博士（薬学）, 北海道大学

snRNP

pre-mRNAスプライシング

Cajal body

非膜オルガネラ

分子生物学

ケミカルバイオロジー

Life Science, Pharmaceutical hygiene and biochemistry, Molecular biology

Bachelor's degree program, School of Pharmaceutical Sciences and Pharmacy

Master's degree program, Graduate School of Life Science

Doctoral (PhD) degree program, Graduate School of Life Science

Balancing RNA processing and innate immune response: Possible roles for SMN condensates in snRNP biogenesis. (review)
Hiroshi Maita; Shinichi Nakagawa
Biochimica et biophysica acta. General subjects, 1869, 3, 130764, 130764, Mar. 2025, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, Biomolecular condensates like U-bodies are specialized cellular structures formed through multivalent interactions among intrinsically disordered regions. U-bodies sequester small nuclear ribonucleoprotein complexes (snRNPs) in the cytoplasm, and their formation in mammalian cells depends on stress conditions. Because of their location adjacent to P-bodies, U-bodies have been considered potential sites for snRNP storage or turnover. SMN, a chaperone for snRNP biogenesis, forms condensates through its Tudor domain. In fly models, defects in SMN trigger innate immune responses similar to those observed with excess cytoplasmic snRNA during viral infection in mammalian cells. Additionally, spinal muscular atrophy (SMA), caused by SMN deficiency, is associated with inflammation. Therefore, SMN may help prevent innate immune aberrant activation due to defective snRNP biogenesis by forming U-bodies to sequester these molecules. Further studies on U-body functions may provide therapeutic insights for diseases related to RNA metabolism.

Identification of BAY61-3606 Derivatives With Improved Activity in Splicing Modulation That Induces Inclusion of Cassette Exons Similar to the Splicing Factor 3B Subunit 1 Mutation.
Matsumaru T; Iwamatsu T; Ishigami K; Inai M; Kanto W; Ishigaki A; Toyoda A; Shuto S; Maenaka K; Nakagawa S; HIROSHI MAITA
Chemical biology & drug design, Oct. 2024, [Peer-reviewed], [Corresponding author]
Scientific journal, Splicing modulation by a small compound offers therapeutic potential for diseases caused by splicing abnormality. However, only a few classes of compounds that can modulate splicing have been identified. We previously identified BAY61-3606, a multiple kinase inhibitor, as a compound that relaxes the splicing fidelity at the 3' splice site recognition. We have also reported the synthesis of derivatives of BAY61-3606. In this study, we tested those compounds for their splicing modulation capabilities and identified two contrasting compounds. These compounds were further investigated for their effects on the whole transcriptome, and analysis of changes in transcription and splicing revealed that the highly active derivative in the splicing reporter assay also showed significantly higher activity in modulating the splicing of endogenously expressed genes. Particularly, cassette exon inclusion was highly upregulated by this compound, and clustering analysis revealed that these effects resembled those in splicing factor 3b subunit 1 (SF3B1) K700E mutant cells but contrasted with those of the splicing inhibitor H3B-8800. Additionally, a group of serine/arginine-rich (SR) protein genes was identified as representatively affected, likely via modulation of poison exon inclusion. This finding could guide further analysis of the mode of action of these compounds on splicing, which could be valuable for developing drugs for diseases associated with splicing abnormalities.

Dissecting the role of SMN multimerization in its dissociation from the Cajal body using harmine as a tool compound
Saki Ohazama; Akiko Fujimoto; Daisuke Konda; Ryota Yokoyama; Shinichi Nakagawa; Hiroshi Maita
Journal of Cell Science, Sep. 2024, [Peer-reviewed], [Corresponding author]
Scientific journal

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, [Peer-reviewed], [Corresponding author], [Domestic magazines]
English, Scientific journal, 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.

Development of a Cell-Based Assay Using a Split-Luciferase Reporter for Compound Screening.
Satoshi Sato; Hiroyoshi Ariga; Hiroshi Maita
Biological & pharmaceutical bulletin, 46, 7, 1010, 1014, 2023, [Peer-reviewed], [Corresponding author], [Domestic magazines]
English, Scientific journal, Recently, the finding of recurrent mutations in the spliceosome components in cancer has indicated that the spliceosome is a potential target for cancer therapy. However, the number of small molecules known to affect the cellular spliceosome is currently limited probably because of the lack of a robust cell-based approach to identify small molecules that target the spliceosome. We have previously reported the development of a genetic reporter to detect the cellular levels of small nuclear ribonucleoproteins (snRNPs), which are subunits of the spliceosome, using a split luciferase. However, the original protocol was designed for small scale experiments and was not suitable for compound screening. Here, we found that the use of cell lysis buffer used in blue native polyacrylamide gel electrophoresis (BN-PAGE) dramatically improved the sensitivity and the robustness of the assay. Improved assay conditions were used to discover a small molecule that altered the reporter activity. Our method may be used with other cellular macromolecular complexes and may assist in the discovery of small bioactive molecules.

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, Jan. 2022, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

Identification of the cell-type-specific ER membrane protein Tanmp expressed in hypothalamic tanycytes and subsets of neurons
Osamu Takahashi; Mayuko Tanahashi; Saori Yokoi; Mari Kaneko; Tomoko Tokuhara; Kaori Yanaka; Shinichi Nakagawa; Hiroshi Maita
Cold Spring Harbor Laboratory, 06 Jul. 2021
AbstractGenomes 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. During a screening of novel noncoding RNAs abundantly expressed in mouse brains, we incidentally identified a gene termed Tanmp, which encoded an endoplasmic reticulum (ER) protein without known functional domains. Tanmp is specifically expressed in the nervous system, and the highest expression was observed in a specialized cell type called tanycyte that aligns the ventral wall of the third ventricle in the hypothalamus. Immunostaining of Tanmp revealed the fine morphology of tanycytes with highly branched apical ER membranes. Immunoprecipitation revealed that Tanmp associates with mitochondrial ATPase at least in vitro, and ER and mitochondrial signals occasionally overlapped in tanycytes. Mutant mice lacking Tanmp did not exhibit overt phenotypes, suggesting that Tanmp is not essential in mice reared under normal laboratory conditions. We also found that RNA probes that are predicted to uniquely detect Tanmp 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.

What is the switch for coupling transcription and splicing? RNA Polymerase II C-terminal domain phosphorylation, phase separation and beyond.
Hiroshi Maita; Shinichi Nakagawa
Wiley interdisciplinary reviews. RNA, 11, 1, e1574, Jan. 2020, [Peer-reviewed], [Invited], [Corresponding author], [International Magazine]
English, 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.

Divergent synthesis of kinase inhibitor derivatives, leading to discovery of selective Gck inhibitors
Takanori Matsumaru; Makoto Inai; Kana Ishigami; Toshiki Iwamatsu; Hiroshi Maita; Satoko Otsuguro; Takao Nomura; Akira Matsuda; Satoshi Ichikawa; Masahiro Sakaitani; Satoshi Shuto; Katsumi Maenaka; Toshiyuki Kan
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 27, 10, 2144, 2147, May 2017, [Peer-reviewed]
English, Scientific journal

A Splicing Reporter Tuned to Non-AG Acceptor Sites Reveals that Luteolin Enhances the Recognition of Non-canonical Acceptor Sites
Masanori Chiba; Hiroyoshi Ariga; Hiroshi Maita
CHEMICAL BIOLOGY & DRUG DESIGN, 87, 2, 275, 282, Feb. 2016, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Epidermal Growth Factor-dependent Activation of the Extracellular Signal-regulated Kinase Pathway by DJ-1 Protein through Its Direct Binding to c-Raf Protein
Kazuko Takahashi-Niki; Izumi Kato-Ose; Hiroaki Murata; Hiroshi Maita; Sanae M. M. Iguchi-Ariga; Hiroyoshi Ariga
JOURNAL OF BIOLOGICAL CHEMISTRY, 290, 29, 17838, 17847, Jul. 2015, [Peer-reviewed]
English, Scientific journal

A split luciferase-based reporter for detection of a cellular macromolecular complex
Hiroshi Maita; Kenji Tomita; Hiroyoshi Ariga
ANALYTICAL BIOCHEMISTRY, 452, 1, 9, May 2014, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Prefoldin prevents aggregation of alpha-synuclein
Mariko Takano; Erika Tashiro; Akira Kitamura; Hiroshi Maita; Sanae M. M. Iguchi-Ariga; Masataka Kinjo; Hiroyoshi Ariga
BRAIN RESEARCH, 1542, 186, 194, Jan. 2014, [Peer-reviewed]
English, Scientific journal

Prefoldin plays a role as a clearance factor in preventing proteasome inhibitor-induced protein aggregation
Akira Abe; Kazuko Takahashi-Niki; Yuka Takekoshi; Takashi Shimizu; Hirotake Kitaura; Hiroshi Maita; Sanae M. M. Iguchi-Ariga; Hiroyoshi Ariga
Journal of Biological Chemistry, 288, 39, 27764, 27776, 27 Sep. 2013, [Peer-reviewed]
English, Scientific journal

Monomer DJ-1 and Its N-Terminal Sequence Are Necessary for Mitochondrial Localization of DJ-1 Mutants
Chinatsu Maita; Hiroshi Maita; Sanae M. M. Iguchi-Ariga; Hiroyoshi Ariga
PLoS ONE, 8, 1, e54087, 17 Jan. 2013, [Peer-reviewed]
English, Scientific journal

Oxidized DJ-1 Inhibits p53 by Sequestering p53 from Promoters in a DNA-Binding Affinity-Dependent Manner
Izumi Kato; Hiroshi Maita; Kazuko Takahashi-Niki; Yoshiro Saito; Noriko Noguchi; Sanae M. M. Iguchi-Ariga; Hiroyoshi Ariga
MOLECULAR AND CELLULAR BIOLOGY, 33, 2, 340, 359, Jan. 2013, [Peer-reviewed]
English, Scientific journal

Neuroprotective function of dj-1 in Parkinson's disease
Hiroyoshi Ariga; Kazuko Takahashi-Niki; Izumi Kato; Hiroshi Maita; Takeshi Niki; Sanae M. M. Iguchi-Ariga
Oxidative Medicine and Cellular Longevity, 2013:683920, 2013, [Peer-reviewed]
English

Neuroprotective effect of a new DJ-1-binding compound against neurodegeneration in Parkinson's disease and stroke model rats
Yoshihisa Kitamura; Shotaro Watanabe; Masanobu Taguchi; Kentaro Takagi; Takuya Kawata; Kazuko Takahashi-Niki; Hiroyuki Yasui; Hiroshi Maita; Sanae M. M. Iguchi-Ariga; Hiroyoshi Ariga
MOLECULAR NEURODEGENERATION, 6, 1, 48, Jul. 2011, [Peer-reviewed]
English, Scientific journal

Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits
Makoto Miyazawa; Erika Tashiro; Hirotake Kitaura; Hiroshi Maita; Hiroo Suto; Sanae M. M. Iguchi-Ariga; Hiroyoshi Ariga
JOURNAL OF BIOLOGICAL CHEMISTRY, 286, 22, 19191, 19203, Jun. 2011, [Peer-reviewed]
English, Scientific journal

DJ-1-Mediated Protective Effect of Protocatechuic Aldehyde Against Oxidative Stress in SH-SY5Y Cells
Jian-Wei Gao; Takuya Yamane; Hiroshi Maita; Shizuma Ishikawa; Sanae M. M. Iguchi-Ariga; Xiao-Ping Pu; Hiroyoshi Ariga
JOURNAL OF PHARMACOLOGICAL SCIENCES, 115, 1, 36, 44, Jan. 2011, [Peer-reviewed]
English, Scientific journal

Spliceosome discards intermediates via the DEAH box ATPase Prp43p
Rabiah M. Mayas; Hiroshi Maita; Daniel R. Semlow; Jonathan P. Staley
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 107, 22, 10020, 10025, Jun. 2010, [Peer-reviewed]
English, Scientific journal

DJ-1 binds to mitochondrial complex I and maintains its activity
Takuya Hayashi; Chikako Ishimori; Kazuko Takahashi-Niki; Takahiro Taira; Yun-chul Kim; Hiroshi Maita; Chinatsu Maita; Hiroyoshi Ariga; Sanae M. M. Iguchi-Ariga
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 390, 3, 667, 672, Dec. 2009, [Peer-reviewed]
English, Scientific journal

Oxidative Status of DJ-1-dependent Activation of Dopamine Synthesis through Interaction of Tyrosine Hydroxylase and 4-Dihydroxy-L-phenylalanine (L-DOPA) Decarboxylase with DJ-1
Shizuma Ishikawa; Takahiro Taira; Takeshi Niki; Kazuko Takahashi-Niki; Chinatsu Maita; Hiroshi Maita; Hiroyoshi Ariga; Sanae M. M. Iguchi-Ariga
JOURNAL OF BIOLOGICAL CHEMISTRY, 284, 42, 28832, 28844, Oct. 2009, [Peer-reviewed]
English, Scientific journal

Tenascin-X Induces Cell Detachment through p38 Mitogen-Activated Protein Kinase Activation
Shinpei Fujie; Hiroshi Maita; Hiroyoshi Ariga; Ken-ichi Matsumoto
BIOLOGICAL & PHARMACEUTICAL BULLETIN, 32, 10, 1795, 1799, Oct. 2009, [Peer-reviewed]
English, Scientific journal

Secretion of DJ-1 into the serum of patients with Parkinson's disease.
Maita C; Tsuji S; Yabe I; Hamada S; Ogata A; Maita H; Iguchi-Ariga SM; Sasaki H; Ariga H
Neuroscience letters, 431, 86, 89, 1, 04 Dec. 2007, [Peer-reviewed]
Scientific journal, DJ-1 was initially identified by us as a novel oncogene and has later been found to be a causative gene for familial Parkinson's disease PARK7. DJ-1 plays role in transcriptional regulation and in oxidative stress function, and loss of its function is thought to be related to onset age, mode of progression and clinical severity of both familial and sporadic forms of Parkinson's disease (PD). DJ-1 is localized both in the cytoplasm and nucleus, and it has been reported to be secreted into the serum or plasma of patients with breast cancer, melanoma, familial amyloidotic polyneuropathy and stroke. In this study, levels of DJ-1 secreted into the serum of healthy controls and patients with sporadic PD were examined by using a DJ-1 ELISA kit, and the level of oxidative stress in the serum was also measured. The results showed that DJ-1 was secreted into the serum of both healthy controls and PD patients. There was no significant difference between the levels of secreted DJ-1 in two groups, and correlations of levels of secreted DJ-1 with age, clinical severity of PD and level of oxidative stress were not found.

Specific cleavage of DJ-1 under an oxidative condition
Hiromasa Ooe; Chinatsu Maita; Hiroshi Maita; Sanae M. M. Iguchi-Ariga; Hiroyoshi Ariga
NEUROSCIENCE LETTERS, 406, 3, 165, 168, Oct. 2006, [Peer-reviewed]
English, Scientific journal

Exon ligation is proofread by the DExD/H-box ATPase Prp22p
Rabiah M. Mayas; Hiroshi Maita; Jonathan P. Staley
Nature Structural and Molecular Biology, 13, 6, 482, 490, 26 Jun. 2006, [Peer-reviewed]
English, Scientific journal

CIR, a corepressor of CBF1, binds to PAP-1 and effects alternative splicing
H Maita; H Kitaura; H Ariga; SMM Iguchi-Ariga
EXPERIMENTAL CELL RESEARCH, 303, 2, 375, 387, Feb. 2005, [Peer-reviewed]
English, Scientific journal

Association of PAP-1 and Prp3p, the products of causative genes of dominant retinitis pigmentosa, in the tri-snRNP complex
H Maita; H Kitaura; H Ariga; SMM Iguchi-Ariga
EXPERIMENTAL CELL RESEARCH, 302, 1, 61, 68, Jan. 2005, [Peer-reviewed]
English, Scientific journal

PAP-1, the mutated gene underlying the RP9 form of dominant retinitis pigmentosa, is a splicing factor
H Maita; H Kitaura; TJ Keen; CF Inglehearn; H Ariga; SMM Iguchi-Ariga
EXPERIMENTAL CELL RESEARCH, 300, 2, 283, 296, Nov. 2004, [Peer-reviewed]
English, Scientific journal

A novel nucleolar protein, PAPA-1, induces growth arrest as a result of cell cycle arrest at the G1 phase
TS Kuroda; H Maita; T Tabata; T Taira; H Kitaura; H Ariga; SMM Iguchi-Ariga
GENE, 340, 1, 83, 98, Sep. 2004, [Peer-reviewed]
English, Scientific journal

Pim-1 translocates sorting nexin 6/TRAF4-associated factor 2 from cytoplasm to nucleus
Y Ishibashi; H Maita; M Yano; N Koike; K Tamai; H Ariga; SMM Iguchi-Ariga
FEBS LETTERS, 506, 1, 33, 38, Sep. 2001, [Peer-reviewed]
English, Scientific journal

PAP-1, a novel target protein of phosphorylation by Pim-1 kinase
H Maita; Y Harada; D Nagakubo; H Kitaura; M Ikeda; K Tamai; K Takahashi; H Ariga; SMM Iguchi-Ariga
EUROPEAN JOURNAL OF BIOCHEMISTRY, 267, 16, 5168, 5178, Aug. 2000, [Peer-reviewed]
English, Scientific journal

Identification of heterochromatin protein 1 (HP1) as a phosphorylation target by Pim-1 kinase and the effect of phosphorylation on the transcriptional repression function of HP1
N Koike; H Maita; T Taira; H Ariga; SMM Iguchi-Ariga
FEBS LETTERS, 467, 1, 17, 21, Feb. 2000, [Peer-reviewed]
English, Scientific journal

スプライシングの脆弱性は創薬につながるか？ がん細胞の新たな弱点
実験医学, 34, 13, 2179, 2180, Aug. 2016

バイオミディア "スプライソソームの意外な利用法"
生物工学会誌, 92, 2, 82, 2014

Winding and Tangling. An Initial Phase of Membrane-Less Organelle Formation. (Phase separation in living cells : benefits and risks)
Hiroshi Maita; Shinichi Nakagawa
Springer, 2023, 9789819948857, viii, 318 p., English, [Contributor]

実験医学増刊 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
羊土社, 2021, [Contributor]

Redox Stress-Induced Modification of SMN Promotes Cajal Body Dispersion
Hiroshi Maita
第26回日本RNA学会年会, Jul. 2025, Oral presentation

細胞周期依存的なU-body様構造体形成機構の解析
嶋田 雄大○; 中川 真一; 米田 宏
第143回日本薬学会, Mar. 2023, Oral presentation

Regulation of Cajal body by Arginine Dimethylation of Coilin
大峽 咲希○; 中川 真一; 米田 宏
第45回日本分子生物学会, Dec. 2022, Nominated symposium

天然物を用いたsnRNP形成機構の解明
第23回天然薬物の開発と応用シンポジウム（札幌）, 17 Oct. 2021, Nominated symposium

Analysis of the mechanism how CDK9 inhibitors modulate pre-mRNA splicing
第43回日本分子生物学会年会, Dec. 2020, Nominated symposium

Chemical approach to elucidate the regulatory mechanisms of snRNP biogenesis
第42回日本分子生物学会年会, Dec. 2019, Nominated symposium

A chemical approach to study how membrane-less organelles control RNA processing
第41回日本分子生物学会年会, Nov. 2018, Nominated symposium

Identification of small chemical compounds that target RNA processing machineries
THE 4TH JAPAN-TAIWAN JOINT SYMPOSIUM FOR PHARMACEUTICAL SCIENCES, 31 Aug. 2018, Nominated symposium

How does the cell balance fidelity and flexibility of splice site recognition?
第40回日本分子生物学会年会, Dec. 2017, Nominated symposium

変異スプライス部位でのスプライシングを誘導する化合物の発見と解析
第12回日本ケミカルバイオロジー学会, Jun. 2017, Oral presentation

生命薬学特論, 2024年, 修士課程, 生命科学院

先端生物科学実験法Ⅰ, 2024年, 学士課程, 薬学部

先端生物科学実験法Ⅱ, 2024年, 学士課程, 薬学部

分子生物学Ⅰ, 2024年, 学士課程, 薬学部

分子生物学Ⅱ, 2024年, 学士課程, 薬学部

一般教育演習(ﾌﾚｯｼｭﾏﾝｾﾐﾅｰ), 2024年, 学士課程, 全学教育

微生物学, 2024年, 学士課程, 薬学部

生化学実習Ⅲ, 2024年, 学士課程, 薬学部

スプライソソーム構成因子の改変によるスプライシング制御法の開発
科学研究費助成事業
01 Apr. 2023 - 31 Mar. 2026
米田 宏
本研究ではスプライシング制御の方策として、スプライソソームの骨格であるsnRNAの機能改変に注目している。snRNAはスプライソソーム構成因子を集合させるための足場となり、5種のsnRNAそれぞれが特異的な結合タンパク質とsnRNPと呼ばれるサブユニットを形成する。snRNAはsnRNPの足場となるだけでなく、配列の相補性を利用して5種のsnRNP同士の会合や、基質となるpre-mRNA配列の認識、結合も司る。そのため、snRNAに様々な配列改変を施すことで、改変snRNAを持つスプライソソームの機能を調整できる可能性がある。また、snRNAを中心とした分子間結合ネットワークはsnRNAの塩基修飾によって相互作用の強弱が調節され、適切なスプライシングを行うための微調整を担っていると予想される。本研究では我々がこれまで低分子化合物で誘導してきたスプライソソームの基質認識の正確性低下をsnRNAの改変により達成することを目指しているが、上記の通り、snRNAに変異導入した場合、修飾の変動も結果の解釈に重要となる。そこで、snRNAの配列改変と修飾変化を同時に検出するために、ナノポアシーケンサーを用いたsnRNAの状態変化の検出法の構築を試みている。まず多量のU2 snRNAをマウス肝臓から精製し、ナノポアシーケンサーで解読した配列データを元にして、配列決定を試みた。その結果、すでに生化学的手法で同定されている修飾部位が高頻度で誤読として検出された。この誤読を修飾塩基の変動検出に利用できる可能性があり、現在検討を行っている。
日本学術振興会, 基盤研究(C), 北海道大学, 23K06093

スプライソソーム形成障害を標的とした低分子薬の探索と解析
科学研究費助成事業
01 Apr. 2020 - 31 Mar. 2023
米田 宏
多くのスプライソソーム構成因子に疾患原因変異が発見されているが、疾患原因変異が引き起こすスプライソソームの機能異常をどのように治療していくか明確な方策はない。また、スプライソソームを直接的に標的とする化合物の場合、スプライシング反応の重要性を鑑みると、バランスよく特定の効果のみを発揮させる難しさも予想される。我々はスプライソソームの量的な変動も活性制御に重要と仮定し、スプライソソームのサブユニットであるsnRNP量を変動させる化合物のスクリー二ングを行ってきた。そのような化合物には間接的にスプライシングに有用な変化をもたらすものも含まれると予想され、実際、通常ではスプライシングされることのない変異イントロンのスプライシングを回復させる化合物を見出した。これらの化合物のスプライシングへの作用には阻害された状態のCDK9蛋白質が必要であるが、その作用機序は不明であった。一方でこの化合物がスクリーニングで取れてきた経緯である、snRNPへの影響は詳しく検討していなかったが、U5 snRNPの構成因子に対する抗体を用いて化合物存在下でU5 snRNPを回収すると、同時に回収されるU4、U6 snRNAのうち、U4 snRNAのみ増加が認められた。近年のスプライソソーム形成機構の理解を踏まえると、この変化はスプライソソームのB complexがBact complexに進む際に必要なU4 snRNPの解離の遅延もしくは抑制と解釈できる。次年度以降はU4 snRNPの解離がこれらの化合物で低下する機構の解明を目指していく。
日本学術振興会, 基盤研究(C), 北海道大学, 20K07028

相分離構造体とプロリン異性化の連携機構の解析と可視化ツールの開発
科学研究費助成事業
01 Apr. 2020 - 31 Mar. 2022
米田 宏
核内構造体Cajal body（CB）はスプライソソームのサブユニットsnRNPが成熟、集積する場で、様々な環境変化に応答してサイズや数が調節される。我々は細胞内のsnRNP量を変動させる化合物探索を行なっており、陽性化合物にはCBに影響する化合物も含まれると考え、CB観察による二次スクリーニングを行なってきた。その結果、デヒドロコスツスラクトン（DCL）で細胞を処理した場合にCBが分散し、その際にはCajal body形成の必須因子で、欠損が脊髄性筋萎縮症の原因となるSMN蛋白質も核内で分散し、またSDS-PAGEでの移動度が変化することを見出した。さらにこの移動度変化がSMNのプロリン異性化によることを変異体を用いた実験で確認し、当該プロリンのアラニン置換変異体ではSMNの二量体、多量体の形成能が温度依存的に低下した。しかし、実際にプロリンの異性化が起こっていることを示すには、本来のSMNの当該プロリンの状態がCis型であることを実証する必要があり、今年度は共同研究によりその検討を進めてきた。まず、SMN蛋白質を大腸菌で発現させたところ、ヒト細胞での発現時と同様に、野生型の蛋白質と標的プロリンをアラニンに置換した変異体ではSDS-PAGEの移動度に大きな差が見られた。このことから、プロリン異性化がSDS-PAGEで移動度変化として検出できる理由を大腸菌の組換え蛋白質で検討できることが明らかとなり、今後プロリン異性化を検出する新たな方策につながると期待される結果であった。
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, 20H04687

Chemical approach to elucidate the molecular mechanism for the splice site selection
Grants-in-Aid for Scientific Research
01 Apr. 2016 - 31 Mar. 2020
Maita Hiroshi
In our previous study, we have developed a reporter gene that contains the splice site mutation. We have screened small compounds using the reporter gene to identify hit compounds that can correct the splicing pattern of the reporter gene. A compound was successfully found and then we analyzed changes of transcriptome by RNA-seq. Transcriptome analysis revealed that the compound significantly reduced the normally retained introns, which may be interpreted as a result of transcription repression because generally half-life of introns are shorter than mature mRNA. In addition, this compound has been reported to block CDK9, a kinase stimulating transcription elongation by phosphorylating RNA polymerase II. Therefore, we hypothesized that the splicing modulating activity of the compound is based on inhibition of CDK9 and further analysis confirmed that CDK9 inhibitors have indeed an activity to modulate splicing, which suggest the novel approach to modulate splicing.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 16K08225

Suppression of the splicing mutation based on the molecular mechanism of pre-mRNA splicing fidelity
Grants-in-Aid for Scientific Research
01 Apr. 2013 - 31 Mar. 2017
MAITA Hiroshi
We have developed an assay to find a compound that affect the spliceosome and have obtained several hit compounds by the automated screening of a small compound library. In this study we made a genetic reporter to detect the compound-mediated recovery of defected splicing that is caused by a mutation in the 3’ splice site. Some of our hit compounds have shown that they can induce splicing of the mutated intron at the correct site.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 25460057

Analysis of the assembly and functional defect of the spliceosome associated with Retinitis pigmentosa
Grants-in-Aid for Scientific Research
2011 - 2012
MAITA Hiroshi
The spliceosome is a highly dynamic macromolecular ribonucleoprotein (RNP) machine that catalyzes pre-mRNA splicing by assembling U1, U2, U4, U5, and U6 small nuclear (sn)RNPs. To process large numbers of introns, synthesis and recycling of snRNPs must be maintained within an appropriate range to avoid their shortage, which would cause a severe disease such as Retinitis pigmentosa. However, the mechanism that maintains cellular snRNP levels is unknown. We constructed an expression library of a luciferase fragment fused to core components of snRNPs and used it to isolate PRPF6 and U5-40K that specifically reconstitute luciferase activity in the U5 snRNP complex. We revealed that the reporter detects the effects of small molecules on the levels of the U5 snRNP-reporter protein complex. Our assay will be useful to identify the small molecule that can control the snRNP level in an appropriate range.
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Hokkaido University, 23790068

A study to elucidate how AdRP-associated mutations in splicing factors affect the spliceosome formation pathway
Grants-in-Aid for Scientific Research
2009 - 2010
MAITA Hiroshi
Although autosomal dominant retinitis pigmentosa (AdRP) is a tissue specific disease, disease associated mutations were found in several constitutive splicing factors consisting the spliceosome as well as genes in photo signal transduction pathway. To elucidate the mechanism how abnormality of general splicing factors triggers AdRP, we tried to detect the effect of the mutations on the cellular splicoeosme levels using a newly developed reporter. The reporter experiment suggested that the cell may regulate the spliceosome level to adjust the surrounding environment.
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Hokkaido University, 21790056

網膜色素変性症原因遺伝子PAP-1を介した新たなPim-1シグナル伝達経路の解析
科学研究費助成事業
2006 - 2007
米田 宏
研究計画に基づき、PAP-1遺伝子の発現レベル変動を培養細胞を用いて各種条件下で検討した。Pim-1と違い、PAP-1には顕著な発現誘導や発現抑制を示す条件がないことから、PAP-1機能の調節には翻訳後調節やそれに伴う結合因子の変化が重要な役割を果たしていると考えた。そこで、ストレプトタグを付加したPAP-1遺伝子の恒常発現株を前立線ガン由来ヒト培養細胞を親株として作成し、PAP-1を含むタンパク複合体をタグを用いて単離することで、3種類のPAP-1結合因子を同定した。これら分子の発現ベクターを作成し、細胞内でのPAP-1との結合を確認したところ、いずれもPAP-1と結合した。これらのうちの1つは網膜色素変性症で遺伝子変異が報告されている分子の結合タンパク質であり、PAP-1も網膜色素変性症の原因遺伝子であることから、その機能的相関に興味がもたれた。一方、昨年度の研究から、我々はU2OS細胞のPAP-1発現抑制株ではPim-1によるレポーター遺伝子のスプライシングパターンへの効果に変化が生じていることを見出している。本年度はさらに誘導型のPAP-1の発現抑制細胞株をヒト胎児腎由来細胞株を用いて作成し、この細胞株が上述のレポーターと同じ遺伝子を発現していることから、そのスプライシングパターンを検討した。しかし、PAP-1単独での発現量の変動はU2OS細胞での結果と同じくスプライシングパターンには影響を与えなかった。また、この細胞株を用いてPim-1のスプライシングへ与える影響へのPAP-1発現抑制の効果も検討したが、大きな変化は見られなかった。この実験には遺伝子導入効率が大きく影響することから、今後は導入方法を工夫するか、別の細胞株を用いて、より効率的にPim-1の効果を検討できる実験系を作成する必要があると考えられた。
日本学術振興会, 若手研究(スタートアップ), 北海道大学, 18890001