA, NEAT1 (nuclear-enriched abundant transcript 1), which assembles various protein components including RNA-binding proteins of the DBHS (Drosophila behavior and human splicing) family. Paraspeckles have been proposed to control several biological processes, such as stress responses and cellular differentiation, but their function at the whole animal level remains unclear. In this review, we summarize a series of studies on paraspeckles that have been carried out in the decade since their discovery and discuss their physiological function and molecular mechanism.

Malat1 is not an essential component of nuclear speckles in mice
Shinichi Nakagawa, Joanna Y. Ip, Go Shioi, Vidisha Tripathi, Xinying Zong, Tetsuro Hirose, Kannanganattu V. Prasanth
RNA-A PUBLICATION OF THE RNA SOCIETY, 18, 8, 1487, 1499, COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2012年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Malat1 is an abundant long, noncoding RNA that localizes to nuclear bodies known as nuclear speckles, which contain a distinct set of pre-mRNA processing factors. Previous studies in cell culture have demonstrated that Malat1 interacts with pre-mRNA splicing factors, including the serine-and arginine-rich (SR) family of proteins, and regulates a variety of biological processes, including cancer cell migration, synapse formation, cell cycle progression, and responses to serum stimulation. To address the physiological function of Malat1 in a living organism, we generated Malat1-knockout (KO) mice using homologous recombination. Unexpectedly, the Malat1-KO mice were viable and fertile, showing no apparent phenotypes. Nuclear speckle markers were also correctly localized in cells that lacked Malat1. However, the cellular levels of another long, noncoding RNA-Neat1-which is an architectural component of nuclear bodies known as paraspeckles, were down-regulated in a particular set of tissues and cells lacking Malat1. We propose that Malat1 is not essential in living mice maintained under normal laboratory conditions and that its function becomes apparent only in specific cell types and under particular conditions.

Long non-coding RNAs in nuclear bodies
Joanna Y. Ip, Shinichi Nakagawa
DEVELOPMENT GROWTH & DIFFERENTIATION, 54, 1, 44, 54, WILEY-BLACKWELL, 2012年01月, ［査読有り］
英語, High-throughput analyses of mammalian transcriptomes have revealed that more than half of the transcripts produced by RNA polymerase II are non-protein-coding. One class of these non-coding transcripts is the long non-coding RNAs (lncRNAs), which are more than 200 nucleotides in length and are molecularly indistinguishable from other protein-coding mRNAs. Although the molecular functions of these lncRNAs have long remained unknown, emerging evidence implicates the functional involvement of lncRNAs in the regulation of gene expression through the modification of chromatin, maintenance of subnuclear structures, transport of specific mRNAs, and control of pre-mRNA splicing. Here, we discuss the functions of a distinct group of vertebrate-specific lncRNAs, NEAT1/MENe/beta/VINC, MALAT1/NEAT2, and Gomafu/RNCR2/MIAT, which accumulate abundantly within the nucleus as RNA components of specific nuclear bodies.

Tsukushi functions as a Wnt signaling inhibitor by competing with Wnt2b for binding to transmembrane protein Frizzled4
Kunimasa Ohta, Ayako Ito, Sei Kuriyama, Giuseppe Lupo, Mitsuko Kosaka, Shin-Ichi Ohnuma, Shinichi Nakagawa, Hideaki Tanaka
Proceedings of the National Academy of Sciences of the United States of America, 108, 36, 14962, 14967, 2011年09月06日, ［査読有り］
英語, 研究論文（学術雑誌）, The Wnt signaling pathway is essential for the development of diverse tissues during embryogenesis. Signal transduction is activated by the binding of Wnt proteins to the type I receptor lowdensity lipoprotein receptor-related protein 5/6 and the seven-pass transmembrane protein Frizzled (Fzd), which contains a Wntbinding site in the form of a cysteine-rich domain. Known extracellular antagonists of the Wnt signaling pathway can be subdivided into two broad classes depending on whether they bind primarily to Wnt or to low-density lipoprotein receptor-related protein 5/6. We show that the secreted protein Tsukushi (TSK) functions as a Wnt signaling inhibitor by binding directly to the cysteine-rich domain of Fzd4 with an affinity of 2.3 × 10 -10 M and competing with Wnt2b. In the developing chick eye, TSK is expressed in the ciliary/iris epithelium, whereasWnt2b is expressed in the adjacent anterior rimof the optic vesicle, where it controls the differentiation of peripheral eye structures, such as the ciliary body and iris. TSK overexpression effectively antagonizes Wnt2b signaling in chicken embryonic retinal cells both in vivo and in vitro and represses Wnt-dependent specification of peripheral eye fates. Conversely, targeted inactivation of the TSK gene in mice causes expansion of the ciliary body and upregulation of Wnt2b and Fzd4 expression in the developing peripheral eye. Thus, we uncover a crucial role for TSK as a Wnt signaling inhibitor that regulates peripheral eye formation.

Revisiting the function of nuclear scaffold/matrix binding proteins in X chromosome inactivation
Yuko Hasegawa, Shinichi Nakagawa
RNA BIOLOGY, 8, 5, 735, 739, LANDES BIOSCIENCE, 2011年09月, ［査読有り］
英語, Mammalian females repress gene expression from one of their two X chromosomes to compensate for the gene dosage difference between females and males, via a process called X chromosome inactivation (XCI). Since the first discovery of XCI 50 years ago, the knowledge of this phenomenon has greatly contributed to a better understanding of the molecular mechanism that controls the epigenetic regulation of gene expression. The key molecule that organizes the chromatin-level repression is an X-linked 17-kb non-coding RNA named Xist. The transcripts of Xist are localized along the entire length of the X chromosome and subsequently recruit a chromatin remodeling complex that introduces the repressive epigenetic modifications. In the present review, we will highlight the recent findings that have illustrated the close relationship between XCI and the structural component of the nucleus called the nuclear scaffold/matrix, with an emphasis on the function of the bonafide scaffold/matrix-binding protein hnRNP U/SAF-A.

eXIST with matrix-associated proteins
Shinichi Nakagawa, Kannanganattu V. Prasanth
TRENDS IN CELL BIOLOGY, 21, 6, 321, 327, ELSEVIER SCIENCE LONDON, 2011年06月, ［査読有り］
英語, X-chromosome inactivation has long served as an experimental model system for understanding the epigenetic regulation of gene expression. Central to this phenomenon is the long, non-coding RNA Xist that is specifically expressed from the inactive X chromosome and spreads along the entire length of the chromosome in cis. Recently, two of the proteins originally identified as components of the nuclear scaffold/matrix (S/MAR-associated proteins) have been shown to control the principal features of X-chromosome inactivation; specifically, context-dependent competency and the chromosome-wide association of Xist RNA. These findings implicate the involvement of nuclear S/MAR-associated proteins in the organization of epigenetic machinery. Here, we describe a model for the functional role of S/MAR-associated proteins in the regulation of key epigenetic processes.

Competition between a noncoding exon and introns: Gomafu contains tandem UACUAAC repeats and associates with splicing factor-1
Hitomi Tsuiji, Rei Yoshimoto, Yuko Hasegawa, Masaaki Furuno, Minoru Yoshida, Shinichi Nakagawa
GENES TO CELLS, 16, 5, 479, 490, WILEY-BLACKWELL, 2011年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Gomafu (also referred to as RNCR2/MIAT) was originally identified as a noncoding RNA expressed in a particular set of neurons. Unlike protein-coding mRNAs, the Gomafu RNA escapes nuclear export and stably accumulates in the nucleus, making a unique nuclear compartment. Although recent studies have revealed the functional relevance of Gomafu in a series of physiological processes, the underlying molecular mechanism remains largely uncharacterized. In this report, we identified a chicken homologue of Gomafu using a comparative genomic approach to search for functionally important and conserved sequence motifs among evolutionarily distant species. Unexpectedly, we found that all Gomafu RNA examined shared a distinctive feature: tandem repeats of UACUAAC, a sequence that has been identified as a conserved intron branch point in the yeast Saccharomyces cerevisiae. The tandem UACUAAC Gomafu RNA repeats bind to the SF1 splicing factor with a higher affinity than the divergent branch point sequence in mammals, which affects the kinetics of the splicing reaction in vitro. We propose that the Gomafu RNA regulates splicing efficiency by changing the local concentration of splicing factors within the nucleus.

Paraspeckles are subpopulation-specific nuclear bodies that are not essential in mice
Shinichi Nakagawa, Takao Naganuma, Go Shioi, Tetsuro Hirose
JOURNAL OF CELL BIOLOGY, 193, 1, 31, 39, ROCKEFELLER UNIV PRESS, 2011年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Nuclei of higher organisms are well structured and have multiple, distinct nuclear compartments or nuclear bodies. Paraspeckles are recently identified mammal-specific nuclear bodies ubiquitously found in most cells cultured in vitro. To investigate the physiological role of paraspeckles, we examined the in vivo expression patterns of two long noncoding RNAs, NEAT1_1 and NEAT1_2, which are essential for the architectural integrity of nuclear bodies. Unexpectedly, these genes were only strongly expressed in a particular subpopulation of cells in adult mouse tissues, and prominent paraspeckle formation was observed only in the cells highly expressing NEAT1_2. To further investigate the cellular functions of paraspeckles, we created an animal model lacking NEAT1 by gene targeting. These knockout mice were viable and fertile under laboratory growth conditions, showing no apparent phenotypes except for the disappearance of paraspeckles. We propose that paraspeckles are nonessential, subpopulation-specific nuclear bodies formed secondary to particular environmental triggers.

Ectopic Mitf in the Embryonic Chick Retina by Co-transfection of beta-Catenin and Otx2
Peter D. Westenskow, Jon B. McKean, Fumi Kubo, Shinichi Nakagawa, Sabine Fuhrmann
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 51, 10, 5328, 5335, ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2010年10月, ［査読有り］
英語, 研究論文（学術雑誌）, PURPOSE. Development of the retinal pigment epithelium (RPE) is controlled by intrinsic and extrinsic regulators including orthodenticle homeobox 2 (Otx2) and the Wnt/beta-catenin pathway, respectively. Otx2 and beta-catenin are necessary for the expression of the RPE key regulator microphthalmia-associated transcription factor (Mitf); however, neither factor is sufficient to promote Mitf expression in vivo. The study was conducted to determine whether Otx2 and beta-catenin act in a combinatorial manner and tested whether co-expression in the presumptive chick retina induces ectopic Mitf expression.
METHODS. The sufficiency of Wnt/beta-catenin activation and/or Otx2 expression to induce RPE-specific gene expression was examined in chick optic vesicle explant cultures or in the presumptive neural retina using in ovo-electroporation. Luciferase assays were used to examine the transactivation potentials of Otx2 and beta-catenin on the Mitf-D enhancer and autoregulation of the Mitf-D and Otx2T0 enhancers.
RESULTS. In optic vesicles explant cultures, RPE-specific gene expression was activated by lithium chloride, a Wnt/beta-catenin agonist. However, in vivo, Mitf was induced only in the presumptive retina if both beta-catenin and Otx2 are co-expressed. Furthermore, both Mitf and Otx2 can autoregulate their own enhancers in vitro.
CONCLUSIONS. The present study provides evidence that beta-catenin and Otx2 are sufficient, at least in part, to convert retinal progenitor cells into presumptive RPE cells expressing Mitf. Otx2 may act as a competence factor that allows RPE specification in concert with additional RPE-promoting factors such as beta-catenin. (Invest Ophthalmol Vis Sci. 2010;51:5328-5335) DOI:10.1167/iovs.09-5015

The Matrix Protein hnRNP U Is Required for Chromosomal Localization of Xist RNA
Yuko Hasegawa, Neil Brockdorff, Shinji Kawano, Kimiko Tsutui, Ken Tsutui, Shinichi Nakagawa
DEVELOPMENTAL CELL, 19, 3, 469, 476, CELL PRESS, 2010年09月, ［査読有り］
英語, 研究論文（学術雑誌）, In XX female mammals, one of the two X chromosomes is epigenetically inactivated to equalize gene expression with XY males. The formation of the inactive X chromosome (Xi) is regulated by an X-linked long noncoding RNA Xist, which accumulates on the entire length of the chromosome in cis and induces heterochromatin formation. However, the mechanism by which Xist RNA "paints" the Xi has long remained elusive. Here, we show that a matrix protein hnRNP U/SP120/SAF-A is required for the accumulation of Xist RNA on the Xi. Xist RNA and hnRNP U interact and upon depletion of hnRNP U, Xist RNA is detached from the Xi and diffusely localized into the nucleoplasm. In addition, ES cells lacking hnRNP U expression fail to form the Xi. We propose that the association with matrix proteins is an essential step in the epigenetic regulation of gene expression by Xist RNA.

Cath6, a bHLH Atonal Family Proneural Gene, Negatively Regulates Neuronal Differentiation in the Retina
Fumi Kubo, Shinichi Nakagawa
DEVELOPMENTAL DYNAMICS, 239, 9, 2492, 2500, WILEY-LISS, 2010年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Basic helix-loop-helix (bHLH) transcription factors play important roles in cell type specification and differentiation during the development of the nervous system. In this study, we identified a chicken homolog of Atonal 8/ath6 (Cath6) and examined its role in the developing retina. Unlike other Atonal-family proneural genes that induce neuronal differentiation, Cath6 was expressed in stem cell-like progenitor cells in the marginal region of the retina, and its overexpression inhibited neuronal differentiation. A Cath6 fused with a VP16 transactivation domain recapitulated the inhibitory effect of Cath6 on neuronal differentiation, indicating that Cath6 functions as a transcription activator. These results demonstrate that Cath6 constitutes a unique member of the Atonal-family of genes in that it acts as a negative regulator of neuronal differentiation. Developmental Dynamics 239:2492-2500, 2010. (c) 2010 Wiley-Liss, Inc.

LIM family transcription factors regulate the subtype-specific morphogenesis of retinal horizontal cells at post-migratory stages
Akiko Suga, Masanori Taira, Shinichi Nakagawa
DEVELOPMENTAL BIOLOGY, 330, 2, 318, 328, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2009年06月, ［査読有り］
英語, 研究論文（学術雑誌）, In the nervous system, transcription factor expression in progenitor and/or nascent neurons regulates cell type specification. Although the functions of these transcription factors at early stages are well established, whether or not they are required during late developmental periods remains an open question. To address this issue, we conditionally manipulated gene expression using a recently developed transposon-mediated gene transfer system combined with in ovo electroporation. In chicken retinas, horizontal cells are classified into three subtypes according to their characteristic neuronal morphology. Two LIM family transcription factors, Lim1 and Isl1, begin to be expressed in a distinct subset of nascent retinal neurons, which results in complementary expression of these genes in mature retinas in type I and type II/III horizontal cells, respectively. Overexpression of Isl1 in post-migratory horizontal cells represses endogenous Lim1 expression and increases the number of neurons with a dendritic morphology characteristic of type II horizontal cells, which normally express Isl1. Inhibition of Lim1 function by expression of a dominant negative form Lim1 perturbs axonal morphogenesis of type I horizontal cells. Therefore, we propose that LIM family transcription factors are required for subtype-specific morphogenesis of horizontal cells at later stages of retinal development. (C) 2009 Elsevier Inc. All rights reserved.

Hairy1 acts as a node downstream of Wnt signaling to maintain retinal stem cell-like progenitor cells in the chick ciliary marginal zone
Fumi Kubo, Shinichi Nakagawa
DEVELOPMENT, 136, 11, 1823, 1833, COMPANY OF BIOLOGISTS LTD, 2009年06月, ［査読有り］
英語, 研究論文（学術雑誌）, In the vertebrate retina, stem cell-like progenitor cells are maintained in a distinct region called the ciliary marginal zone (CMZ). Canonical Wnt signaling regulates the maintenance of the progenitor cells in the CMZ. However, its downstream molecular mechanisms have remained largely unclear. Here, we show that chick Hairy1, an established Notch signaling effector, mediates the Wnt-dependent maintenance of CMZ progenitor cells in chicken. Interestingly, unlike other developmental contexts in which Hes gene expression is regulated by Notch signaling, Hairy1 expression in the CMZ is regulated by Wnt signaling. Hairy1 is necessary and sufficient for the expression of a set of molecular markers characteristic of the CMZ, and Wnt2b fails to induce CMZ markers when Hairy1 activity is inhibited. Furthermore, microarray analysis identifies multiple Wnt-responsive transcription factors that activate Hairy1 expression. We thus propose that Hairy1 functions as a node downstream of Wnt signaling to maintain progenitor cells in the chick CMZ.

Wnt signaling in retinal stem cells and regeneration
Fumi Kubo, Shinichi Nakagawa
DEVELOPMENT GROWTH & DIFFERENTIATION, 50, 4, 245, 251, WILEY-BLACKWELL, 2008年05月, ［査読有り］
英語, In the vertebrate retina, stem cells with prolonged proliferative capacities reside in the most peripheral region, the ciliary marginal zone (CMZ), and they persist even after the functional eye has formed. These stem cells contribute to the formation of the retinal structures during the postnatal period in vivo, or can expand as neurospheres in vitro. Despite the wealth of anatomical descriptions of the characteristics of CMZ cells, molecular mechanisms for their specification or maintenance have long been uncharacterized. Recent studies provide evidence that certain secreted signaling molecules act as key regulators at multiple steps during these processes. In this review, we discuss the molecular basis for the regulation of retinal stem cells and their related cell types, especially focusing on the role of Wnt signaling.

Transposon-mediated stable integration and tetracycline-inducible expression of electroporated transgenes in chicken embryos
Yoshiko Takahashi, Tadayoshi Watanabe, Shinichi Nakagawa, Koichi Kawakami, Yuki Sato
AVIAN EMBRYOLOGY, 2ND EDITION, 87, 271, +, ELSEVIER ACADEMIC PRESS INC, 2008年, ［査読有り］
英語, 論文集(書籍)内論文

Tsukushi is a Frizzled4 ligand that regulates the proliferation of retinal stem cells in competition with Wnt2b
Kunimasa Ohta, Ayako Ito, Sei Kuriyama, Rika Nakayama, Naoko Oshima, Mitsuko Kosaka, Shinichi Ohnuma, Shinichi Nakagawa, Hideaki Tanaka
NEUROSCIENCE RESEARCH, 61, S42, S42, ELSEVIER IRELAND LTD, 2008年, ［査読有り］
英語

Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis
Dai Chida, Shinichi Nakagawa, So Nagai, Hiroshi Sagara, Harumi Katsumata, Toshihiro Imaki, Harumi Suzuki, Fumiko Mitani, Tadashi Ogishima, Chikara Shimizu, Hayato Kotaki, Shigeru Kakuta, Katsuko Sudo, Takao Koike, Mitsurnasa Kubo, Yoichiro Iwakura
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 104, 46, 18205, 18210, NATL ACAD SCIENCES, 2007年11月, ［査読有り］
英語, 研究論文（学術雑誌）, ACTH (i.e., corticotropin) is the principal regulator of the hypothalamus-pituitary-adrenal axis and stimulates steroidogenesis in the adrenal gland via the specific cell-surface melanocortin 2 receptor (MC2R). Here, we generated mice with an inactivation mutation of the MC2R gene to elucidate the roles of MC2R in adrenal development, steroidogenesis, and carbohydrate metabolism. These mice, the last of the knockout (KO) mice to be generated for melanocortin family receptors, provide the opportunity to compare the phenotype of proopiomelanocortin KO mice with that of MCIR-MC5R KO mice. We found that the MC2R KO mutation led to neonatal lethality in three-quarters of the mice, possibly as a result of hypoglycemia. Those surviving to adulthood exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata, whereas the zona glomerulosa and the medulla remained fairly intact. Mutations of MC2R have been reported to be responsible for 25% of familial glucocorticoid deficiency (FGD) cases. Adult MC2R KO mice resembled FGD patients in several aspects, such as undetectable levels of corticosterone despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia after prolonged (36 h) fasting. However, MC2R KO mice differ from patients with MC2R-null mutations in several aspects, such as low aldosterone, levels and unaltered body length. These results indicate that MC2R is required for postnatal adrenal development and adrenal steroidogenesis and that MC2R KO mice provide a useful animal model by which to study FGD.

The mRNA-like noncoding RNA Gomafu constitutes a novel nuclear domain in a subset of neurons
Masamitsu Sone, Tetsutaro Hayashi, Hiroshi Tarui, Kiyokazu Agata, Masatoshi Takeichi, Shinichi Nakagawa
JOURNAL OF CELL SCIENCE, 120, 15, 2498, 2506, COMPANY OF BIOLOGISTS LTD, 2007年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Recent transcriptome analyses have revealed that a large body of noncoding regions of mammalian genomes are actually transcribed into RNAs. Our understanding of the molecular features of these noncoding RNAs is far from complete. We have identified a novel mRNA-like noncoding gene, named Gomafu, which is expressed in a distinct set of neurons in the mouse nervous system. Interestingly, spliced mature Gomafu RNA is localized to the nucleus despite its mRNA-like characteristics, which usually act as potent export signals to the cytoplasm. Within the nucleus, Gomafu RNA is detected as numerous spots that do not colocalize with known nuclear domain markers. Gomafu RNA is extremely insoluble and remains intact after nuclear matrix preparation. Furthermore, heterokaryon assays revealed that Gomafu RNA does not shuttle between the nucleus and cytoplasm, but is retained in the nucleus after its transcription. We propose that Gomafu RNA represents a novel family of mRNA-like noncoding RNA that constitutes a cell-type-specific component of the nuclear matrix.

Tsukushi inhibits the proliferation of retinal stem/progenitor cells
Kunimasa Ohta, Ayako Ito, Sei Kuriyama, Shinichi Ohnuma, Mitsuko Kosaka, Shinichi Nakagawa, H. Tanaka
DEVELOPMENTAL BIOLOGY, 306, 1, 391, 391, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2007年06月, ［査読有り］
英語

Tet-on inducible system combined with in ovo electroporation dissects multiple roles of genes in somitogenesis of chicken embryos
Tadayoshi Watanabe, Daisuke Saito, Koji Tanabe, Rinako Suetsugu, Yukiko Nakaya, Shinichi Nakagawa, Yoshiko Takahashi
DEVELOPMENTAL BIOLOGY, 305, 2, 625, 636, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2007年05月, ［査読有り］
英語, 研究論文（学術雑誌）, The in ovo electroporation technique in chicken embryos has enabled investigators to uncover the functions of numerous developmental genes. In this technique, the ubiquitous promoter, CAGGS (CMV base), has often been used for overexpression experiments. However, if a given gene plays a role in multiple steps of development and if overexpression of this gene causes fatal consequences at the time of electroporation, its roles in later steps of development would be overlooked. Thus, a technique with which expression of an electroporated DNA can be controlled in a stage-specific manner needs to be formulated. Here we show for the first time that the tetracycline-controlled expression method, "tet-on" and "tet-off', works efficiently to regulate gene expression in electroporated chicken embryos. We demonstrate that the onset or termination of expression of an electroporated DNA can be precisely controlled by timing the administration of tetracycline into an egg. Furthermore, with this technique we have revealed previously unknown roles of RhoA, cMeso-1 and Pax2 in early somitogenesis. In particular, cMeso-1 appears to be involved in cell condensation of a newly forming somite by regulating Pax2 and NCAM expression. Thus, the novel molecular technique in chickens proposed in this study provides a useful tool to investigate stage-specific roles of developmental genes. (c) 2007 Elsevier Inc. All rights reserved.

Stable integration and conditional expression of electroporated transgenes in chicken embryos
Yuki Sato, Toshiharu Kasai, Shinichi Nakagawa, Koji Tanabe, Tadayoshi Watanabe, Koichi Kawakami, Yoshiko Takahashi
DEVELOPMENTAL BIOLOGY, 305, 2, 616, 624, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2007年05月, ［査読有り］
英語, 研究論文（学術雑誌）, The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon To12, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with To12 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the To12 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals. (c) 2007 Elsevier Inc. All rights reserved.

Cadherin is required for dendritic morphogenesis and synaptic terminal organization of retinal horizontal cells
Koji Tanabe, Yoshiko Takahashi, Yuki Sato, Koichi Kawakami, Masatoshi Takeichi, Shinichi Nakagawa
DEVELOPMENT, 133, 20, 4085, 4096, COMPANY OF BIOLOGISTS LTD, 2006年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Dendrite morphology of neurons provides a structural basis for their physiological characteristics, and is precisely regulated in a cell type-dependent manner. Using a unique transposon-mediated gene transfer system that enables conditional and cell-type specific expression of exogenous genes, we investigated the role of cadherin on dendritic morphogenesis of horizontal cells in the developing chicken retina. We first visualized single horizontal cells by overexpressing membrane-targeted EGFP, and confirmed that there were three subtypes of horizontal cells, the dendritic terminals of which projected to distinct synaptic sites in the outer plexiform layer. Expression of a dominant-negative cadherin decreased the dendritic field size, and perturbed the termination of dendritic processes onto the photoreceptor cells. The cadherin blockade also impaired the accumulation of GluR4, a postsynaptic marker, at the cone pedicles. We thus provide in vivo evidence that cadherin is required for dendrite morphogenesis of horizontal cells and subsequent synapse formation with photoreceptor cells in the vertebrate retina.

Embryonic stem cells that differentiate into RPE cell precursors in vitro develop into RPE cell monolayers in vivo
H Aoki, A Hara, S Nakagawa, T Motohashi, M Hirano, Y Takahashi, T Kunisada
EXPERIMENTAL EYE RESEARCH, 82, 2, 265, 274, ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD, 2006年02月, ［査読有り］
英語, 研究論文（学術雑誌）, A culture system to generate eye-like structures consisting of lens, neural retina, and retinal pigmented epithelium (RPE) cells from undifferentiated embryonic stem cells has been established. Precursors of RPE cells that differentiated in the cultures were responsive to Wnt2b signaling and identified retrospectively to form secondary colonies consisting of only RPE-like cells in eye-like structures. These transplanted eye-like structures were capable of populating the developing chick eye as neuronal retina and RPE cells. The outgrowth of a single cell layer of mature RPE cells from the grafted eye-like structures confirmed the existence of precursors for RPE cells. These results suggest that the eye-like structures resulted from the normal developmental pathway responsible for generating eyes in vivo. If a functional effect of these cells can be established, such eye-like structures may be potentially used to establish therapy models for various eye diseases. (c) 2005 Elsevier Ltd. All rights reserved.

Wnt2b inhibits differentiation of retinal progenitor cells in the absence of Notch activity by downregulating the expression of proneural genes
F Kubo, M Takeichi, S Nakagawa
DEVELOPMENT, 132, 12, 2759, 2770, COMPANY OF BIOLOGISTS LTD, 2005年06月, ［査読有り］
英語, 研究論文（学術雑誌）, During the development of the central nervous system, cell proliferation and differentiation are precisely regulated. In the vertebrate eye, progenitor cells located in the marginal-most region of the neural retina continue to proliferate for a much longer period compared to the ones in the central retina, thus showing stem-cell-like properties. Wnt2b is expressed in the anterior rim of the optic vesicles, and has been shown to control differentiation of the progenitor cells in the marginal retina. In this paper, we show that stable overexpression of Wnt2b in retinal explants inhibited cellular differentiation and induced continuous growth of the tissue. Notably, Wnt2b maintained the undifferentiated progenitor cells in the explants even under the conditions where Notch signaling was blocked. Wnt2b downregulated the expression of multiple proneural bHLH genes as well as Notch. In addition, expression of Cath5 under the control of an exogenous promoter suppressed the negative effect of Wnt2b on neuronal differentiation. Importantly, Wnt2b inhibited neuronal differentiation independently of cell cycle progression. We propose that Wnt2b maintains the naive state of marginal progenitor cells by attenuating the expression of both proneural and neurogenic genes, thus preventing those cells from launching out into the differentiation cascade regulated by proneural genes and Notch.

Identification of a nonchordate-type classic cadherin in vertebrates: Chicken Hz-cadherin is expressed in horizontal cells of the neural retina and contains a nonchordate-specific domain complex
K Tanabe, M Takeichi, S Nakagawa
DEVELOPMENTAL DYNAMICS, 229, 4, 899, 906, WILEY-LISS, 2004年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Classic cadherins mediate calcium-dependent cell-cell adhesion in a variety of animals, but there are marked differences in their domain structures between chordate and nonchordate animals. The extracellular domain of chordate-type classic cadherins (type I and type 11 classic cadherins) consists of five tandem repeats of conserved sequences called EC domains, whereas that of nonchordate-type classic cadherins (designated as type III classic cadherin) contains a variable number of EC domains, followed by a characteristic domain complex made of laminin-A globular domains and EGF-like repeats. In the present study, we identified a novel vertebrate type III cadherin showing high sequence similarity to Drosophila N-cadherin, and named this molecule chicken Hz-cadherin (cHz-cadherin), because of the distinct expression in horizontal cells of the neural retina. cHz-cadherin functioned as an adhesion molecule when introduced into cultured cells. Database search revealed one cHz-cadherin homologue in zebrafish and two in puffer fish, but none in other vertebrate species examined. These observations indicate that type III classic cadherins have been conserved in vertebrate species, being expressed by limited cells types, but lost in particular phylogenic groups of the vertebrates. Developmental Dynamics 229.899-906, 2004. (C) 2004 Wiley-Liss, Inc.

Identification of the laminar-inducing factor: Wnt-signal from the anterior rim induces correct laminar formation of the neural retina in vitro
S Nakagawa, S Takada, R Takada, M Takeichi
DEVELOPMENTAL BIOLOGY, 260, 2, 414, 425, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2003年08月, ［査読有り］
英語, 研究論文（学術雑誌）, To study the molecular mechanism that controls the laminar organization of the retina, we utilized reaggregation cultures of dissociated retina] cells prepared from chicken embryos. These cells cannot generate laminated structures by themselves and, instead, form rosettes within the reaggregates. However, the dissociated cells can organize into a correctly laminated structure when cultured in the presence of a putative laminar inducing factor coming from particular tissue or cells, but its molecular identity of this factor has long remained elusive. In this study, we found that the anterior rim of the retina sends a signal to rearrange the rosette-forming cells into a neuroepithelial structure characteristic of the undifferentiated retinal layer. This activity of the anterior rim was mimicked by Wnt-2b expressed in this tissue, and was neutralized by a soluble form of Frizzled, which works as a Wnt antagonist. Furthermore, the neuroepithelial structure induced by Wnt-2b subsequently developed into correctly laminated retinal layers. These observations suggest that the anterior rim functions as a layer-organizing center in the retina, by producing Wnt-2b. (C) 2003 Elsevier Inc. All rights reserved.

Wnt2b controls retinal cell differentiation at the ciliary marginal zone
F Kubo, M Takeichi, S Nakagawa
DEVELOPMENT, 130, 3, 587, 598, COMPANY OF BIOLOGISTS LTD, 2003年02月, ［査読有り］
英語, 研究論文（学術雑誌）, The ciliary marginal zone of the vertebrate retina contains undifferentiated progenitor cells that continue to proliferate and add new neurons and glia peripherally during the embryonic stages - even after the formation of a functional retina. To understand the molecular mechanism that controls the prolonged progenitor cell proliferation in the ciliary marginal zone, we employed a candidate molecule approach, focusing on Wnt2b (formerly know as Wnt13), which is expressed in the marginal most tip of the retina. Frizzled 4 and 5, seven-pass transmembrane Writ receptors, were expressed in the peripheral and central part of the retina, respectively. LEF1, a downstream Writ signaling component, was expressed at high levels in the ciliary marginal zone with expression gradually decreasing towards the central retina. The LEF1-expressing region, which is where Writ signaling is supposedly activated, expressed a set of molecular markers that are characteristic of the progenitor cells in the ciliary marginal zone. Overexpression of Wnt2b by use of in ovo electroporation in the central retina inhibited neuronal differentiation and induced the progenitor cell markers. Blocking of the Wnt downstream signaling pathway by a dominant-negative LEF1 inhibited proliferation of the cells in the marginal area, which resulted in their premature neuronal differentiation. The progenitor cells in the ciliary marginal zone differentiated into all the neuronal and glial cell types when cultured in vitro, and they proliferated for a longer period than did centrally located progenitor cells that underwent a limited number of cell divisions. In addition, the proliferation of these progenitor cells was promoted in the presence of Wnt2b. These results suggest that Wnt2b functions to maintain undifferentiated progenitor cells in the ciliary marginal zone, and thus serves as a putative stem cell factor in the retina.

Cadherin-dependent cell-cell adhesion.
Takeichi M, Nakagawa S
Current protocols in cell biology, Chapter 9, Unit 9.3, 2001年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Differential treatment of cells with trypsin can be used to distinguish Ca(2+)-dependent adhesion (CDS) from Ca(2+)-independent adhesion (CIDS). Cadherins appear to be a unique family of molecules whose structure and function as adhesion molecules are protected from trypsin in the presence of Ca(2+). This unit provides protocols for preparation and analysis of cells for cadherin-dependent adhesion in short-term and long-term aggregation assays. The functions of different cadherins can be assessed in mixed aggregate assays. Fluorescence antibody-based assays are used to identify specific cadherins and their associated catenins, and transformation of cells with specific constructs can be used to assay adhesion in cells with loss of cadherin activity.

Patterning of cell assemblies regulated by adhesion receptors of the cadherin superfamily
M Takeichi, S Nakagawa, S Aono, T Usui, T Uemura
PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON SERIES B-BIOLOGICAL SCIENCES, 355, 1399, 885, 890, ROYAL SOC LONDON, 2000年07月, ［査読有り］
英語, 研究論文（学術雑誌）, During morphogenesis, cell cell association patterns are dynamically altered. We are interested in how ce adhesion molecule scan regulate the patterning of cellular assemblies. Cadherins, a group of cell-cell adhesion receptors, are crucial for the organized assembly of many cell types, but they also regulate dynamic aspects of cell association. For example, during neural crest emigration from the neural tube, the cadherin subtypes expressed by crest cells ar switched from one subtype to another. Artificial perturbation of this switch results in blocking of their escape from the neural tube. Intracellular modulations of cadherin activity also seem to play a role in regulation of cell adhesion. We identified p120(ctn) as a regulator of cadherin function in carcinoma cells. With such regulators, cells may make a choice as to whether they should maintain stable cell contacts or disrupt their association. Finally, we found another type of cadherin-mediated cell patterning: Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity in Drosophila imaginal discs. Thus, the cadherin superfamily receptors control the patterning of cell assemblies through a variety of mechanisms.

Morphologic fate of diencephalic prosomeres and their subdivisions revealed by mapping cadherin expression
C Redies, M Ast, S Nakagawa, M Takeichi, M Martinez-de-la-Torre, L Puelles
JOURNAL OF COMPARATIVE NEUROLOGY, 421, 4, 481, 514, WILEY-LISS, 2000年06月, ［査読有り］
英語, The expression of four cadherins (cadherin-6B, cadherin-7, R-cadherin, and N-cadherin) was mapped in the diencephalon of chicken embryos at 11 days and 15 days of incubation and was compared with Nissl stains and radial glial topology. Results showed that each cadherin is expressed in a restricted manner by a different set of embryonic divisions, brain nuclei, and their subregions. An analysis of the segmental organization based on the prosomeric model indicated that, in the mature diencephalon, each prosomere persists and forms a coherent domain of gray matter extending across the entire transverse dimension of the neural tube, from the ventricular surface to the pial surface. Moreover, the results suggest the presence of a novel set of secondary subdivisions for the dorsal thalamus (dorsal, intermediate, and ventral tiers and anteroventral subregion). They also confirm the presence of secondary subdivisions in the pretectum (commissural, juxtacommissural, and precommissural). At most of the borders between the prosomeres and their secondary subdivisions, changes in radial glial fiber density were observed. The diencephalic brain nuclei that derive from each of the subdivisions were determined. In addition, a number of previously less well-characterized gray matter regions of the diencephalon were defined in more detail based on the mapping of cadherin expression. The results demonstrate in detail how the divisions of the early embryonic diencephalon persist and transform into mature gray matter architecture during brain morphogenesis, and they support the hypothesis that cadherins play a role in this process by providing a framework of potentially adhesive specificities. J. Comp. Neurol. 421:481-514, 2000. (C) 2000 Wiley-Liss, Inc.

Blockade of cadherin-6B activity perturbs the distribution of PSD-95 family proteins in retinal neurones
Y Honjo, S Nakagawa, M Takeichi
GENES TO CELLS, 5, 4, 309, 318, BLACKWELL SCIENCE LTD, 2000年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Background: Synaptic junctions have cadherin-catenin complexes, but their functions are poorly understood. Using retinal neurones, we investigated the role of this adhesion machinery in synaptic organization.
Results: In cultures of chicken retinal cells, cadherin-6B (cad6B) and cadherin-7 (cad7) are expressed by distinct neurones, each being distributed in a punctate pattern along their neurites as well as in the soma. Double-immunostaining for cad6B and PSD-95/SAP90 or other PSD-95 family members, known to localize in the postsynaptic density, showed that their distributions overlapped each other. To assess the role for cad6B, we incubated retinal cells with antibodies that could specifically block cad6B-mediated adhesion. In the antibody-treated neurones, the localization pattern of PSD-95 family proteins was altered, that is, their staining signals tended to be reduced or disarranged. We then examined whether cadherins interacted molecularly with PSD-95: Cadherin immunoprecipitates from brain lysates did not contain PSD-95; nevertheless, this protein was co-precipitated with alpha N- and beta-catenins. When PSD-95 proteins were ectopically expressed in epithelial cells, some of these molecules were concentrated in cell-cell junctions, co-localizing with E-cadherin, and this junctional localization of PSD-95 was abolished by blocking of E-cadherin activity.
Conclusion: These results suggest that cadherins play a role in the subcellular organization of postsynaptic density components through some, perhaps indirect, molecular interactions.

Ephrin-B regulates the ipsilateral routing of retinal axons at the optic chiasm
S Nakagawa, C Brennan, KG Johnson, D Shewan, WA Harris, CE Holt
NEURON, 25, 3, 599, 610, CELL PRESS, 2000年03月, ［査読有り］
英語, 研究論文（学術雑誌）, In Xenopus tadpoles, all retinal ganglion cells (RGCs) send axons contralaterally across the optic chiasm. At metamorphosis, a subpopulation of EphB-expressing RGCs in the ventrotemporal retina begin to project ipsilaterally. However, when these metamorphic RGCs are grafted into embryos, they project contralaterally, suggesting that the embryonic chiasm lacks signals that guide axons ipsilaterally. Ephrin-B is expressed discretely at the chiasm of metamorphic but not premetamorphic Xenopus. When expressed prematurely in the embryonic chiasm, ephrin-B causes precocious ipsilateral projections from the EphB-expressing RGCs. Ephrin-B is also found in the chiasm of mammals, which have ipsilateral projections, but not in the chiasm of fish and birds, which do not. These results suggest that ephrin-B/EphB interactions play a key role in the sorting of axons at the vertebrate chiasm.

p120(ctn) acts as an inhibitory regulator of cadherin function in colon carcinoma cells
S Aono, S Nakagawa, AB Reynolds, M Takeichi
JOURNAL OF CELL BIOLOGY, 145, 3, 551, 562, ROCKEFELLER UNIV PRESS, 1999年05月, ［査読有り］
英語, 研究論文（学術雑誌）, p120(ctn) binds to the cytoplasmic domain of cadherins but its role is poorly understood. Cole 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin-dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120(ctn) to a faster position. To investigate whether p120(ctn) plays a role in this cadherin reactivation process, we transfected Cole 205 cells with a series of p120(ctn) deletion constructs. Notably, expression of NH2-terminally deleted p120(ctn) induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120(ctn)-binding site was introduced into Cole 205 cells, this molecule also induced cell aggregation, indicating that cadherins can function normally if they do not bind to p120(ctn). These findings suggest that in Cole 205 cells, a signaling mechanism exists to modify a biochemical state of p120(ctn) and the modified p120(ctn) blocks the cadherin system. The NH2 terminus-deleted p120(ctn) appears to compete with the endogenous p120(ctn) to abolish the adhesion-blocking action.

Combinatorial expression of cadherins in the tectum and the sorting of neurites in the tectofugal pathways of the chicken embryo
JCP Wohrn, S Nakagawa, M Ast, M Takeichi, C Redies
NEUROSCIENCE, 90, 3, 985, 1000, PERGAMON-ELSEVIER SCIENCE LTD, 1999年, ［査読有り］
英語, 研究論文（学術雑誌）, The expression of four cadherins (N-cadherin, R-cadherin, cadherin-6B and cadherin-7) was mapped in the developing tectal system of the chicken embryo from four to 19 days of incubation. Each of the cadherins is expressed in a restricted fashion in specific tectal layers, with partial overlap between the cadherins. In some layers, subpopulations of neurons differentially express the cadherins, e.g., in the stratum griseum centrale. Double labeling demonstrates that many of the projection neurons in this layer co-express at least two cadherins. Fibers of the efferent (tectofugal) pathways originating in these neurons also differentially express the cadherins, most prominently at around 11 days of incubation. While the different subpopulations of cadherin-expressing projection neurons are dispersed and mixed within the tectum, their neurites sort out and fasciculate according to which cadherin they express, as they collect in the major output of the tectum, the brachium colliculi superioris. From here, cadherin-expressing fascicles follow separate paths to their respective target areas, some of which also express the respective cadherins, in a matching fashion.
We propose that the preferentially homophilic binding of cadherins provides a potential adhesive basis for the sorting and selective fasciculation of specific subpopulations of neurites, similar to the well-established sorting and aggregation of cells expressing cadherins. The combinatorial expression of cadherins by the tectal projection neurons may contribute to the complexity and specificity of functional connections in this system. (C) 1999 IBRO. Published by Elsevier Science Ltd.

Neural crest emigration from the neural tube depends on regulated cadherin expression
S Nakagawa, M Takeichi
DEVELOPMENT, 125, 15, 2963, 2971, COMPANY OF BIOLOGISTS LTD, 1998年08月, ［査読有り］
英語, 研究論文（学術雑誌）, During the emergence of neural crest cells from the neural tube, the expression of cadherins dynamically changes. In the chicken embryo, the early neural tube expresses two cadherins, N-cadherin and cadherin-6B (cad6B), in the dorsal-most region where neural crest cells are generated. The expression of these two cadherins is, however, downregulated in the neural crest cells migrating from the neural tube; they instead begin expressing cadherin-7 (cad7). As an attempt to investigate the role of these changes in cadherin expression, we overexpressed various cadherin constructs, including N-cadherin, cad7, and a dominant negative N-cadherin (cN390 Delta), in neural crest-generating cells. This was achieved by injecting adenoviral expression vectors encoding these molecules into the lumen of the closing neural tube of chicken embryos at stage 14, In neural tubes injected with the viruses, efficient infection was observed at the neural crest-forming area, resulting in the ectopic cadherin expression also in migrating neural crest cells. Notably, the distribution of neural crest cells with the ectopic cadherins changed depending on which constructs were expressed. Many crest cells failed to escape from the neural tube when N-cadherin or cad7 overexpressed. Moreover, none of the cells with these ectopic cadherins migrated along the dorsolateral (melanocyte) pathway. When these samples were stained for Mitf, an early melanocyte marker, positive cells were found accumulated within the neural tube, suggesting that the failure of their migration was not due to differentiation defects. In contrast to these phenomena, cells expressing non-functional cadherins exhibited a normal migration pattern. Thus, the overexpression of a neuroepithelial cadherin (N-cadherin) and a crest cadherin (cad7) resulted in the same blocking effect on neural crest segregation from neuroepithelial cells, especially for melanocyte precursors. These findings suggest that the regulation of cadherin expression or its activity at the neural crest-forming area plays a critical role in neural crest emigration from the neural tube.

Cadherin expression in the retina and retinofugal pathways of the chicken embryo
JCP Wohrn, L Puelles, S Nakagawa, M Takeichi, C Redies
JOURNAL OF COMPARATIVE NEUROLOGY, 396, 1, 20, 38, WILEY-LISS, 1998年06月, ［査読有り］
英語, 研究論文（学術雑誌）, The expression of two calcium-dependent adhesion molecules of the cadherin superfamily (cadherin-6B and cadherin-7) was mapped in the embryonic neural retina and retinofugal pathways of the chicken embryo and compared with the expression of R-cadherin, N-cadherin, and B-cadherin, studied previously. Whereas B-cadherin is only found in Muller glia, the other four cadherins are each expressed by specific subpopulations of retinal neurons. For example, different (but partly overlapping) populations of bipolar cells express R-cadherin, cadherin-6B, and cadherin-7. Cadherin-6B and cadherin-7 are also expressed by subsets of amacrine cells. In the inner plexiform layer, cadherin-6B and cadherin-7 immunoreactivities are restricted to specific sublaminae associated with synapsin-I-positive nerve terminals. In addition, cadherin-6B and cadherin-7 are expressed by a subset of ganglion cells that project to several retinorecipient nuclei forming part of the accessory optic system (e.g., nucleus of the basal optic root and external pretectal nucleus). Together with their connecting fiber tracts, these nuclei also express cadherin-6B and cadherin-7 in their neurons and neuropile. The expression patterns of the two cadherins overlap but show distinct differences. Some other visual nuclei express cadherin-7 but not cadherin-6B. The expression patterns differ from those previously described for N- and R-cadherin. Together, these results demonstrate that cadherins could provide a system of adhesive cues that specify developing retinal circuits and other functional connections and subsystems in the embryonic chicken visual system. (C) 1998 Wiley-Liss, Inc.

Cadherin-defined segments and parasagittal cell ribbons in the developing chicken cerebellum
K Arndt, S Nakagawa, M Takeichi, C Redies
MOLECULAR AND CELLULAR NEUROSCIENCE, 10, 5-6, 211, 228, ACADEMIC PRESS INC, 1998年04月, ［査読有り］
英語, 研究論文（学術雑誌）, In the developing chicken cerebellar cortex, three cadherins (Cad6B, Cad7, and R-cadherin) are expressed in distinct parasagittal segments that are separated from each other by ribbons of migrating interneurons and granule cells which express R-cadherin and Cad7, respectively. The segment/ribbon pattern is respected by the expression of other types of molecules, such as engrailed-2 and SC1/BEN/DM-GRASP. The cadherin-defined segments contain young Purkinje cells which are connected to underlying nuclear zones expressing the same cadherin, thereby forming parasagittal cortico-nuclear zones of topographically organized connections. In addition, R-cadherin-positive mossy fiber terminals display a periodic pattern in the internal granular layer. In this layer, Cad7 and R-cadherin are associated with synaptic complexes. These results suggest that cadherins play a pivotal role in the formation of functional cerebellar architecture by providing a three-dimensional scaffold of adhesive information.

Cadherin-defined segments and parasagittal cell ribbons in the developing chicken cerebellum
K Arndt, S Nakagawa, M Takeichi, C Redies
MOLECULAR AND CELLULAR NEUROSCIENCE, 10, 5-6, 211, 228, ACADEMIC PRESS INC, 1998年04月, ［査読有り］
英語, 研究論文（学術雑誌）, In the developing chicken cerebellar cortex, three cadherins (Cad6B, Cad7, and R-cadherin) are expressed in distinct parasagittal segments that are separated from each other by ribbons of migrating interneurons and granule cells which express R-cadherin and Cad7, respectively. The segment/ribbon pattern is respected by the expression of other types of molecules, such as engrailed-2 and SC1/BEN/DM-GRASP. The cadherin-defined segments contain young Purkinje cells which are connected to underlying nuclear zones expressing the same cadherin, thereby forming parasagittal cortico-nuclear zones of topographically organized connections. In addition, R-cadherin-positive mossy fiber terminals display a periodic pattern in the internal granular layer. In this layer, Cad7 and R-cadherin are associated with synaptic complexes. These results suggest that cadherins play a pivotal role in the formation of functional cerebellar architecture by providing a three-dimensional scaffold of adhesive information.

N-cadherin is crucial for heart formation in the chick embryo
S Nakagawa, M Takeichi
DEVELOPMENT GROWTH & DIFFERENTIATION, 39, 4, 451, 455, BLACKWELL SCIENCE, 1997年08月, ［査読有り］
英語, 研究論文（学術雑誌）, The developing heart primordium strongly expresses N-cadherin. In order to investigate the role of this adhesion molecule in heart morphogenesis, chicken embryos were cultured al stages 5-12, and injected with anti-N-cadherin antibodies that can specifically block the activity of this cadherin. In the injected embryos, the epimyocardial layers, which develop bilaterally from the splanchnic mesoderm, did not fuse to form a single cardiac tube. Moreover, each of the unfused layers became fragmented into epithelioid clusters. At the cellular level, large intercellular gaps were observed in the antibody-treated myocardial layers. These disorganized myocardial layers beat to some extent, suggesting that their differentiation was not blocked; however, their contraction was not coordinated. Morphogenesis of other tissues, not only N-cadherin-negative but also N-cadherin-positive tissues, such as the neural tube and notochord, proceeded normally even in the presence of anti-N-cadherin antibodies. These results suggest that N-cadherin is indispensable for heart formation, but not for morphogenesis of the other tissues, at the developmental stages examined. For the latter processes, expression of other cadherin subtypes presumably compensated for the loss of N-cadherin activity.

In vivo evidence of the critical role of cadherin-5 in murine vascular integrity.
N Matsuyoshi, K Toda, Y Horiguchi, T Tanaka, S Nakagawa, M Takeichi, S Imamura
Proceedings of the Association of American Physicians, 109, 4, 362, 71, 1997年07月, ［国際誌］
英語, 研究論文（学術雑誌）, Vascular endothelial cell-cell adhesion is crucial for the regulation of vascular functions and is associated with many circulatory disorders. We isolated a rat monoclonal antibody (VECD1) recognizing the mouse vascular endothelial cell adhesion molecule and found that it inhibited vascular endothelial cell-cell association. We sequenced a full-length cDNA of the antigen that was identical to mouse cadherin-5. L-cells transfected with its cDNA acquired cell-cell adhesiveness, and these transfectants reacted with VECD1 at cell-cell contact areas. We studied the role of mouse cadherin-5 in vascular functions. The addition of VECD1 antibody to a cultured vascular endothelial cell line (F-2) caused the detachment of each cell. Although normal F-2 cells formed tubular structures on Matrigel, VECD1 disturbed the tubulogenesis. VECD1 also increased the permeability through the F-2 cell layer. To clarify the in vivo function of mouse cadherin-5, we intraperitoneally injected the hybridomas producing VECD1 into adult mice. Severe venous stasis and subcutaneous hemorrhage were induced within several days after the injection, resulting in the early death of the animals. These findings are evidence of an essential role of cadherin-5 in the regulation of vascular endothelial cell-cell adhesion in vivo.

11 Selective Aggregation Assays for Embryonic Brain Cells and Cell lines
Shinichi Nakagawa, Hiroaki Matsunami, Masatoshi Takeichi
Current Topics in Developmental Biology, 36, C, 197, 210, 1997年01月01日, ［査読有り］
英語, 研究論文（学術雑誌）, This chapter illustrates how to assay the role of adhesion mechanisms in selective adhesion of embryonic brain cells and fibroblastic cell lines. The methods described in the chapter include how to dissociate cells while leaving a particular set of adhesion mechanisms intact, how to let them reaggregate, and how to evaluate selective cell adhesion. Cell-cell adhesion mechanisms are complex, and comprise multiple systems, Animals cells are known to exhibit selective adhesiveness. When cells derived from different tissues are mixed, they sort out. This property of cells is thought to play a key role in the organization of complex tissue architecture. Studies suggest that the selective adhesiveness of cells also contributes to regionalization in the embryonic brain. To understand the molecular basis of patterning in the developing brain, therefore, it is critical to accumulate much more information on the adhesion properties of the cells that belong to each compartment. There are many ways to measure and quantify cell-cell adhesiveness. A classic method is to dissociate cell masses into single cells, and observe their reaggregation process. This chapter presents an updated version of classic methods, illustrates how to dissociate cells, how to allow them to reaggregate, and how to assay their selective adhesiveness. © 1998 Academic Press, Inc.

NEURAL CREST CELL-CELL ADHESION CONTROLLED BY SEQUENTIAL AND SUBPOPULATION-SPECIFIC EXPRESSION OF NOVEL CADHERINS
S NAKAGAWA, M TAKEICHI
DEVELOPMENT, 121, 5, 1321, 1332, COMPANY OF BIOLOGISTS LTD, 1995年05月, ［査読有り］
英語, 研究論文（学術雑誌）, We identified two cadherins, c-cad6B and c-cad7, expressed by neural crest cells at their premigratory and migratory stages, respectively, in chicken embryos, cDNA transfection experiments showed that both were homophilic adhesion endowing cells with molecules, specific adhesiveness. During development, c-cad6B appeared in the neural fold, localizing at the future neural crest area. This expression was maintained during neural tube closure, but disappeared after neural crest cells had left the neural tube, suggesting its role in neural fold fusion and/or in the formation and maintenance of the presumptive neural crest domain in the neural plate/tube. Crest cells emerging from the neural tube lost c-cad6B, and a subpopulation of them began to express c-cad7. This subpopulation-specific expression of c-cad7 persisted during their migration. The migrating c-cad7-positive cells clustered together, and eventually populated restricted regions including the dorsal and ventral roots but very little ganglia. The latter was populated with N-cadherin-positive crest cells, Migrating neural crest cells expressed alpha- and beta-catenin at cell-cell contacts, indicating that their cadherins are functioning. These results suggest that the migrating crest cells are grouped into subpopulations expressing different cadherins. The cadherin-mediated specific interaction between crest cells likely plays a role in intercellular signaling between homotypic cells as well as in sorting of heterotypic cells.

NEAT1のRNAドメインによって決定されるシェル局在タンパク質はパラスペックルの核スペックルとの分離を制御する
高桑央, 高桑央, 山崎智弘, SOUQUERE Sylvie, 足達俊吾, 藤原奈央子, 吉野彪羅, 山本哲也, 中川真一, 村上正晃, PIERRON Gerard, 廣瀬哲郎, 廣瀬哲郎, 廣瀬哲郎, 日本分子生物学会年会プログラム・要旨集(Web), 46th, 2023年

メダカを用いたmalat1の機能解析
横井佐織, 水戸麻里, 岩崎信太郎, 中川真一, 日本分子生物学会年会プログラム・要旨集(Web), 45th, 2022年

メダカをモデルとした行動に関わる性格の分子神経基盤解析
田中豪, 成瀬清, 郷康広, 辰本将司, 豊田敦, 勝村啓史, 中川真一, 横井佐織, 日本神経化学会大会抄録集(Web), 65th, 2022年

【相分離 メカニズムと疾患"膜のないオルガネラ"はいかに機能するか?神経変性疾患・ウイルス感染とどう関わるか?】(第2章)相分離と細胞内構造 核内の非膜オルガネラ Cajal bodyの多機能性と柔軟性の包括的理解
大峽 咲希, 中川 真一, 米田 宏, 実験医学, 39, 10, 1509, 1514, 2021年06月
Cajal bodyは不思議な構造体である。非膜オルガネラのなかでも古典的な部類に入り、構成因子や動態はよく調べられているにもかかわらず、Cajal bodyとは何なのか、その全体像は捉えづらい。この総説ではこれまでの研究をベースに、Cajal bodyを定義する難しさがどこにあるのか、Cajal bodyの分子レベルの機能や生理的意義は何かといった基本的な点を振り返りつつ、なぜこの構造体がcoilinを含むのかという根源的な問いへの答えをCajal bodyが海苔巻きであるという妄想も交えつつ考察したい。(著者抄録), (株)羊土社, 日本語

細胞内ティール組織化:マルチバレント相互作用と相分離が織りなす細胞内プロセスの理解 ティール相互作用によって作られる核内非膜オルガネラの形成機構　　　　　　　　　　　　　　　
中川 真一, 大峽 咲希, 米田 宏, 日本細胞生物学会大会講演要旨集, 72回, W2, 4, 2020年06月
(一社)日本細胞生物学会, 日本語

【イメージング時代の構造生命科学 細胞の動態、膜のないオルガネラ、分子の構造変化をトランススケールに観る】(第2章)構造生命科学からトランススケール・イメージングによる細胞動態学へ トランススケールな解析が待たれる生命科学の未解決課題 RNAを含む非膜構造体の内部微細構造観察と天然変性領域の役割
中川 真一, 実験医学, 38, 5, 762, 768, 2020年03月
細胞内には核小体に代表される、脂質二重膜で囲まれていない構造体が多数存在し、それらは総称して非膜構造体とよばれている。非膜構造体の多くはRNAをその構成成分に含んでいるが、これらのRNAは天然変性領域を有する各種RNA結合タンパク質が集合する足場を提供し、局所的な濃度の上昇によって天然変性領域が仲介する多数の弱い分子間相互作用が働く環境を作ることで、可塑性に富む非膜構造体形成を促進していると考えられている。本稿では、近年相次いで報告されたRNAを含む非膜構造体の内部微細構造観察の動向をまとめ、立体構造に依存しない新しいタイプの分子間相互作用の重要性について議論する。(著者抄録), (株)羊土社, 日本語

配偶者選択におけるオキシトシンの役割の性的二形性
横井佐織, 成瀬清, 亀井保博, 安齋賢, 木下政人, 水戸麻理, 岩崎信太郎, 井ノ上俊太郎, 奥山輝大, 中川真一, YOUNG Larry J., 竹内秀明, 日本分子生物学会年会プログラム・要旨集(Web), 43rd, 2020年

パラスペックル:RNAを骨格として形成されるミクロ相分離構造体
山崎智弘, 山本哲也, 吉野彪羅, 高桑央, 高桑央, SOUQUERE Sylvie, 中川真一, PIERRON Gerard, 廣瀬哲郎, 日本分子生物学会年会プログラム・要旨集(Web), 43rd, 2020年

長鎖ノンコーディングRNAであるGomafuの核内繋留機構におけるSF3b2の寄与の解明　　　　　　　　　　　　　　　
上原 理加, 梅本 銀河, 中川 真一, 吉田 稔, 服部 光治, 築地 仁美, 日本生化学会大会プログラム・講演要旨集, 92回, [2P, 242], 2019年09月
(公社)日本生化学会, 日本語

核内構造体パラスペックルの生理機能　　　　　　　　　　　　　　　
中川 真一, 臨床神経学, 58, Suppl., S56, S56, 2018年12月
(一社)日本神経学会, 日本語

UPA‐Seq:UVへの感受性を利用した機能性lncRNAの同定
中川真一, 小松太和, 水戸麻理, 竹本愛菜, 藤居光一, 岩崎信太郎, 日本RNA学会年会要旨集, 20th, 40, 2018年07月09日
日本語

核内構造体パラスペックルはNEAT1 IncRNAの複数の機能ドメインにより構築される　　　　　　　　　　　　　　　
山崎 智弘, Souquere Sylvie, 木立 尚孝, Fox Archa, 中川 真一, Gerard Pierron, 廣瀬 哲郎, 生命科学系学会合同年次大会, 2017年度, [1P, 0764], 2017年12月
生命科学系学会合同年次大会運営事務局, 日本語

分子共生-RNAタンパク質相互作用による機能創発 UV照射に対する異なる感受性を利用した新規機能的lncRNA候補の同定(Identification of novel functional IncRNA candidates using differential sensitivities to UV-irradiation)　　　　　　　　　　　　　　　
中川 真一, 生命科学系学会合同年次大会, 2017年度, [4AW09, 7], 2017年12月
生命科学系学会合同年次大会運営事務局, 英語

DISTURBED EXPRESSION OF IMMUNOREGULATORY LONG NONCODING RNAS IN CIRCULATING IMMUNE CELLS FROM PATIENTS WITH EARLY MYOCARDIAL INFARCTION
Wolfgang Poller, Martina Gast, Shinichi Nakagawa, Jan Haas, Andrea Stroux, Christian Mueller, Daniela Boernigen, Dirk Westermann, Carsten Skurk, Ursula Rauch-Kroehnert, Kannanganattu Prasanth, Stefan Blankenberg, Benjamin Meder, Ulf Landmesser, Tanja Zeller, JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY, 69, 11, 2036, 2036, 2017年03月
ELSEVIER SCIENCE INC, 英語, 研究発表ペーパー・要旨（国際会議）

DEFICIENCY OF LONG NONCODING RNA MALAT1 CAUSES IMMUNE SYSTEM DEREGULATION AND ACCELERATED ATHEROSCLEROSIS IN APO E-/- MICE
Wolfgang Poller, Martina Gast, Bernhard Rauch, Shinichi Nakagawa, Kannanganattu Prasanth, Andrea Stroux, Jan Haas, Benjamin Meder, Carsten Skurk, Ursula Rauch-Kroehnert, Ulf Landmesser, JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY, 69, 11, 2031, 2031, 2017年03月
ELSEVIER SCIENCE INC, 英語, 研究発表ペーパー・要旨（国際会議）

Lessons from reverse-genetic studies of lncRNAs
Shinichi Nakagawa, Biochimica et Biophysica Acta - Gene Regulatory Mechanisms, 1859, 1, 177, 183, 2016年01月01日
The functions of long noncoding RNAs (lncRNAs) have mainly been studied using cultured cell lines, and this approach has revealed the involvement of lncRNAs in a variety of biological processes, including the epigenetic control of gene expression, post-transcriptional regulation of mRNA, and cellular proliferation and differentiation. Recently, increasing numbers of studies have investigated the functions of lncRNAs using gene-targeted model mice, largely confirming the physiological importance of lncRNA-mediated regulation in individual animals. In some cases, however, the results obtained by studies using knockout mice have been somewhat inconsistent with those of the preceding cell-based analyses. In this review, I will summarize the lessons that we are learning from the reverse-genetic studies of lncRNAs, namely the importance of noncoding DNA elements, the weak correlation between expression level and phenotypic prominence, the existence of tissue- and condition-specific phenotypes and incomplete penetrance, and the function of lncRNAs as precursor molecules. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa., Elsevier, 英語, 書評論文,書評,文献紹介等

Clues to long noncoding RNA taxonomy
Tetsuro Hirose, Shinichi Nakagawa, BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS, 1859, 1, 1, 2, 2016年01月
ELSEVIER SCIENCE BV, 英語, その他

ncRNA研究をはじめよう 6.RNA細胞内局在の高解像度解析
水戸麻理, 中川真一, 実験医学, 33, 20, 3402, 3406, 2015年12月10日
日本語

長鎖ノンコーディングRNAのマイルストーン研究 核内構造体lncRNA 13.染色体機能とlncRNA
若井道仁, 中川真一, 実験医学, 33, 20, 3296, 3297, 2015年12月10日
日本語

長鎖ノンコーディングRNAのマイルストーン研究 その他のlncRNA関連トピック 17.lncRNA複合体解析法の登場
中川真一, 実験医学, 33, 20, 3304, 3305, 2015年12月10日
日本語

長鎖ノンコーディングRNAのマイルストーン研究 その他のlncRNA関連トピック 19.lncRNA解析の問題点と遺伝学的解析
中川真一, 実験医学, 33, 20, 3308, 3309, 2015年12月10日
日本語

RNAと生命現象 長鎖ノンコーディングRNAによる妊孕性の制御　　　　　　　　　　　　　　　
中川 真一, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [2S15, 2], 2015年12月
(公社)日本生化学会, 英語

【ノンコーディングRNAテキストブック 最新の医学・創薬研究、方法論とマイルストーン論文200報】(第2章)長鎖ノンコーディングRNAのマイルストーン研究 総論 長鎖ncRNA研究の歴史と現状
中川 真一, 実験医学, 33, 20, 3266, 3269, 2015年12月
(株)羊土社, 日本語

【ノンコーディングRNAテキストブック 最新の医学・創薬研究、方法論とマイルストーン論文200報】(第2章)長鎖ノンコーディングRNAのマイルストーン研究 核内構造体IncRNA 染色体機能とIncRNA
若井 道仁, 中川 真一, 実験医学, 33, 20, 3296, 3297, 2015年12月
(株)羊土社, 日本語

【ノンコーディングRNAテキストブック 最新の医学・創薬研究、方法論とマイルストーン論文200報】(第2章)長鎖ノンコーディングRNAのマイルストーン研究 その他のIncRNA関連トピック IncRNA複合体解析法の登場
中川 真一, 実験医学, 33, 20, 3304, 3305, 2015年12月
(株)羊土社, 日本語

【ノンコーディングRNAテキストブック 最新の医学・創薬研究、方法論とマイルストーン論文200報】(第2章)長鎖ノンコーディングRNAのマイルストーン研究 その他のIncRNA関連トピック IncRNA解析の問題点と遺伝学的解析
中川 真一, 実験医学, 33, 20, 3308, 3309, 2015年12月
(株)羊土社, 日本語

【ノンコーディングRNAテキストブック 最新の医学・創薬研究、方法論とマイルストーン論文200報】(第4章)ncRNA研究をはじめよう RNA細胞内局在の高解像度解析
水戸 麻理, 中川 真一, 実験医学, 33, 20, 3402, 3406, 2015年12月
RNAの局在を可視化する手法として最も古くから使われている手法が、in situハイブリダイゼーションである。その原理は、固定した細胞に対してラベルしたプローブを高特異性条件下でハイブリダイズさせ、ラベルを検出することで細胞内局在を調べるというきわめてシンプルなものであるが、感度、特異性、ともにいまだこれを上回る技術は開発されていない。本稿ではin situハイブリダイゼーションを行う際の留意点、および、RNA局在を超解像顕微鏡を用いて観察する手法を解説する。(著者抄録), (株)羊土社, 日本語

NEAT1 lncRNAの難溶性を考慮したパラスペックル構造因子の再定量と構造構築原理の探究
中條 岳志, 中川 真一, 川口 哲哉, 内匠 透, 廣瀬 哲郎, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [2T25p, 08(2P0765)], 2015年12月
(公社)日本生化学会, 日本語

RNA制御のケミカルバイオロジー スプライシング阻害剤Spliceostatin Aの感受性を決定づけるイントロンの特徴とは?　　　　　　　　　　　　　　　
芳本 玲, 甲斐田 大輔, 古野 正朗, 鈴木 治和, 林崎 良英, 前田 明, 中川 真一, 吉田 稔, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [4W10, 4], 2015年12月
(公社)日本生化学会, 日本語

Close Up実験法(Series 266) 簡単RNA蛍光in situハイブリダイゼーション実験
川口 哲哉, 廣瀬 哲郎, 中川 真一, 実験医学, 33, 14, 2283, 2289, 2015年09月
(株)羊土社, 日本語

教えて!エコ実験RETURNS 工夫&節約のメリハリ研究術 第4回 染物屋のカイゼン術
中川真一, 実験医学, 33, 11, 1802, 1806, 2015年07月01日
(株)羊土社, 日本語

スプライシング阻害剤Spliceostatin Aの感受性を決定づけるイントロンの特徴とは?
芳本玲, 芳本玲, 甲斐田大輔, 甲斐田大輔, 古野正朗, 鈴木治和, 林崎良英, 前田明, 中川真一, 吉田稔, 日本生化学会大会(Web), 88th, 4W10-P-4 (WEB ONLY), 2015年
日本語

長鎖ノンコーディングRNA:遺伝子発現制御メカニズム別・機能リファレンス【メカニズム 4):mRNAと相互作用するlncRNA】レトロトランスポゾン配列を介して形成される二本鎖RNAによる遺伝子発現制御
石田賢太郎, 中川真一, 細胞工学, 34, 1, 38, 42, 2014年12月22日
日本語

【長鎖ノンコーディングRNA:遺伝子発現制御メカニズム別・機能リファレンス】【メカニズム mRNAと相互作用するIncRNA】レトロトランスポゾン配列を介して形成される二本鎖RNAによる遺伝子発現制御
石田 賢太郎, 中川 真一, 細胞工学, 34, 1, 38, 42, 2014年12月
(株)学研メディカル秀潤社, 日本語

The Long Noncoding MALAT1-MascRNA System is a Novel Regulator of Cardiac Innate Immunity
Wolfgang Poller, Martina Gast, Blanche Schroen, Antje Voigt, Jan Haas, Anna Papageorgiou, Uwe Kuehl, Dirk Lassner, Xiaomin Wang, Madlen Loebel, Sabrina Wilk, Nadine Althof, Tim Peters, Kannanganattu Prasanth, Hugo Katus, Benjamin Meder, Shinichi Nakagawa, Carmen Scheibenbogen, Stephane Heymans, CIRCULATION, 130, 2014年11月
LIPPINCOTT WILLIAMS & WILKINS, 英語, 研究発表ペーパー・要旨（国際会議）

YbによるpiRNA生合成因子/piRNA前駆体の制御機構
室田友紀子, 石津大嗣, 中川真一, 岩崎由香, 芝田晋介, 鎌谷太陽, 齋藤都暁, 岡野栄之, 塩見春彦, 塩見美喜子, 日本RNA学会年会要旨集, 16th, 122, 2014年07月23日
日本語

スプライシングがMalat1 RNAの局在と安定化を保証している!
芳本玲, 古野正明, 鈴木治和, 林崎良英, 中川真一, 吉田稔, 日本RNA学会年会要旨集, 16th, 229, 2014年07月23日
日本語

hnRNP Uにより制御される新規インプリンティング遺伝子の同定
長谷川優子, 中川真一, 日本RNA学会年会要旨集, 16th, 114, 2014年07月23日
日本語

Nuclear lncRNAs as epigenetic regulators-Beyond skepticism
Shinichi Nakagawa, Yuji Kageyama, Biochimica et Biophysica Acta - Gene Regulatory Mechanisms, 1839, 3, 215, 222, 2014年03月
Systematic transcriptome analysis has revealed that a vast majority of the mammalian genome is transcribed into RNA, thus establishing the concept of "pervasive transcription." More than half of these RNAs do not encode proteins, and they are collectively called noncoding RNAs. Although the physiological relevance of the transcription of these noncoding RNAs has remained unclear, it was recently proposed that one of the major roles of long noncoding RNAs (lncRNAs) in the nucleus is the regulation of gene expression at the transcriptional level via histone or DNA modification. In this review, we will summarize the advancement of our understanding of the molecular mechanisms of lncRNAs. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development. © 2013 The Authors., 書評論文,書評,文献紹介等

Global regulation of gene expression by a nuclear noncoding RNA highly homologous to SINE B1
Shinichi Nakagawa, Kentaro Ishida, GENES & GENETIC SYSTEMS, 88, 6, 343, 343, 2013年12月
GENETICS SOC JAPAN, 英語, 研究発表ペーパー・要旨（国際会議）

レトロトランスポゾンSINE B1と相同配列を持つ核内ノンコーディングRNAによるグローバルな遺伝子発現制御
中川真一, 石田賢太郎, 日本遺伝学会大会プログラム・予稿集, 85th, 75, 2013年08月31日
日本語

核内構造体パラスペックルはGAリッチな配列を含むイントロンを繋留している
WEST Jason, 工樂樹洋, KINGSTON Robert, 廣瀬哲郎, 中川真一, 日本RNA学会年会要旨集, 15th, 100, 2013年07月24日
日本語

【生命分子を統合するRNA-その秘められた役割と制御機構 分子進化・サイレンシング・non-coding RNAからRNA修飾・編集・RNA-タンパク質間相互作用まで】(第2章)転移因子・ウイルスと宿主の"せめぎ合い"と遺伝子サイレンシング 概論 転移因子・ウイルスと宿主のせめぎ合いによりもたらされるゲノム進化
中川 真一, 実験医学, 31, 7, 1047, 1054, 2013年05月
日常生活のタイムスケールにおいてゲノムDNAは不変であり、その安定性が生命現象の基盤となっているのは周知の事実である。ところが、数億年という単位で物事が進む進化的なタイムスケールでみるとこの常識は必ずしも真ではなく、むしろ、ゲノムDNAが変化していくことこそ生命の本質であるという見方もできよう。前者のタイムスケールにおいては、情報はDNAからRNAを経てタンパク質へとほぼ一方向に流れていくが、後者のタイムスケールにおいては逆転写反応を介したRNAからDNAへの情報の流れも端倪すべからざる大きな力として働いてくる。本章では、宿主ゲノムDNAとウイルスや内在性の転移因子のせめぎ合いを概観し、その進化的な役割を考察してゆく。(著者抄録), (株)羊土社, 日本語

転移因子・ウイルスと宿主の“せめぎ合い”と遺伝子サイレンシング 概論 転移因子・ウイルスと宿主のせめぎ合いによりもたらされるゲノム進化
中川真一, 実験医学, 31, 7, 1047, 1054, 2013年05月01日
日本語

【生命分子を統合するRNA-その秘められた役割と制御機構 分子進化・サイレンシング・non-coding RNAからRNA修飾・編集・RNA-タンパク質間相互作用まで】(第2章)転移因子・ウイルスと宿主の"せめぎ合い"と遺伝子サイレンシング mRNA非翻訳領域のレトロトランスポゾン挿入配列による転写後遺伝子発現制御 潜在的な遺伝的変化の蓄積と表現型としての多様性の急激な解放
中川 真一, 石田 賢太郎, 実験医学, 31, 7, 1079, 1085, 2013年05月
高等真核生物においてゲノムの大半を占めるレトロトランスポゾンの配列は、遺伝子間領域やイントロンのみならず、mRNAの非翻訳領域にも少なからぬ割合で挿入されている。本稿では転写産物内のレトロトランスポゾン配列によって制御されている遺伝子発現制御に関する知見をまとめるとともに、中立的なレトロトランスポゾン配列の挿入によって蓄積された潜在的な変異が、それらの配列をターゲットにした発現制御システムの登場によって一気に多様な表現型として解放される可能性について議論する。(著者抄録), (株)羊土社, 日本語

プロテアソーム阻害に応答した核内構造体noncoding RNAの転写抑制機能
廣瀬哲郎, 谷川明恵, VIRNICCHI Giorgio, BENARD Marianne, 長沼孝雄, 佐々木保典, 中川真一, FOX Archa, PIRRON Gerard, 日本RNA学会年会要旨集, 14th, 52, 2012年07月18日
日本語

核内構造体パラスペックルの骨格ncRNAであるNEAT1を欠損するマウスは早期に不妊になる
中川真一, 廣瀬哲郎, 日本RNA学会年会要旨集, 14th, 48, 2012年07月18日
日本語

4.5SH RNAによる核内繋留を介した遺伝子発現制御におけるdsRNA結合タンパク質Ilf3の役割
石田賢太郎, 中川真一, 日本RNA学会年会要旨集, 14th, 140, 2012年07月18日
日本語

ショウジョウバエ卵巣体細胞におけるprimary piRNA生合成機構
室田友紀子, 石津大嗣, 芝田晋介, 鎌谷太陽, 齋藤都暁, 中川真一, 岡野栄之, 塩見春彦, 塩見美喜子, 日本分子生物学会年会プログラム・要旨集(Web), 35th, 2P-0287 (WEB ONLY), 2012年
日本語

【細胞核-構造と機能】その他 核内ノンコーディングRNAの役割
石塚 明, 中川 真一, 生体の科学, 62, 5, 502, 503, 2011年10月
(公財)金原一郎記念医学医療振興財団, 日本語

hnRNP UはXist RNA局在制御を通じてX染色体不活性化に関与する
長谷川 優子, 筒井 公子, 筒井 研, 鈴木 勉, 中川 真一, 日本生化学会大会プログラム・講演要旨集, 84回, 1S1p, 4, 2011年09月
(公社)日本生化学会, 日本語

【ゲノムの"ダークマター"長鎖ncRNAが制御する多彩な生命現象】概論 長鎖ncRNA研究のはじまりの終わり
中川 真一, 影山 裕二, 実験医学, 29, 11, 1702, 1708, 2011年07月
ヒトやマウスのゲノムからタンパク質をコードしないノンコーディングRNA(ncRNA)が大量に転写されているということが明らかとなってから、かれこれ10年近くが経とうとしている。ncRNAのなかでも長鎖のncRNAの生理機能は長らく不明であったが、近年になってようやく個々の遺伝子について詳細な解析がなされるようになり、これらの分子が制御する生命機能の全体像がおぼろげながら見えるようになってきた。この概論では、現時点における長鎖ncRNA研究の現状と問題点を解説したい。(著者抄録), (株)羊土社, 日本語

ゲノムの“ダークマター”長鎖ncRNAが制御する多彩な生命現象 長鎖ncRNA研究のはじまりの終わり
中川真一, 影山裕二, 実験医学, 29, 11, 1702, 1708, 2011年07月01日
日本語

【ノンコーディングRNAによるエピジェネティック制御機構:設計図にはない多様な細胞機能を生み出す仕組み】核内構造体に活躍の場を求めたノンコーディングRNAたち
中川 真一, 細胞工学, 30, 7, 734, 739, 2011年06月
(株)学研メディカル秀潤社, 日本語

90塩基の低分子ノンコーディングRNAによる核内繋留を介した新規遺伝子発現制御(A novel gene expression regulatory mechanism controlled by 90nt nuclear non-coding RNA through the nuclear retention)　　　　　　　　　　　　　　　
石田 賢太郎, 中川 真一, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 83回・33回, 4P, 0796, 2010年12月
(公社)日本生化学会, 英語

非コードRNA作用マシナリー 動作原理の分子基盤と生理機能 大量に蓄積する核内ノンコーディングRNAの機能解析 Gomafuを例にとって
中川 真一, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 83回・33回, 2W8, 6, 2010年12月
(公社)日本生化学会, 日本語

核内構造構築に必要なnoncoding RNAの選択的3'末端プロセシングの分子機構
長沼 孝雄, 中川 真一, 青木 一真, 五島 直樹, 佐々木 保典, 廣瀬 哲郎, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 83回・33回, 3P, 0795, 2010年12月
(公社)日本生化学会, 日本語

核内構造体noncoding RNAの選択的プロセシング制御機構
長沼孝雄, 中川真一, 青木一真, 五島直樹, 佐々木保典, 廣瀬哲郎, 日本RNA学会年会要旨集, 12th, 72, 2010年07月27日
日本語

核内長鎖ノンコーディングRNA Gomafuは動物の行動を制御する
曽根正光, 築地仁美, 芳本玲, 吉田稔, 北市清幸, 山本経之, 阿部高也, 高雄啓三, 宮川剛, 中川真一, 日本RNA学会年会要旨集, 12th, 70, 2010年07月27日
日本語

拡大・進展を続けるRNA研究の最先端 長鎖noncoding RNA・small RNAからRNA修飾・編集・品質管理まで 第1章 noncoding RNAの分子機構と生命現象,疾患との関わり 1.進化的に「新しい」バイオマテリアル―核内mRNA型noncoding RNAに何ができるのか
中川真一, 実験医学, 28, 10, 1496-1501,1490(1), 2010年06月10日
日本語

【拡大・進展を続けるRNA研究の最先端 長鎖noncoding RNA・small RNAからRNA修飾・編集・品質管理まで】noncoding RNAの分子機構と生命現象、疾患との関わり 進化的に「新しい」バイオマテリアル 核内mRNA型noncoding RNAに何ができるのか
中川 真一, 実験医学, 28, 10, 1496, 1501, 2010年06月
さまざまな生物のゲノムが解読されて明らかとなったのは、多細胞生物間でタンパク質をコードしている遺伝子の数がそれほど変わらないという事実であった。一方、高等真核生物では大量のnoncoding RNAがゲノムから読み出されている。これらのRNAの働きで高等な生物に見られる複雑さを説明できるのか?本稿ではnoncoding RNAの中でも、特に哺乳類に特異的に存在する4種類の核内mRNA型noncoding RNAに焦点を当て、進化的に「新しい」それらの遺伝子が制御する核内プロセスや生命現象を概説していきたい。(著者抄録), (株)羊土社, 日本語

Emerging world of nuclear long noncoding RNAs
Shinichi Nakagawa, Seikagaku, 82, 1, 42, 46, 2010年, ［査読有り］
1, 日本語

中枢神経系の層形成時に細胞タイプ特異的に発現する新規遺伝子の機能解析
中川真一, 脳の神経回路の機能解明 平成16-21年度 No.16069101, 118, 119, 2010年
日本語

核内高分子ノンコーディングRNAの世界
中川 真一, 生化学, 82, 1, 42, 46, 2010年01月
これまでに知られている機能性核内高分子ノンコーティングRNAの主な機能について、転移座からみてcisに働いて転写サイト付近の転写を制御するもの、transに働いて核内構造体を形成するものの二つに分けて概説した。パラスペックルの構造維持に必要なMENε/β、核スペックルに局在するMalat1、および著者等によって同定された、神経系に特異的に発現する新規核内構造物を形成するGomafuなどの発現、構造、機能などについて述べた。, (公社)日本生化学会, 日本語

ノンコーディングRNAが制御する多様な機能 新規核内高分子ノンコーディングRNA Gomafuの機能解析
中川 真一, 日本生化学会大会プログラム・講演要旨集, 82回, 2S12a, 1, 2009年09月
(公社)日本生化学会, 日本語

Tsukushi is a Frizzled ligand that, in competition with Wnt2b, regulates the proliferation of retinal stem/progenitor cells
Kunimasa Ohta, Ayako Ito, Sei Kuriyama, Giuseppe Lupo, Rika Nakayama, Naoko Ohshima, Mitsuko Kosaka, Shin-ichi Ohnuma, Shinichi Nakagawa, Hideaki Tanaka, MECHANISMS OF DEVELOPMENT, 126, S273, S273, 2009年08月
ELSEVIER SCIENCE BV, 英語, 研究発表ペーパー・要旨（国際会議）

齧歯類特異的な核内低分子ノンコーディングRNA4.5S RNA_Hによる新規遺伝子発現抑制機構
石田賢太郎, 中川真一, 日本RNA学会年会要旨集, 11th, 42, 2009年07月27日
日本語

Molecular function of Tsukushi on neuronal stem cells
Kunimasa Ohta, Ayako Ito, Sei Kuriyama, Giuseppe Lupo, Rika Nakayama, Naoko Ohshima, Mitsuko Kosaka, Shin-ichi Ohnuma, Shinichi Nakagawa, Hideaki Tanaka, NEUROSCIENCE RESEARCH, 65, S91, S91, 2009年
ELSEVIER IRELAND LTD, 英語, 研究発表ペーパー・要旨（国際会議）

核内構造構築を司る長鎖noncoding RNAの選択的3’末端形成を介した機能発現機構
長沼孝雄, 佐々木保典, 中川真一, 廣瀬哲郎, 日本分子生物学会年会講演要旨集, 32nd, Vol.1, 24, 2009年
日本語

大量の核内mRNA型ノンコーディングRNAが語るもの (特集 ノンコーディングRNA--現状と問題点)
中川真一, 石田賢太郎, 蛋白質核酸酵素, 53, 15, 1932, 1939, 2008年12月
共立出版

[Lessons from abundant nuclear mRNA-like noncoding RNAs]
Nakagawa S, Ishida K, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 53, 1932, 1939, 2008年12月, ［査読有り］
15

【ノンコーディングRNA:現状と問題点】大量の核内mRNA型ノンコーディングRNAが語るもの
中川 真一, 石田 賢太郎, 蛋白質・核酸・酵素, 53, 15, 1932, 1939, 2008年12月
近年のトランスクリプトーム解析によって,ゲノムの大部分が転写されていること,その多くがmRNAとしての特徴をもちながら蛋白質をコードしていないmRNA型ノンコーディングRNAであることが明らかとなり,特定のものについてはその機能もわかりつつある.本稿では,とくに最近,あいついで報告された,核内に大量に蓄積するmRNA型ノンコーディングRNAに焦点をあて,その研究の現状を解説したい.また,ノンコーディングRNA全般について,洪水のように発表されつづけている論文から少し距離をおいた議論をしてみたい.(著者抄録), 共立出版(株), 日本語

非コードRNA研究の新領域 新規mRNA様非コードRNA Gomafuの機能解析(Functional analysis of a novel mRNA-like noncoding RNA Gomafu)　　　　　　　　　　　　　　　
中川 真一, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 81回・31回, 1S3, 1, 2008年11月
(公社)日本生化学会, 英語

神経特異的mRNA様non‐codingRNAであるGomafuの核内繋留メカニズムの解析
曽根正光, 築地仁美, 甲斐田大輔, 吉田稔, 中川真一, 日本RNA学会年会要旨集, 10th, 4, 2008年07月23日
日本語

機能性RNAの生理学 7.核内mRNA型non‐coding RNA
中川真一, 実験医学, 26, 10, 1552-1556,HYOSHI,1474(2), 2008年06月10日
日本語

【RNAの機能解明と医療応用 最新の解析戦略と機能性RNAの知見から、RNA異常と疾患の関わり、RNA医薬・診断への応用まで】機能性RNAの生理学 核内mRNA型non-coding RNA
中川 真一, 実験医学, 26, 10, 1552, 1556, 2008年06月
一般にタンパク質をコードしているmRNAは細胞質に局在するが、mRNA型non-coding RNAの中には細胞質へと運ばれず核内に局在するものがある。これら核内mRNA型non-coding RNAの既知の生理的機能として染色体レベルでの発現調節や刷り込みの制御等が知られているが、核内で特定の「足場」を作っていると思われる新たな分子群が同定されつつある。これら新規mRNA型non-coding RNAの機能解析を通じて全く新たな研究領域が発掘される可能性がある。(著者抄録), (株)羊土社, 日本語

【命名から20年を超えて Wnt研究の新機軸】 概論 Wnt研究の現在 基礎研究の広がりと臨床応用への予兆　　　　　　　　　　　　　　　
中川 真一, 実験医学, 26, 3, 356, 360, 2008年02月
(株)羊土社, 日本語

hnRNP UはXist RNAの不活性化X染色体上への局在を制御する
長谷川優子, 河野真二, 筒井公子, 筒井研, 中川真一, 生化学, 2T5-2, 2008年
日本語

核内に局在する4.5S RNA_Hの遺伝子発現調整
石田賢太郎, 中川真一, 生化学, 1P-0918, 2008年
日本語

mRNA型ノンコーディングRNAであるGomafuの生化学的性質の検討
築地仁美, 曽根正光, 中川真一, 日本RNA学会年会要旨集, 9th, 234, 2007年07月28日
日本語

核に局在するmRNA‐like ncRNA Gomafuの機能解析
曽根正光, 稲垣智子, 竹市雅俊, 中川真一, 日本RNA学会年会要旨集, 9th, 231, 2007年07月28日
日本語

精子形成における哺乳類piRNAメチル化酵素の機能解析
長岡真, 大原智也, 中川真一, 加藤敬行, 五島直樹, 野村信夫, 鈴木健夫, 坂口裕理子, 鈴木勉, 日本RNA学会年会要旨集, 9th, 22, 2007年07月28日
日本語

網膜幹細胞の未分化性を制御する分子メカニズム
中川真一, 竹市雅俊, 久保郁, 久保郁, 日本眼科学会雑誌, 111, 101, 2007年03月15日
日本語

網膜細胞分化の分子メカニズム 網膜幹細胞の未分化性を制御する分子メカニズム　　　　　　　　　　　　　　　
中川 真一, 竹市 雅俊, 久保 郁, 日本眼科学会雑誌, 111, 臨増, 101, 101, 2007年03月
(公財)日本眼科学会, 日本語

Tsukushi inhibits proliferation of retinal stem cells by Wnt signalling inhibition
Kunimasa Ohta, Ayako Ito, Sei Kuriyama, Shin-Ichi Ohnuma, Mitsuko Kosaka, Shin-Ichi Nakagawa, Hideaki Tanaka, NEUROSCIENCE RESEARCH, 58, S56, S56, 2007年
ELSEVIER IRELAND LTD, 英語, 研究発表ペーパー・要旨（国際会議）

メラノコルチン2型受容体は,副腎皮質の発生,ステロイド合成,糖新生に必要である
千田大, 中川真一, 相良洋, 鈴木春巳, 久保光正, 岩倉洋一郎, 生化学, 3P-1139, 2007年
日本語

網膜水平細胞の形態形成における,サブタイプ特異的な転写因子の機能解析
須賀晶子, 中川真一, 生化学, 4P-0753, 2007年
日本語

核に局在する新規ノンコーディングRNAgomafuの同定
曽根正光, 竹市雅俊, 中川真一, 日本RNA学会年会要旨集, 8th, 37, 2006年07月18日
日本語

The molecular function of Tsukushi in retinal progenitor cells
Kunimasa Ohta, Ayako Ito, Ryu Gejima, Sei Kuriyama, Mitsuyo Kosaka, Shin-ichi Nakagawa, Hideaki Tanaka, NEUROSCIENCE RESEARCH, 55, S53, S53, 2006年
ELSEVIER IRELAND LTD, 英語, 研究発表ペーパー・要旨（国際会議）

Molecular network controlling retinal stem cell differentiation
Shinichi Nakagawa, Masatoshi Takeichi, Fumi Kubo, NEUROSCIENCE RESEARCH, 55, S18, S18, 2006年
ELSEVIER IRELAND LTD, 英語, 研究発表ペーパー・要旨（国際会議）

新規核スペックル構造を形成するノンコーディングRNA遺伝子の同定
曽根正光, 竹市雅俊, 中川真一, 日本分子生物学会年会講演要旨集, 28th, 74, 2005年11月25日
日本語

Wntシグナルによる培養条件下での層形成の誘導と幹細胞の維持活性 (特集 視聴覚の再生)
中川真一, 再生医療, 4, 2, 277, 282,200, 2005年05月
メディカルレビュー社

【視聴覚の再生】 Wntシグナルによる培養条件下での層形成の誘導と幹細胞の維持活性
中川 真一, 再生医療, 4, 2, 277, 282, 2005年05月
(株)メディカルレビュー社, 日本語

Wntシグナルによる培養条件下での層形成の誘導と幹細胞の維持活性
中川真一, 再生医療, 4, 2, 277-282,200, 2005年05月01日
日本語

眼の器官形成におけるWntの役割
中川真一, 生化学, 76, 12, 1573, 1576, 2004年12月25日
(公社)日本生化学会, 日本語

The role of Wnt signaling in eye organogenesis
S Nakagawa, SEIKAGAKU, 76, 12, 1573, 1576, 2004年12月, ［査読有り］
JAPANESE BIOCHEMICAL SOC, 日本語, 書評論文,書評,文献紹介等

網膜の組織形成とWntの活性 (特集 Wntシグナルとその多彩な機能--細胞極性・細胞運動・幹細胞の自己複製・発癌)
中川真一, 細胞工学, 23, 6, 651, 654, 2004年06月
秀潤社, 日本語

Wntシグナルとその多彩な機能:細胞極性・細胞運動・幹細胞の自己複製・発癌 網膜の組織形成とWntの活性
中川真一, 細胞工学, 23, 6, 651, 654, 2004年06月
日本語

網膜層形成における細胞体移動を制御する遺伝子のスクリーニング
曽根正光, 竹市雅俊, 中川真一, 日本発生生物学会大会発表要旨集, 37th, 77, 2004年05月10日
日本語

【Wntシグナルとその多彩な機能 細胞極性・細胞運動・幹細胞の自己複製・発癌】 網膜の組織形成とWntの活性　　　　　　　　　　　　　　　
中川 真一, 細胞工学, 23, 6, 651, 654, 2004年05月
発生期の網膜の細胞はいったんばらばらにしても再集合して組織塊を形成することができるが,そこでは特徴的な網膜の層構造は決して再生されず,内腔面に細胞が同心円状に並んだロゼットと呼ばれる異常な構造体ができてしまう.最近になって,Wntがこのロゼット形成を阻害し,正しい層構造を持った組織の再生を誘導することが明らかになった.この層形成誘導因子研究の歴史的背景と網膜の辺縁部の特殊性に焦点を当てて解説した, (株)学研メディカル秀潤社, 日本語

Axon sorting at the chiasm in xenopus
CE Holt, S Nakagawa, EUROPEAN JOURNAL OF NEUROSCIENCE, 12, 184, 184, 2000年
BLACKWELL SCIENCE LTD, 英語, 研究発表ペーパー・要旨（国際会議）

The metamorphosis antidote
S Nakagawa, WA Harris, NEURON, 24, 4, 763, 764, 1999年12月
CELL PRESS, 英語, その他

p120はカドヘリンの負の制御因子である
青野真也, 中川真一, 竹市雅俊, 日本細胞生物学会大会講演要旨集, 52nd, 36, 1999年
日本語

組換えアデノウイルスを用いた神経冠細胞への遺伝子強制発現
中川 真一, 森吉 弘毅, 中西 重忠, 竹市 雅敏, 日本分子生物学会年会プログラム・講演要旨集, 19, 675, 675, 1996年08月01日
日本語

Reexamination of the properties of epimorphin and its possible roles
Yohei Hirai, Shin-ichi Nakagawa, Masatoshi Takeichi, Cell, 73, 3, 426, 427, 1993年05月07日
英語, 速報，短報，研究ノート等（学術雑誌）

ノンコーディングRNAテキストブック : 最新の医学・創薬研究、方法論とマイルストーン論文200報
塩見 美喜子, 中川 真一, 浅原 弘嗣
羊土社, 2015年, 9784758103510, 日本語

Preferential association of prion-like-domain-containing RNA binding proteins to functional lncRNA candidates　　　　　　　　　　　　　　　
中川 真一
2018年11月30日, 英語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

Prediction of functional lncRNAs using UPA-seq　　　　　　　　　　　　　　　
中川 真一
CSHL Asia, 2018年10月31日, 英語, 口頭発表（招待・特別）
［国際会議］

UPA‐Seq:UVへの感受性を利用した機能性lncRNAの同定
中川真一, 小松太和, 水戸麻理, 竹本愛菜, 藤居光一, 岩崎信太郎
日本RNA学会年会要旨集, 2018年07月09日, 日本語

Attempts towards identification of novel functional lncRNAs　　　　　　　　　　　　　　　
中川 真一
EMBO workshop, 2018年04月11日, 英語, 口頭発表（招待・特別）
［国際会議］

Towards Identification of Novel Functional Long noncoding RNAs　　　　　　　　　　　　　　　
中川 真一
Kumamoto Key Forum, 2018年01月12日, 英語, 口頭発表（招待・特別）
［国内会議］

核内構造体パラスペックルの生理機能
中川真一
日本神経学会学術大会プログラム・抄録集, 2018年, 日本語

Identification of novel functional lncRNA candidates using differential sensitivities to UV-irradiation　　　　　　　　　　　　　　　
中川 真一
ConBio 2017, 2017年12月09日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

長鎖ノンコーディングRNAの個体レベルでの機能解析　　　　　　　　　　　　　　　
中川 真一
第6回生命医薬情報学連合大会, 2017年09月29日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

UV‐Phenol RNA‐seqによる機能性長鎖非コードRNAの同定
小松太和, 小松太和, 中川真一
日本RNA学会年会要旨集, 2017年07月19日, 日本語

Observation of sub-micron size nuclear bodies using super-resolution microscopy　　　　　　　　　　　　　　　
中川 真一
The 43th 内藤コンファレンス「Noncoding RNA: Biology, Chemistry, & Diseases」, 2017年06月29日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

Fine structural analyses of nuclear body paraspeckle using SIM　　　　　　　　　　　　　　　
中川 真一
第50回 日本発生生物学会年会, 2017年05月10日, 英語, 口頭発表（一般）
［国内会議］

「長鎖ノンコーディングRNAにできること~マウスからのメッセージ」
中川真一
千里ライフサイエンスセミナー講演要旨集, 2017年, 日本語

Observation of nuclear bodies using super-resolution microscopy　　　　　　　　　　　　　　　
Shinichi Nakagawa
第39回 日本分子生物学会年会, 2016年12月01日, 英語, 口頭発表（招待・特別）
Yokohama, ［招待講演］, ［国内会議］

核内構造体パラスペックルの 生理機能と微細構造解析　　　　　　　　　　　　　　　
中川 真一
第53回日本生化学会北海道支部例会, 2016年07月08日, 日本語, 口頭発表（基調）
［国内会議］

RNAと生命現象 長鎖ノンコーディングRNAによる妊孕性の制御　　　　　　　　　　　　　　　
中川 真一
日本生化学会大会・日本分子生物学会年会合同大会, 2015年12月, 英語

核内構造体を形成する長鎖ノンコーディングRNAの生理機能解析
中川真一
日本核酸医薬学会年会講演要旨集, 2015年11月15日, 日本語

超解像顕微鏡による核内構造体パラスペックルの微細構造観察
中川真一, 水戸麻理, 廣瀬哲郎
日本RNA学会年会要旨集, 2015年07月15日, 日本語

hnRNP Uにより制御される新規インプリンティング遺伝子の同定
長谷川優子, 中川真一
日本RNA学会年会要旨集, 2014年07月23日, 日本語

レトロトランスポゾンSINE B1と相同配列を持つ核内ノンコーディングRNAによるグローバルな遺伝子発現制御
中川真一, 石田賢太郎
日本遺伝学会大会プログラム・予稿集, 2013年08月31日, 日本語

核内構造体パラスペックルはGAリッチな配列を含むイントロンを繋留している
WEST Jason, 工樂樹洋, KINGSTON Robert, 廣瀬哲郎, 中川真一
日本RNA学会年会要旨集, 2013年07月24日, 日本語

長鎖非コードRNAの局在と機能に対するRNA結合タンパク質hnRNP Uの役割
長谷川優子, 中川真一
日本分子生物学会年会プログラム・要旨集(Web), 2013年, 日本語

核内構造体パラスペックルの骨格ncRNAであるNEAT1を欠損するマウスは早期に不妊になる
中川真一, 廣瀬哲郎
日本RNA学会年会要旨集, 2012年07月18日, 日本語

4.5SH RNAによる核内繋留を介した遺伝子発現制御におけるdsRNA結合タンパク質Ilf3の役割
石田賢太郎, 中川真一
日本RNA学会年会要旨集, 2012年07月18日, 日本語

核内ノンコーディングRNA Gomafuは新規核内構造体であるCelf3/SF1体と相互作用する
石塚明, 長谷川優子, 中川真一
日本分子生物学会年会プログラム・要旨集(Web), 2012年, 日本語

ショウジョウバエ卵巣由来体細胞におけるprimary piRNA生合成機構
室田友紀子, 石津大嗣, 齋藤都暁, 小谷葉月, 中川真一, 塩見春彦, 塩見美喜子
日本分子生物学会年会プログラム・要旨集(Web), 2011年, 日本語

非コードRNA作用マシナリー 動作原理の分子基盤と生理機能 大量に蓄積する核内ノンコーディングRNAの機能解析 Gomafuを例にとって　　　　　　　　　　　　　　　
中川 真一
日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 2010年12月, 日本語

90塩基の低分子ノンコーディングRNAによる核内繋留を介した新規遺伝子発現制御(A novel gene expression regulatory mechanism controlled by 90nt nuclear non-coding RNA through the nuclear retention)　　　　　　　　　　　　　　　
石田 賢太郎, 中川 真一
日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 2010年12月, 英語

hnRNP UはXist RNAの不活性化X染色体上への局在を制御する
長谷川優子, BROCKDORFF Neil, 筒井公子, 筒井研, 中川真一
日本RNA学会年会要旨集, 2010年07月27日, 日本語

核内構造体noncoding RNAの選択的プロセシング制御機構
長沼孝雄, 中川真一, 青木一真, 五島直樹, 佐々木保典, 廣瀬哲郎
日本RNA学会年会要旨集, 2010年07月27日, 日本語

核内長鎖ノンコーディングRNA Gomafuは動物の行動を制御する
曽根正光, 築地仁美, 芳本玲, 吉田稔, 北市清幸, 山本経之, 阿部高也, 高雄啓三, 宮川剛, 中川真一
日本RNA学会年会要旨集, 2010年07月27日, 日本語

ノンコーディングRNAが制御する多様な機能 新規核内高分子ノンコーディングRNA Gomafuの機能解析　　　　　　　　　　　　　　　
中川 真一
日本生化学会大会プログラム・講演要旨集, 2009年09月, 日本語

hnRNP UはXist RNAの不活性化X染色体上への局在を制御する
長谷川優子, 河野真二, 筒井公子, 筒井研, 鈴木勉, 中川真一
日本RNA学会年会要旨集, 2009年07月27日, 日本語

mRNA様non‐coding RNAであるGomafuによるスプライシング制御
築地仁美, 芳本玲, 吉田稔, 中川真一
日本分子生物学会年会講演要旨集, 2009年, 日本語

非コードRNA研究の新領域 新規mRNA様非コードRNA Gomafuの機能解析(Functional analysis of a novel mRNA-like noncoding RNA Gomafu)　　　　　　　　　　　　　　　
中川 真一
日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 2008年11月, 英語

核に局在するmRNA‐like ncRNA Gomafuの機能解析
曽根正光, 稲垣智子, 竹市雅俊, 中川真一
日本RNA学会年会要旨集, 2007年07月28日, 日本語

mRNA型ノンコーディングRNAであるGomafuの生化学的性質の検討
築地仁美, 曽根正光, 中川真一
日本RNA学会年会要旨集, 2007年07月28日, 日本語

核内ノンコーディングRNAの探索及び解析
石田賢太郎, 中川真一
日本RNA学会年会要旨集, 2007年07月28日, 日本語

Wntシグナル伝達の新たな展望 Wntシグナル伝達下流における網膜幹細胞維持の毛様体境界帯でのNotchシグナル伝達とは独立したC-hairy1による制御(New horizon of Wnt signaling c-hairy1 controls retinal stem cell maintenance downstream of Wnt signaling, independently of Notch singnaling at the ciliary marginal zone)　　　　　　　　　　　　　　　
久保 郁, 竹市 雅俊, 中川 真一
日本発生生物学会・日本細胞生物学会合同大会要旨集, 2007年05月, 英語

核に局在する新規ノンコーディングRNAgomafuの同定
曽根正光, 竹市雅俊, 中川真一
日本RNA学会年会要旨集, 2006年07月18日, 日本語

核に局在する新規ノンコーディングRNAの探索
石田賢太郎, 中川真一
日本RNA学会年会要旨集, 2006年07月18日, 日本語

新規核スペックル構造を形成するノンコーディングRNA遺伝子の同定
曽根正光, 竹市雅俊, 中川真一
日本分子生物学会年会講演要旨集, 2005年11月25日, 日本語

カドヘリンは網膜水平細胞の樹状突起形態形成を制御する
田辺光志, 竹市雅俊, 中川真一
日本分子生物学会年会講演要旨集, 2005年11月25日, 日本語

網膜水平細胞における樹状突起の形態形成
田辺光志, 竹市雅俊, 中川真一
日本発生生物学会大会発表要旨集, 2005年05月, 日本語

Notchシグナルと独立なWntシグナルによる網膜前駆細胞の分化抑制機構
中川真一
日本分子生物学会年会プログラム・講演要旨集, 2004年11月25日, 日本語

網膜層形成における細胞体移動を制御する遺伝子のスクリーニング
曽根正光, 竹市雅俊, 中川真一
日本発生生物学会大会発表要旨集, 2004年05月10日, 日本語

網膜の再集合実験から見えてきたWnt2bの役割
中川真一
日本発生生物学会大会発表要旨集, 2004年05月10日, 日本語

網膜の幹細胞の維持と層形成におけるWnt‐2bの役割
久保郁, 竹市雅俊, 中川真一
日本発生生物学会大会発表要旨集, 2002年, 日本語

組換えアデノウイルスを用いた神経冠細胞への遺伝子強制発現
中川真一, 森吉弘毅, 中西重忠, 竹市雅敏
日本分子生物学会年会プログラム・講演要旨集, 1996年08月01日

組換えアデノウイルスを用いた神経冠細胞への遺伝子強制発現
中川真一, 森吉弘毅, 中西重忠, 竹市雅俊
日本分子生物学会年会プログラム・講演要旨集, 1996年07月, 日本語

神経管,冠の形成時のc‐cad6B,7の発現とその機能解析
中川真一, 竹市雅俊
日本発生生物学会大会発表要旨集, 1996年, 日本語

神経冠細胞のふるまいに応じたカドヘリンの発現変化
中川真一, 竹市雅俊
日本分子生物学会年会プログラム・講演要旨集, 1994年11月, 日本語

神経冠細胞に発現する新しいカドヘリンの同定
中川真一, 竹市雅俊
日本発生生物学会大会発表要旨集, 1994年, 日本語

分子遺伝学特論 I　　　　　　　　　　　　　　　
埼玉大学理工学研究科

分子生物学 II　　　　　　　　　　　　　　　
北海道大学薬学部

分子生物学 I　　　　　　　　　　　　　　　
北海道大学薬学部

スプライシングを制御するsrRNAの作用機序の解明と新規srRNAの同定
科学研究費助成事業
2024年04月01日 - 2029年03月31日
中川 真一, 芳本 玲
日本学術振興会, 基盤研究(A), 北海道大学, 24H00546

マウス変異体を用いた非ドメイン型RNAの生理機能解析
科学研究費助成事業
2021年09月10日 - 2026年03月31日
中川 真一, 荒木 喜美
日本学術振興会, 学術変革領域研究(A), 北海道大学, 21H05274

非ドメイン型バイオポリマー領域の組織運営
科学研究費助成事業
2021年09月10日 - 2026年03月31日
中川 真一, 廣瀬 哲郎, 尾山 大明, 稲田 利文, 荒川 和晴, 甲斐 歳恵, 依田 隆夫, 伊藤 拓宏, 泊 幸秀
本領域では、種間で高度に保存された配列を持たずに機能を発揮するRNAやタンパク質を非ドメイン型バイオポリマーと定義し、その生理的な役割から分子レベルの分子機構まで階層横断的な研究を進め、新たな動作原理に基づく画期的な生体制御技術を開発することを目指している。
今年度は領域立ち上げの初年度に当たり、班員間の連携を深めるために、領域班会議を10月9-10日の二日間にわたって北広島のクラッセホテルにてハイブリッド形式にて開催した。また、研究者間の交流をより深めるために、ワールドポスター形式でのディスカッションを同日程でオンラインで行った。対面での総括班会議では今後の活動方針について確認を行い、技術講習会についてはZoomを利用したオンライン形式でのハンズオンセミナーを取り入れてゆくこと、若手支援の海外ミーティング参加支援は新型コロナウイルス感染症の流行の状況を見ながら、渡航可能になり次第開始すること、公募班を含めた次回の領域会議は東京で行うことを確認した。
班員間のコミュニケーションを深めるために、Slackのワークスペースを立ち上げた。各計画研究ごとのスレッドを作製して研究代表者、分担者、領域代表者で打ち合わせを随時行ったほか、汎用的な技術に関するスレッドも作製し、領域全体で共同研究のノウハウを共有できるプラットフォームを立ち上げた。また、変異マウスの作製の共同研究ならびに近位ビオチンラベルによる相互作用因子の質量分析による解析の共同研究をアレンジし、それら共同研究に係る経費の一部を支援した。
若手育成に関してはRNAフロンティアミーティングの会場費の支援を予定していたが、オンライン開催になったため、班員の研究室からの積極的な参加という形で活動の支援を行った。
日本学術振興会, 学術変革領域研究(A), 北海道大学, 21H05273

非ドメイン型バイオポリマーの生物学：生物の柔軟な機能獲得戦略
科学研究費助成事業
2021年09月10日 - 2026年03月31日
中川 真一
日本学術振興会, 学術変革領域研究(A), 21A304

超天然変性タンパク質の個体レベルでの生理機能解析
科学研究費助成事業
2021年07月09日 - 2024年03月31日
中川 真一, 泊 幸秀
本研究は全長に渡って天然変性領域を持つ超天然変性タンパク質に注目し、その変異体を作製することで、機能未知の超天然変性タンパク質の生理機能を明らかにすることを目的としている。これまでの研究によって、病原性の凝集体形成を阻害したりタンパク質を変性から保護する活性を持つ超天然変性タンパク質が少なくとも6種類あることが明らかとなっていた。そこでこれらの遺伝子全てについてゲノム編集マウスを作製したところ、Hero11とHero45の二種類の遺伝子のホモ個体の生体は観察されず、胎生致死になることが予想された。さらに詳細な解析を行ったところ、Hero11は胎生5.5日目から7.5日目の間に、Hero45は胎生14日目以降に致死になることが明らかとなった。また、Hero11のノックアウトマウスから樹立したES細胞は野生型のES細胞に比べて増殖が大きく低下することが明らかとなった。さらに、Hero45のメスのヘテロマウスは、交尾をして妊娠しても正常に出産できない個体が頻繁に見られることが分かった。
Hero11とHero45の分子動作機構を明らかにするために、これらのタンパク質にタグを付けた分子を培養細胞に発現させたところ、Hero11は核に局在し、核小体に特に濃縮すること、Hero45は主として細胞質に局在することが明らかとなった。また、Hero11がタンパク質の凝集体形成を抑制する際に、その細胞内局在が変化することも明らかとなった。
これらに加え、小胞体に局在し、細胞内ドメインが天然変性領域からなる機能未知タンパク質UGS148についても変異マウスを作製し、その表現型解析を行った。その結果、UGS148は間脳脳室腹側に存在するタニサイト細胞の小胞体に特異的に分布するほかオレキシンニューロンにも強い発現が見られること、しかしながらノックアウトマウスは大きな異常を示さないことが明らかとなった。
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 21K19246

反応場に着目したpiRNA経路の生化学的解析
科学研究費助成事業
2018年06月11日 - 2023年03月31日
泊 幸秀, 中川 真一
これまでに、遺伝学的スクリーニングと、タンパク質-タンパク質相互作用の探索によって、膨大な数のpiRNA関連因子が存在することが明らかになっている、その中で生化学活性が特定されているものは、我々が発見したpiRNA成熟のためのエクソヌクレアーゼTrimmerや、その前段階ではたらくエンドヌクレアーゼZucchiniを含めてごく少数に限られている。我々は、アメリカの研究室との共同研究と独自の生化学系の利用によって、GTSF1 (Gametocyte Specific Factor 1)と呼ばれる因子が、PIWIタンパク質と相互作用し、その標的RNA切断活性を大きく向上させる機能を持っているということを見いだした。さらに、別のアメリカの研究室との共同研究により、RNase κ と呼ばれるミトコンドリアに局在するヌクレアーゼが、piRNAの効率的な生産に重要であることを発見した。また、piRNA関連の新規因子を探索する解析を通じて、カイコが持つ2つのPIWIタンパク質、SiwiとBmAgo3のうち、BmAgo3をノックダウンした時だけ、Siwiと特異的に相互作用する機能未知の新規因子を発見した。さらには、次世代シーケンサーを駆使することにより、カイコBmN4細胞ゲノムの中に存在する最も代表的なpiRNAクラスターであるtorimochiの配列および性状解析を進めた。また、piRNA経路と同様に、トランスポゾンやウイルスなどの遺伝的侵略者からゲノムを守る役割を果たしているsiRNA経路については、その生合成において重要な働き果たすDicer(ショウジョウバエ由来のDicer-2)が長い二本鎖RNAを切断しながらsiRNAを次々と産生してゆく過程を一分子レベルで観察することに成功し、従来提案されていたモデルに修正を与えることとなった。
日本学術振興会, 基盤研究(S), 東京大学, 18H05271

逆遺伝学スクリーニングを用いた新規長鎖ノンコーディングRNAの生理機能解析
科学研究費助成事業
2017年04月01日 - 2022年03月31日
中川 真一, 横井 佐織
ヒトやマウスを含む高等生物のゲノムからはタンパク質をコードしないノンコーディングRNAが大量に転写されている。本研究では、膨大な数の未解析lncRNA群の中から生理機能を持つ候補遺伝子を体系的に選び出し、その機能解析を効率良く進める手法を確立することを目指した。その結果、UV照射後のRNAの回収量を指標に、タンパク質と強固な複合体を形成している機能性lncRNAを予測することができることが明らかとなった。また、簡易ゲノム編集法であるiGONAD法を用いて、lncRNAの機能を欠失するゲノム編集マウスを効率よく作製する手法を確立した。
日本学術振興会, 基盤研究(B), 北海道大学, 17H03604

ノンコーディングRNAネオタクソノミの実現を加速する国際活動支援
科学研究費助成事業
2015年11月06日 - 2019年03月31日
廣瀬 哲郎, 中川 真一, 淺原 弘嗣, 泊 幸秀, 鈴木 勉, 塩見 美喜子, 影山 裕二
領域研究の推進と成果発信を目的とした海外研究者との交流活動を支援するために、主に３つの活動、国際共同研究の支援、海外研究機関の訪問支援、海外著名研究者の招聘を推進した。特に本領域特有のリソースであるncRNA遺伝子改変マウスに関する国際共同研究の支援、また若手研究者の国際研究機関への短期滞在を含めた国際活動支援を推進した。その結果、若手研究者を含めた領域班員による多数の国際共同研究が活発に行われ、34報もの国際共著論文に結びついた。
日本学術振興会, 新学術領域研究(研究領域提案型), 北海道大学, 15K21720

ネオタクソノミに応じたncRNAの生理機能の解明
科学研究費助成事業
2014年07月10日 - 2019年03月31日
中川 真一
ゲノムからはタンパク質をコードしないノンコーディングRNAが数多く転写されている。ncRNAは大別すると、RNAサイレンシングに関わる「小さなRNA」と、長さが200塩基以上の「長鎖ノンコーディングRNA（lncRNA）」に分けられるが、2万種類を越すlncRNAのうちどれぐらいが実際の生理機能を持っているのかは不明である。そこで、既知の機能性lncRNAがマイルドなUV照射によって高効率でタンパク質に架橋される性質を利用し、新規機能性lncRNA候補遺伝子を同定する手法を開発した。また、lncRNAが形成する微細構造を超解像顕微鏡を用いて解析する手法も確立した。
日本学術振興会, 新学術領域研究(研究領域提案型), 26113005

非コードRNA作用マシナリー
科学研究費助成事業
2009年07月23日 - 2014年03月31日
泊 幸秀, 影山 裕二, 鈴木 健夫, 程 久美子, 中澤 敬信, 和田 猛, 佐渡 敬, 塩見 美喜子, 宮川 さとみ, 中川 真一, 竹下 文隆, 小田 健昭, 山下 暁朗, 西村 教子
H21～25年度の新学術領域研究「非コードRNA作用マシナリー」領域からは非コードRNA動作原理に関する優れた研究成果が多数生み出され、本領域はホームページや雑誌企画、ワークショップ、一般向けのプレスリリースなど、外部に対して積極的に情報を発信し、得られた研究成果を社会に還元するように努めてきた。これまで、当該分野で顕著な成果を上げている海外の研究者と共に国際シンポジウムを開催することで、国際的にもこの分野を牽引する日本の非コードRNA研究を世界に向けて発信してきた。平成26年度の本研究取りまとめにおいては、過去の実績を踏まえ、本領域の研究期間である５年間を通して得られた研究成果を取りまとめ、その全体像を当該学問領域のみならず、一般国民に分かりやすく還元すると共に情報の発信を継続することを目的として下記の計画を遂行した。
①本分野をリードする著名な海外の研究者を第37回日本分子生物学会年会シンポジウムおよびワークショップに招聘するとともに、本領域主催の「非コードRNA作用マシナリー」をテーマとした国際シンポジウムの開催し、領域の研究成果を国内外に発信すると共に海外の研究者との情報交換を強化した。②領域の研究成果を広く社会・国民に発信するためのアウトリーチ活動の一環として、領域班員がRNA関連研究を伝える活動を地域の教育・研究機関で行った。③領域の研究成果や上記イベント等を広報するために、引き続きncrna.jpドメインを維持し、ホームページやブログを継続公開・更新した。④研究成果をわかりやすくまとめた成果報告書を作成し、本領域外の科学分野面にも広く配布した。
以上のように、本領域の研究成果を当該分野のみならず、広く一般国民に還元することができたと考える。
日本学術振興会, 新学術領域研究(研究領域提案型), 東京大学, 21115001

レトロトランスポゾン挿入配列によるmRNAの局在・代謝制御機構の解析
科学研究費補助金(挑戦的萌芽研究)
2012年 - 2012年
中川 真一
mRNAの非翻訳領域のレトロトランスポゾン挿入配列の機能を調べるために、レトロトランスポゾンSINE B1と高い相同性を持つ核内ノンコーディングRNA、4.5SHに注目した解析を行った。SINE B1がアンチセンス方向に挿入された転写産物は4.5SHと核内で二本鎖RNA構造を形成し、核内に繋留されていた。4.5SHをノックダウンすると、アンチセンス挿入SINE B1を持つ転写産物の核内繋留が解除された。また、4.5SHを介した核内繋留には二本鎖RNA結合蛋白質であるNF110が必要であった。これらの結果から、分子間二本鎖RNA構造形成による新規遺伝子発現制御機構を明らかとなった。
文部科学省, 挑戦的萌芽研究, 独立行政法人理化学研究所, 研究代表者, 競争的資金, 24657123

核マトリクス・ノンコーディングRNA複合体によるエピジェネティックな発現制御解析
科学研究費補助金(基盤研究(B))
2011年 - 2012年
中川 真一
ヒトやマウスのゲノムからは大量のタンパク質をコードしないノンコーディングRNAが転写されている。それらの多くは、クロマチン制御因子と複合体を形成し、エピジェネティックな遺伝子発現を制御しているのではないかと考えられている。本研究ではX染色体の不活性化を制御するノンコーディングRNAであるXistの染色体局在を制御する核マトリクスタンパク質、hnRNP Uに注目した機能解析を行い、hnRNP UがXist以外のノンコーディングRNAによるエピゲノム制御にも関わっていること、hnRNP Uをノックダウンした細胞では複数の相同染色体特異的な遺伝子発現が影響を受けることなどを明らかにした。
文部科学省, 基盤研究(B), 独立行政法人理化学研究所, 研究代表者, 競争的資金, 23370093

スプライシング因子の新機能に関する化学遺伝学研究
科学研究費補助金(基盤研究(S))
2009年 - 2012年
吉田 稔, 中川 真一
抗癌剤候補化合物として発見されたFR901464を改変して作成した安定誘導体Spliceostatin A (SSA)はスプライシング因子複合体SF3bに結合してスプライシング反応を阻害し、その結果pre-mRNAからタンパク質が合成され、イントロンの配列の翻訳を引き起こす。スプライシング阻害とイントロン配列翻訳のメカニズムを明らかにするため、in vitroスプライシングに対する効果を詳細に調べたところ、U1、SF1、U2AFなどが結合するE complexの形成には影響がなく、SF1とU2が入れ替わってA complexが形成されるステップで阻害が起こっていることがわかった。また、スプライシング阻害の程度は、イントロン内のBranch point配列に依存していることも明らかになった。さらにイントロン内にMS2配列を導入したプローブを用いたイメージング法によっても同様の結果が得られた。次にSSAの標的分子であるSF3bによって安定性が制御されていることが示唆されていた非コードRNAのXistの局在を制御する分子を同定するために、RNA結合ドメインを持つタンパク質に対するカスタムsiRNAライブラリーを作製し、特定の遺伝子をノックダウンした細胞におけるXistの局在を調べた。その結果、hnRNP Uというタンパク質がXistの染色体上への局在に必要なこと、hnRNP Uの機...
文部科学省, 基盤研究(S), 独立行政法人理化学研究所, 連携研究者, 競争的資金, 21228003

遺伝学的アプローチによる高分子非コードRNAマシナリーの生理機能解析
科学研究費補助金(新学術領域研究(研究領域提案型))
2009年 - 2012年
影山 裕二, 中川 真一
21年度までの解析により、ショウジョウバエMRE32は胚発生期において中枢神経系特異的に発現するnon-coding RNAであることが明らかになっている。また、MRE32遺伝子の欠失変異体は発生に遅れが生じることから、MRE32が神経系において重要な機能を担っていることが推察されている。ショウジョウバエMRE32遺伝子の発現組織・細胞を同定するため、in situハイブリダイゼーションによる解析を行ったところ、従来明らかになっていた胚発生期の中枢神経系に加え、成虫視葉の二次狭窄を構成する神経細胞にもMRE32の発現が見られた。また、それ以外の多くの中枢神経細胞にも発現が見られたが、これらについては細胞の同定のためにさらなる解析が必要であると考えられる。胚発生期の発現細胞については、前胸腺特異的な分子マーカとの二重染色により、少なくとも食道神経節細胞の一部で発現していることが推察された。また、MRE32の転写領域を確定するため、RT-PCRによるマッピングを行ったところ、逆向きのトランスポゾン配列(roo element、約10kb)を含む約30kbがMRE32遺伝子被転写領域であると考えられた。この領域では、RNA-seq法により複数のタグが検出されており、転写活性は低いものの、何らかの転写産物が合成されているという従来の知見とよく一致する。なお、雌特異的と考えられたMRE...
文部科学省, 新学術領域研究(研究領域提案型), 大学共同利用機関法人自然科学研究機構(共通施設)->神戸大学, 連携研究者, 競争的資金, 21115007

網膜の幹細胞を維持する分子メカニズムの解明
科学研究費補助金(基盤研究(C))
2007年 - 2009年
中川 真一
網膜の最もマージン側の領域には長期間にわたって多分化能と分裂能を保つ網膜幹細胞が存在しており、それらは分泌性のシグナル分子であるWnt2bによって維持されている。本研究においては、Wntの下流で働いている転写因子に注目し、それらが作るネットワーク構造を解明することを目指した。その結果、Wntによって活性化されるβ-catenin/LEF1複合体がc-mycを含む複数の転写因子群を活性化していること、それらが協調して転写抑制因子であるc-hairy1を活性化していること、c-hairy1はWntの幹細胞維持活性を細胞に伝えるためのノードとして働いている事などを明らかにした。
文部科学省, 基盤研究(C), 独立行政法人理化学研究所, 研究代表者, 競争的資金, 19570216

網膜の予定神経節細胞の核に局在するノンコーディングRNAの機能解析
科学研究費補助金(特定領域研究)
2005年 - 2006年
中川 真一
我々は、中枢神経系の層構造の形成時に細胞タイプ特異的な振る舞いを制御する遺伝子を同定するために、それぞれの細胞タイプで特異的に発現している遺伝子を単一細胞サブトラクション法を用いて同定して来た。その過程で、通常のmRNAと異なり転写産物が核マトリクスに多数のスポットとして観察される、全長9kbのユニークなmRNA型ノンコーディングRNAを同定し、その特徴的な核内局在からこの遺伝子をGomafu(胡麻斑)と名付けた。本年度はGomafuの性質についてより詳細な解析を行なった。まず、GomafuをHelaやNeuro2Aなどの培養細胞に強制発現したところ、それらの細胞でも生体内と同様に核内に局在する事が分かった。また、これらの細胞を、Gomafuを発現していない親株と融合させてヘテロカリオンを作製したところ、Gomafu RNAはトランスフェクタント由来の核でのみ観察された事から、Gomafu RNAは転写後細胞質に輸送される事なく核内に保持されている事が分かった。また、これまでに知られているXist、Air、Evflなどの核内に局在するmRNA型ノンコーディングRNAと局在を比較したところ、GomafUはユニークな場所に局在する事が分かった。また、興味深い事に、Gomafu RNAは熱処理や翻訳阻害などのストレスによって消失する事が分かった。熱処理によっても新規タンパク質の合成...
文部科学省, 特定領域研究, 独立行政法人理化学研究所, 研究代表者, 競争的資金, 17026040

中枢神経系の層形成を制御する分子メカニズムの研究
科学研究費補助金(基盤研究(C))
2004年 - 2006年
中川 真一
器官形成の過程では細胞の増殖・分化は正確に制御されており、生み出された細胞が個々の形質に応じて適切な位置に配置されることで機能的な組織が形づくられる。我々はこれまでの研究において、分泌性のシグナル分子であるWnt2bが網膜の再集合培養の系で正しい層形成を誘導する事が出来る事、さらに実際の生体内では網膜幹細胞の維持に関わっている事などを明らかにして来た。本研究においては、これらの成果をさらに発展させ、どのようなシグナルが細胞内に伝わる事で幹細胞としての性質が維持されるかについて詳細な解析を行なった。神経細胞の分化はプロニューラル遺伝子と呼ばれる一群の転写因子によって促進され、その活性はNotch受容体からのシグナルで抑制されていることが知られている。そこで網膜幹細胞においてWntのシグナルとこれらの細胞分化抑制機構がどのような関係にあるかを調べたところ、興味深い事に、WntにはNotchの活性に非依存的にプロニューラル遺伝子の活性を抑制できることが明らかとなった。さらにWntの下流で働いている遺伝子を明らかにするためにWntのシグナルが活性化している細胞とそうでない細胞の間でサグトラクションスクリーニングを行ない、複数の遺伝子を得る事が出来た。これらのWnt応答性遺伝子の発現を実際の生体内において調べたところ、そのいくつかは網膜の幹細胞で特異的に発現しており、幹細胞においてWn...
文部科学省, 基盤研究(C), 独立行政法人理化学研究所, 研究代表者, 競争的資金, 16570184

中枢神経系の層形成時に細胞タイプ特異的に発現する新規遺伝子の機能解析
科学研究費補助金(特定領域研究)
2005年 - 2005年
中川 真一
中枢神経系の層形成の過程において、それぞれの細胞がどのようにして細胞タイプに応じた特定の層へ移動してゆくのかを調べるために、これまでに網膜をモデルシステムとして用いて、発生期に細胞タイプ特異的な発現を示す遺伝子を複数同定して来た。本年度はそれらの候補遺伝子を網膜全体に強制発現させ、それぞれの細胞の振る舞いに変化が起きるかを調べたが、顕著な異常を観察する事は出来なかった。一方、網膜への遺伝子導入技術を改良している過程で、時期特異的、かつ細胞タイプ特異的に遺伝子を発現する系を立ち上げる事に成功した。この技術を用いて網膜の水平細胞をEGFPでラベルしたところ、樹状突起の形態および軸索の有無から明確に区別出来る3種類の水平細胞が存在する事を見出した。さらに、これらの樹状突起の形態形成を制御する分子メカニズムを明らかにするために、神経系における主要な細胞間接着分子の一つであるカドヘリンの機能阻害実験を試みた。その結果、カドヘリンの機能を阻害すると、樹状突起の伸展が大きく阻害される事が分かった。しかしながら、短いながらもそれぞれの樹状突起の末端は適切な視細胞の末端に投射していた。ただし、そこでのシナプスマーカーの集積は阻害されていた。これらの事から、カドヘリンは樹状突起の伸展、および最終的なシナプス形成の過程を制御しているが、局所的な標的認識過程には関与していない事が明らかとなった。
文部科学省, 特定領域研究, 独立行政法人理化学研究所, 研究代表者, 競争的資金, 17023053

中枢神経系の層形成過程においてニューロンが特定の層を認識する分子メカニズムの研究
科学研究費補助金(特定領域研究)
2003年 - 2004年
中川 真一
中枢神経系の様々な領域で、特定の機能を持ったニューロンおよびグリア細胞が決められた位置に一列に並んだような、規則的な層構造が見られる。この層構造が形成される過程でそれぞれの細胞が細胞タイプに応じた目的地をどのように認識しそこへ移動してゆくのかを調べるために、我々は網膜をモデルシステムとして研究を進めてきた。網膜の神経節細胞と錐体光受容細胞は発生の初期、ほぼ同じ時期におなじ神経上皮のアピカル側で最終分裂を行うが、その後生み出された細胞は正反対の方向へ移動してゆく。すなわち神経節細胞は基底膜側に移動してゆくのに対し、錐体光受容細胞はアピカル側に戻ってきてそこで細胞層を形成する。我々はこのような振る舞いの違いはそれぞれの細胞タイプで特異的に発現している遺伝子によって制御されていると考え、以下のような実験を行った。まず、これらの細胞が移動をしているような時期のマウスの網膜を単一の細胞に解離し、それぞれの細胞からPCRによるバイアスを抑えるような条件のもとでcDNAを合成した。次にサザンプロット上でマーカー遺伝子の発現を調べることで、それぞれのcDNAがどの細胞タイプに由来するものかを遡及的に同定した。このようにして選んだ神経節細胞由来のcDNAと錐体光受容細胞由来のcDNAを用いてサブトラクションライブラリーを作製し、それぞれの細胞由来のプローブでディファレンシャルスクリーニングを行...
文部科学省, 特定領域研究, 特殊法人理化学研究所->独立行政法人理化学研究所, 研究代表者, 競争的資金, 15029262

中枢神経系に見られる層構造の形成機構の解析
科学研究費補助金(基盤研究(C))
2001年 - 2003年
中川 真一
我々は、脊椎動物の中枢神経系に見られる層構造の形成が、どのような分子メカニズムによって制御されているか調べるために、特に網膜に注目して研究を行っている。未分化な網膜の細胞を単一細胞に乖離してから培養条件下で再集合させると、それらは組織塊を形成し、網膜に見られるすべての種類の神経細胞およびグリア細胞を作り出すことができる。しかしながらそのような組織には正常網膜に見られるような層構造は見られず、ロゼットと呼ばれる異常な構造体が形成される。我々は、この異常なロゼット形成を阻害するような領域が発生期の眼胞組織に存在するかどうかを調べるために、網膜の様々な領域から組織片を作製し、乖離した網膜前駆細胞と共培養を行った。その結果、レンズに隣接する網膜のもっともマージン側の領域にロゼット形成を阻害する活性があることが分かった。さらに、この領域で発現しているシグナル分子Wnt2bにも同様のロゼット形成阻害活性があることを見い出し、このタンパク質の存在下では単一細胞に乖離した網膜前駆細胞からも正しい層構造を持った網膜が再生することが分かった。次に、実際に発生過程におけるWnt2bの役割を調べるために、網膜全体でこの分子を発現させたところ、神経細胞への分化が抑制されることが分かった。また、ドミナントネガティブ分子を発現することによってWntの下流のシグナルを阻害すると、網膜のマージン側の領域で意志...
文部科学省, 基盤研究(C), 京都大学->特殊法人理化学研究所->独立行政法人理化学研究所, 研究代表者, 競争的資金, 13680804