Researcher Profile and Settings

Affiliation

• Research Faculty of Agriculture　Fundamental AgriScience Research　Applied Bioscience

Job Title

• Professor

Contact information

• onouchiagr.hokudai.ac.jp

URL

http://arabi4.agr.hokudai.ac.jp/arabi.html

J-Global ID

• 200901032536120618

Research Interests

• 分子生物学   植物生理学   Plant physiology   Molecular genetics   Molecular biology   

Research Areas

• Life sciences / Molecular biology
• Life sciences / Plants: molecular biology and physiology

Educational Organization

•  Bachelor's degree program　School of Agriculture
•  Master's degree program　Graduate School of Agriculture
•  Doctoral (PhD) degree program　Graduate School of Agriculture

Academic & Professional Experience

• 2021/07 - Today Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture Professor
• 2007/04 - 2021/06 Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture Associate Professor
• 2006/04 - 2007/03 Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture Associate Professor
• 2005 - 北海道大学大学院農学研究科・助教授 助教授
• 2005 - Associate Professor
• 2000 - 北海道大学大学院農学研究科・助手 助手
• 2000 - Research Associate
• 1996 -
• 1996 - Postdoctoral Fellowships of Japan Society for the Promotion of Science

Education

•        - 1995  Nagoya University
•        - 1995  Nagoya University  Graduate School, Division of Natural Science
•        - 1992  Nagoya University
•        - 1992  Nagoya University  Graduate School, Division of Natural Science
•        - 1990  Nagoya University  School of Science
•        - 1990  Nagoya University  Faculty of Science

Association Memberships

• 日本分子生物学会   日本植物生理学会   日本植物学会   RNA学会   

Research Activities

Published Papers

• Phylogeny-linked occurrence of ribosome stalling on the mRNAs of Arabidopsis unfolded protein response factor bZIP60 orthologs in divergent plant species.

  Tomoya Imamichi, Nao Kusumoto, Haruka Aoyama, Seidai Takamatsu, Yugo Honda, Shiori Muraoka, Yuka Hagiwara-Komoda, Yukako Chiba, Hitoshi Onouchi, Yui Yamashita, Satoshi Naito

  Nucleic acids research 2024/02/16 

  The bZIP60, XBP1 and HAC1 mRNAs encode transcription factors that mediate the unfolded protein response (UPR) in plants, animals and yeasts, respectively. Upon UPR, these mRNAs undergo unconventional cytoplasmic splicing on the endoplasmic reticulum (ER) to produce active transcription factors. Although cytoplasmic splicing is conserved, the ER targeting mechanism differs between XBP1 and HAC1. The ER targeting of HAC1 mRNA occurs before translation, whereas that of XBP1 mRNA involves a ribosome-nascent chain complex that is stalled when a hydrophobic peptide emerges from the ribosome; the corresponding mechanism is unknown for bZIP60. Here, we analyzed ribosome stalling on bZIP60 orthologs of plants. Using a cell-free translation system, we detected nascent peptide-mediated ribosome stalling during the translation elongation of the mRNAs of Arabidopsis, rice and Physcomitrium (moss) orthologs, and the termination-step stalling in the Selaginella (lycopod) ortholog, all of which occurred ∼50 amino acids downstream of a hydrophobic region. Transfection experiments showed that ribosome stalling contributes to cytoplasmic splicing in bZIP60u orthologs of Arabidopsis and Selaginella. In contrast, ribosome stalling was undetectable for liverwort, Klebsormidium (basal land plant), and green algae orthologs. This study highlights the evolutionary diversity of ribosome stalling and its contribution to ER targeting in plants.
• Upstream open reading frame-mediated upregulation of ANAC082 expression in response to nucleolar stress in Arabidopsis

  Shun Sasaki, Toru Murakami, Miharu Yasumuro, Ayaka Makita, Yutaro Oi, Yuta Hiragori, Shun Watanabe, Rin Kudo, Noriya Hayashi, Iwai Ohbayashi, Munetaka Sugiyama, Yui Yamashita, Satoshi Naito, Hitoshi Onouchi

  Plant Biotechnology 40 (1) 21 - 30 1342-4580 2023/03/25
• Genome-wide identification of Arabidopsis non-AUG-initiated upstream ORFs with evolutionarily conserved regulatory sequences that control protein expression levels.

  Yuta Hiragori, Hiro Takahashi, Taihei Karino, Atsushi Kaido, Noriya Hayashi, Shun Sasaki, Kodai Nakao, Taichiro Motomura, Yui Yamashita, Satoshi Naito, Hitoshi Onouchi

  Plant molecular biology 2022/08/31 [Refereed]
   

  KEY MESSAGE: This study identified four novel regulatory non-AUG-initiated upstream ORFs (uORFs) with evolutionarily conserved sequences in Arabidopsis and elucidated the mechanism by which a non-AUG-initiated uORF promotes main ORF translation. Upstream open reading frames (uORFs) are short ORFs found in the 5'-untranslated regions (5'-UTRs) of eukaryotic transcripts and can influence the translation of protein-coding main ORFs (mORFs). Recent genome-wide ribosome profiling studies have revealed that hundreds or thousands of uORFs initiate translation at non-AUG start codons. However, the physiological significance of these non-AUG uORFs has so far been demonstrated for only a few of them. In this study, to identify physiologically important regulatory non-AUG uORFs in Arabidopsis, we took an approach that combined bioinformatics and experimental analysis. Since physiologically important non-AUG uORFs are likely to be conserved across species, we first searched the Arabidopsis genome for non-AUG-initiated uORFs with evolutionarily conserved sequences. Then, we examined the effects of the conserved non-AUG uORFs on the expression of the downstream mORFs using transient expression assays. As a result, three inhibitory and one promotive non-AUG uORFs were identified. Among the inhibitory non-AUG uORFs, two exerted repressive effects on mORF expression in an amino acid sequence-dependent manner. These two non-AUG uORFs are likely to encode regulatory peptides that cause ribosome stalling, thereby enhancing their repressive effects. In contrast, one of the identified regulatory non-AUG uORFs promoted mORF expression by alleviating the inhibitory effect of a downstream AUG-initiated uORF. These findings provide insights into the mechanisms that enable non-AUG uORFs to play regulatory roles despite their low translation initiation efficiencies.
• De novo reconstitution of chromatin using wheat germ cell‐free protein synthesis

  Yaeta Endo, Nobuaki Takemori, Szilvia K. Nagy, Kei‐ichi Okimune, Rohinton Kamakaka, Hitoshi Onouchi, Taichi E. Takasuka

  FEBS Open Bio 11 (6) 1552 - 1564 2211-5463 2021/06 [Refereed][Not invited]
   

  DNA is packaged with histones to form chromatin that impinges on all nuclear processes, including transcription, replication and repair, in the eukaryotic nucleus. A complete understanding of these molecular processes requires analysis of chromatin context in vitro. Here, Drosophila four core histones were produced in a native and unmodified form using wheat germ cell-free protein synthesis. In the assembly reaction, four unpurified core histones and three chromatin assembly factors (dNAP-1, dAcf1 and dISWI) were incubated with template DNA. We then assessed stoichiometry with the histones, nucleosome arrays, supercoiling and the ability of the chromatin to serve as a substrate for histone-modifying enzymes. Overall, our method provides a new avenue to produce chromatin that can be useful in a wide range of chromatin research.
• Exhaustive identification of conserved upstream open reading frames with potential translational regulatory functions from animal genomes

  Hiro Takahashi, Shido Miyaki, Hitoshi Onouchi, Taichiro Motomura, Nobuo Idesako, Anna Takahashi, Masataka Murase, Shuichi Fukuyoshi, Toshinori Endo, Kenji Satou, Satoshi Naito, Motoyuki Itoh

  Scientific Reports 10 (1) 2020/10 [Refereed][Not invited]
   

  Upstream open reading frames (uORFs) are present in the 5′-untranslated regions of many eukaryotic mRNAs, and some peptides encoded by these regions play important regulatory roles in controlling main ORF (mORF) translation. We previously developed a novel pipeline, ESUCA, to comprehensively identify plant uORFs encoding functional peptides, based on genome-wide identification of uORFs with conserved peptide sequences (CPuORFs). Here, we applied ESUCA to diverse animal genomes, because animal CPuORFs have been identified only by comparing uORF sequences between a limited number of species, and how many previously identified CPuORFs encode regulatory peptides is unclear. By using ESUCA, 1517 (1373 novel and 144 known) CPuORFs were extracted from four evolutionarily divergent animal genomes. We examined the effects of 17 human CPuORFs on mORF translation using transient expression assays. Through these analyses, we identified seven novel regulatory CPuORFs that repressed mORF translation in a sequence-dependent manner, including one conserved only among Eutheria. We discovered a much higher number of animal CPuORFs than previously identified. Since most human CPuORFs identified in this study are conserved across a wide range of Eutheria or a wider taxonomic range, many CPuORFs encoding regulatory peptides are expected to be found in the identified CPuORFs.
• Comprehensive genome-wide identification of angiosperm upstream ORFs with peptide sequences conserved in various taxonomic ranges using a novel pipeline, ESUCA.

  Hiro Takahashi, Noriya Hayashi, Yuta Hiragori, Shun Sasaki, Taichiro Motomura, Yui Yamashita, Satoshi Naito, Anna Takahashi, Kazuyuki Fuse, Kenji Satou, Toshinori Endo, Shoko Kojima, Hitoshi Onouchi

  BMC genomics 21 (1) 260 - 260 2020/03/30 [Refereed][Not invited]
   

  BACKGROUND: Upstream open reading frames (uORFs) in the 5'-untranslated regions (5'-UTRs) of certain eukaryotic mRNAs encode evolutionarily conserved functional peptides, such as cis-acting regulatory peptides that control translation of downstream main ORFs (mORFs). For genome-wide searches for uORFs with conserved peptide sequences (CPuORFs), comparative genomic studies have been conducted, in which uORF sequences were compared between selected species. To increase chances of identifying CPuORFs, we previously developed an approach in which uORF sequences were compared using BLAST between Arabidopsis and any other plant species with available transcript sequence databases. If this approach is applied to multiple plant species belonging to phylogenetically distant clades, it is expected to further comprehensively identify CPuORFs conserved in various plant lineages, including those conserved among relatively small taxonomic groups. RESULTS: To efficiently compare uORF sequences among many species and efficiently identify CPuORFs conserved in various taxonomic lineages, we developed a novel pipeline, ESUCA. We applied ESUCA to the genomes of five angiosperm species, which belong to phylogenetically distant clades, and selected CPuORFs conserved among at least three different orders. Through these analyses, we identified 89 novel CPuORF families. As expected, ESUCA analysis of each of the five angiosperm genomes identified many CPuORFs that were not identified from ESUCA analyses of the other four species. However, unexpectedly, these CPuORFs include those conserved across wide taxonomic ranges, indicating that the approach used here is useful not only for comprehensive identification of narrowly conserved CPuORFs but also for that of widely conserved CPuORFs. Examination of the effects of 11 selected CPuORFs on mORF translation revealed that CPuORFs conserved only in relatively narrow taxonomic ranges can have sequence-dependent regulatory effects, suggesting that most of the identified CPuORFs are conserved because of functional constraints of their encoded peptides. CONCLUSIONS: This study demonstrates that ESUCA is capable of efficiently identifying CPuORFs likely to be conserved because of the functional importance of their encoded peptides. Furthermore, our data show that the approach in which uORF sequences from multiple species are compared with those of many other species, using ESUCA, is highly effective in comprehensively identifying CPuORFs conserved in various taxonomic ranges.
• Reverse genetics-based biochemical studies of the ribosomal exit tunnel constriction region in eukaryotic ribosome stalling: spatial allocation of the regulatory nascent peptide at the constriction

  Seidai Takamatsu, Yubun Ohashi, Noriyuki Onoue, Yoko Tajima, Tomoya Imamichi, Shinya Yonezawa, Kyoko Morimoto, Hitoshi Onouchi, Yui Yamashita, Satoshi Naito

  Nucleic Acids Research 48 (4) 1985 - 1999 0305-1048 2020/02/28 [Refereed][Not invited]
   

  A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which β-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the β-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.
• バイオインフォマティクスによる配列データベース横断解析に基づく機能上流ORFの推定

  髙橋広夫, 伊藤素行, 尾之内均

  生物工学会誌 (公社)日本生物工学会 97 (8) 496 - 499 0919-3758 2019/08 [Not refereed][Invited]
  
• Boron-dependent translational suppression of the borate exporter BOR1 contributes to the avoidance of boron toxicity

  Izumi Aibara, Tatsuya Hirai, Koji Kasai, Junpei Takano, Hitoshi Onouchi, Satoshi Naito, Toru Fujiwara, Kyoko Miwa

  Plant Physiology 177 (2) 759 - 774 1532-2548 2018/06/01 [Refereed][Not invited]
   

  Boron (B) is an essential element for plants however, as high B concentrations are toxic, B transport must be tightly regulated. BOR1 is a borate exporter in Arabidopsis (Arabidopsis thaliana) that facilitates B translocation into shoots under B deficiency conditions. When the B supply is sufficient, BOR1 expression is down-regulated by selective degradation of BOR1 protein, while additional BOR1 regulatory mechanisms are proposed to exist. In this study, we identified a novel B-dependent BOR1 translational suppression mechanism. In vivo and in vitro reporter assays demonstrated that BOR1 translation was reduced in a B-dependent manner and that the 5′-untranslated region was both necessary and sufficient for this process. Mutational analysis revealed that multiple upstream open reading frames in the 5′-untranslated region were required for BOR1 translational suppression, and this process depended on the efficiency of translational reinitiation at the BOR1 open reading frame after translation of the upstream open reading frames. To understand the physiological significance of BOR1 regulation, we characterized transgenic plants defective in either one or both of the BOR1 regulation mechanisms. BOR1 translational suppression was induced at higher B concentrations than those triggering BOR1 degradation. Plants lacking both regulation mechanisms exhibited more severe shoot growth reduction under high-B conditions than did plants lacking BOR1 degradation alone, thus demonstrating the importance of BOR1 translational suppression. This study demonstrates that two mechanisms of posttranscriptional BOR1 regulation, each induced under different B concentrations, contribute to the avoidance of B toxicity in plants.
• Identification of Arabidopsis thaliana upstream open reading frames encoding peptide sequences that cause ribosomal arrest

  Noriya Hayashi, Shun Sasaki, Hiro Takahashi, Yui Yamashita, Satoshi Naito, Hitoshi Onouchi

  NUCLEIC ACIDS RESEARCH 45 (15) 8844 - 8858 0305-1048 2017/09 [Refereed][Not invited]
   

  Specific sequences of certain nascent peptides cause programmed ribosomal arrest during mRNA translation to control gene expression. In eukaryotes, most known regulatory arrest peptides are encoded by upstream open reading frames (uORFs) present in the 5'-untranslated region ofmRNAs. However, to date, a limited number of eukaryotic uORFs encoding arrest peptides have been reported. Here, we searched for arrest peptide-encoding uORFs among Arabidopsis thaliana uORFs with evolutionarily conserved peptide sequences. Analysis of in vitro translation products of 22 conserved uORFs identified three novel uORFs causing ribosomal arrest in a peptide sequence-dependent manner. Stop codon-scanning mutagenesis, in which the effect of changing the uORF stop codon position on the ribosomal arrest was examined, and toeprint analysis revealed that two of the three uORFs cause ribosomal arrest during translation elongation, whereas the other one causes ribosomal arrest during translation termination. Transient expression assays showed that the newly identified arrest-causing uORFs exerted a strong sequence-dependent repressive effect on the expression of the downstream reporter gene in A. thaliana protoplasts. These results suggest that the peptide sequences of the three uORFs identified in this study cause ribosomal arrest in the uORFs, thereby repressing the expression of proteins encoded by the main ORFs.
• The Minimum Open Reading Frame, AUG-Stop, Induces Boron-Dependent Ribosome Stalling and mRNA Degradation.

  Mayuki Tanaka, Naoyuki Sotta, Yusuke Yamazumi, Yui Yamashita, Kyoko Miwa, Katsunori Murota, Yukako Chiba, Masami Yokota Hirai, Tetsu Akiyama, Hitoshi Onouchi, Satoshi Naito, Toru Fujiwara

  The Plant cell 28 (11) 2830 - 2849 1040-4651 2016/11 [Refereed][Not invited]
   

  Upstream open reading frames (uORFs) are often translated ahead of the main ORF of a gene and regulate gene expression, sometimes in a condition-dependent manner, but such a role for the minimum uORF (hereafter referred to as AUG-stop) in living organisms is currently unclear. Here, we show that AUG-stop plays an important role in the boron (B)-dependent regulation of NIP5;1, encoding a boric acid channel required for normal growth under low B conditions in Arabidopsis thaliana High B enhanced ribosome stalling at AUG-stop, which was accompanied by the suppression of translation and mRNA degradation. This mRNA degradation was promoted by an upstream conserved sequence present near the 5'-edge of the stalled ribosome. Once ribosomes translate a uORF, reinitiation of translation must take place in order for the downstream ORF to be translated. Our results suggest that reinitiation of translation at the downstream NIP5;1 ORF is enhanced under low B conditions. A genome-wide analysis identified two additional B-responsive genes, SKU5 and the transcription factor gene ABS/NGAL1, which were regulated by B-dependent ribosome stalling through AUG-stop. This regulation was reproduced in both plant and animal transient expression and cell-free translation systems. These findings suggest that B-dependent AUG-stop-mediated regulation is common in eukaryotes.
• 止まって働くリボソーム 新生ペプチドが司る植物の細胞内恒常性維持機構

  山下由衣, 尾之内均, 内藤哲

  化学と生物 54 (3) 191 - 197 2016/03 [Not refereed][Invited]
  
• 4-2-1 シロイヌナズナのホウ素輸送体、NIP5;1のホウ素に応答したmRNA蓄積のmRNA分解と転写の制御機構の解析（4-2 植物の微量栄養素，2016年度佐賀大会）

  田中 真幸, 反田 直之, 千葉 由佳子, 尾之内 均, 内藤 哲, 藤原 徹

  日本土壌肥料学会講演要旨集 一般社団法人 日本土壌肥料学会 62 57 - 57 2016
• シロイヌナズナCGS1 mRNAの新生ペプチドによる翻訳アレストへのリボソーム出口トンネルの関与

  高松 世大, 大橋 悠文, 尾上 典之, 山下 由衣, 尾之内 均, 内藤 哲

  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 (公社)日本生化学会 88回・38回 [3T25 - 07(3P0741)] 2015/12
• An upstream open reading frame represses expression of a tomato homologue of Arabidopsis ANAC096, a NAC domain transcription factor gene, in a peptide sequence-dependent manner

  Abdul Latif Noh, Shun Watanabe, Hiro Takahashi, Satoshi Naito, Hitoshi Onouchi

  PLANT BIOTECHNOLOGY 32 (2) 157 - U126 1342-4580 2015/06 [Refereed][Not invited]
   

  Many eukaryotic mRNAs contain one or more upstream open reading frames (uORFs) in their 5' untranslated regions (5'-UTRs). Some uORFs encode regulatory peptides that repress translation of the main ORF. To comprehensively identify uORFs encoding regulatory peptides, genome-wide searches for uORFs with evolutionarily conserved amino acid sequences, referred to as conserved peptide uORFs (CPuORFs), have been conducted using bioinfomatic approaches. To date, more than 40 homology groups of CPuORFs have been identified in dicotyledonous plants. The Arabidopsis thaliana ANAC096 gene is one of the CPuORF-containing genes; however, the ANAC096 CPuORF exerts only little peptide sequence-dependent effect on expression of the main ORF. Here, we investigated the effect of the CPuORF sequence of a tomato ANAC096 homologue on expression of the main ORF, because it has a more highly conserved amino acid sequence than the ANAC096 CPuORF. Mutational analyses revealed that the CPuORF of the tomato ANAC096 homologue represses main ORF expression in a peptide sequence-dependent manner, and determined the critical amino acid residues of the CPuORF peptide responsible for the repression. This study identified a novel peptide sequence-dependent regulatory uORF and demonstrated that the level of uORF peptide-mediated repression can differ among closely related homologues.
• Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner

  Isao Ebina, Mariko Takemoto-Tsutsumi, Shun Watanabe, Hiroaki Koyama, Yayoi Endo, Kaori Kimata, Takuya Igarashi, Karin Murakami, Rin Kudo, Arisa Ohsumi, Abdul Latif Noh, Hiro Takahashi, Satoshi Naito, Hitoshi Onouchi

  NUCLEIC ACIDS RESEARCH 43 (3) 1562 - 1576 0305-1048 2015/02 [Refereed][Not invited]
   

  Upstream open reading frames (uORFs) are often found in the 5 '-leader regions of eukaryotic mRNAs and can negatively modulate the translational efficiency of the downstream main ORF. Although the effects of most uORFs are thought to be independent of their encoded peptide sequences, certain uORFs control translation of the main ORF in a peptide sequence-dependent manner. For genome-wide identification of such peptide sequence-dependent regulatory uORFs, exhaustive searches for uORFs with conserved amino acid sequences have been conducted using bioinformatic analyses. However, whether the conserved uORFs identified by these bioinformatic approaches encode regulatory peptides has not been experimentally determined. Here we analyzed 16 recently identified Arabidopsis thaliana conserved uORFs for the effects of their amino acid sequences on the expression of the main ORF using a transient expression assay. We identified five novel uORFs that repress main ORF expression in a peptide sequence-dependent manner. Mutational analysis revealed that, in four of them, the C-terminal region of the uORF-encoded peptide is critical for the repression of main ORF expression. Intriguingly, we also identified one exceptional sequence-dependent regulatory uORF, in which the stop codon position is not conserved and the C-terminal region is not important for the repression of main ORF expression.
• A U-system approach for predicting metabolic behaviors and responses based on an alleged metabolic reaction network

  Kansuporn Sriyudthsak, Yuji Sawada, Yukako Chiba, Yui Yamashita, Shigehiko Kanaya, Hitoshi Onouchi, Toru Fujiwara, Satoshi Naito, Ebernard O. Voit, Fumihide Shiraishi, Masami Yokota Hirai

  BMC SYSTEMS BIOLOGY 8 S4  1752-0509 2014/12 [Refereed][Not invited]
   

  Background: Progress in systems biology offers sophisticated approaches toward a comprehensive understanding of biological systems. Yet, computational analyses are held back due to difficulties in determining suitable model parameter values from experimental data which naturally are subject to biological fluctuations. The data may also be corrupted by experimental uncertainties and sometimes do not contain all information regarding variables that cannot be measured for technical reasons. Results: We show here a streamlined approach for the construction of a coarse model that allows us to set up dynamic models with minimal input information. The approach uses a hybrid between a pure mass action system and a generalized mass action (GMA) system in the framework of biochemical systems theory (BST) with rate constants of 1, normal kinetic orders of 1, and -0.5 and 0.5 for inhibitory and activating effects, named Unity (U)-system. The U-system model does not necessarily fit all data well but is often sufficient for predicting metabolic behavior of metabolites which cannot be simultaneously measured, identifying inconsistencies between experimental data and the assumed underlying pathway structure, as well as predicting system responses to a modification of gene or enzyme. The U-system approach was validated with small, generic systems and implemented to model a large-scale metabolic reaction network of a higher plant, Arabidopsis. The dynamic behaviors obtained by predictive simulations agreed with actually available metabolomic time-series data, identified probable errors in the experimental datasets, and estimated probable behavior of unmeasurable metabolites in a qualitative manner. The model could also predict metabolic responses of Arabidopsis with altered network structures due to genetic modification. Conclusions: The U-system approach can effectively predict metabolic behaviors and responses based on structures of an alleged metabolic reaction network. Thus, it can be a useful first-line tool of data analysis, model diagnostics and aid the design of next-step experiments.
• The N-Terminal Cleavable Pre-Sequence Encoded in the First Exon of Cystathionine gamma-Synthase Contains Two Different Functional Domains for Chloroplast Targeting and Regulation of Gene Expression

  Yuka Hagiwara-Komoda, Tomoya Sugiyama, Yui Yamashita, Hitoshi Onouchi, Satoshi Naito

  PLANT AND CELL PHYSIOLOGY 55 (10) 1779 - 1792 0032-0781 2014/10 [Refereed][Not invited]
   

  Chloroplast transit peptide sequences (cTPs) located in the N-terminal region of nuclear-encoded chloroplast proteins are essential for their sorting, and are generally cleaved from the proteins after their import into the chloroplasts. The Arabidopsis thaliana cystathionine gamma-synthase (CGS), the first committed enzyme of methionine biosynthesis, is a nuclear-encoded chloroplast protein. Arabidopsis CGS possesses an N-terminal extension region that is dispensable for enzymatic activity. This N-terminal extension contains the cTP and several functional domains including an MTO1 region, the cis-element for post-transcriptional feedback regulation of CGS1 that codes for CGS. A previous report suggested that the cTP cleavage site of CGS is located upstream of the MTO1 region. However, the region required for protein sorting has not been analyzed. In this study, we carried out functional analyses to elucidate the region required for chloroplast targeting by using a chimeric protein, Ex1:GFP, in which the CGS1 exon 1 coding region containing the N-terminal extension was tagged with green fluorescent protein. The sequence upstream of the MTO1 region was responsible for efficient chloroplast targeting and for avoidance of missorting to the mitochondria. Our data also showed that the major N-terminus of Ex1: GFP is Ala91, which is located immediately downstream of the MTO1 region, and the MTO1 region is not retained in the mature Ex1: GFP accumulated in the chloroplast. These findings suggest that the N-terminal cleavable pre-sequence harbors dual functions in protein sorting and in regulating gene expression. Our study highlights the unique properties of Arabidopsis CGS cTP among chloroplast-targeted proteins.
• Polyamine-responsive ribosomal arrest at the stop codon of an upstream open reading frame of the AdoMetDC1 gene triggers nonsense-mediated mRNA decay in Arabidopsis thaliana.

  Naoko Uchiyama-Kadokura, Karin Murakami, Mariko Takemoto, Naoto Koyanagi, Katsunori Murota, Satoshi Naito, Hitoshi Onouchi

  Plant & cell physiology 55 (9) 1556 - 67 0032-0781 2014/09 [Refereed][Not invited]
   

  During mRNA translation, nascent peptides with certain specific sequences cause arrest of ribosomes that have synthesized themselves. In some cases, such ribosomal arrest is coupled with mRNA decay. In yeast, mRNA quality control systems have been shown to be involved in mRNA decay associated with ribosomal arrest. However, a link between ribosomal arrest and mRNA quality control systems has not been found in multicellular organisms. In this study, we aimed to explore the relationship between ribosomal arrest and mRNA decay in plants. For this purpose, we used an upstream open reading frame (uORF) of the Arabidopsis thaliana AdoMetDC1 gene, in which the uORF-encoded peptide is involved in polyamine-responsive translational repression of the main coding sequence. Our in vitro analyses revealed that the AdoMetDC1 uORF-encoded peptide caused ribosomal arrest at the uORF stop codon in response to polyamine. Using transgenic calli harboring an AdoMetDC1 uORF-containing reporter gene, we showed that polyamine promoted mRNA decay in a uORF sequence-dependent manner. These results suggest that the polyamine-responsive ribosomal arrest mediated by the uORF-encoded peptide is coupled with mRNA decay. Our results also showed that the polyamine-responsive acceleration of mRNA decay was compromised by defects in factors that are essential for nonsense-mediated mRNA decay (NMD), an mRNA quality control system that degrades mRNAs with premature stop codons, suggesting that NMD is involved in AdoMetDC1 uORF peptide-mediated mRNA decay. Collectively, these findings suggest that AdoMetDC1 uORF peptide-mediated ribosomal arrest at the uORF stop codon induces NMD.
• Ribosomes in a Stacked Array

  Yui Yamashita, Yoshitomo Kadokura, Naoyuki Sotta, Toru Fujiwara, Ichigaku Takigawa, Akiko Satake, Hitoshi Onouchi, Satoshi Naito

  JOURNAL OF BIOLOGICAL CHEMISTRY 289 (18) 12693 - 12704 0021-9258 2014/05 [Refereed][Not invited]
   

  Background:S-Adenosyl-l-methionine arrests ribosomes on CGS1 mRNA at the pre-translocation step, and ribosomes stack behind it. Results: Puromycin reactivity of stacked ribosomes was intermediate between ribosomes at the pre- and post-translocation steps. Conclusion: Ribosomes are stacked at nine-codon intervals, and the majority are stalled at an early step of translocation. Significance: This study is the first report of ribosomal states in a stacked array. Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-l-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state.
• Translation elongation arrest induced by S-adenosyl-l-methionine-sensing nascent peptide in plants

  Yui Yamashita, Noriyuki Onoue, Katsunori Murota, Hitoshi Onouchi, Satoshi Naito

  Regulatory Nascent Polypeptides 187 - 201 2014/04 [Refereed][Invited]
   

  © 2014 Springer Japan. All rights are reserved. The CGS1 gene of the model plant Arabidopsis encodes cystathionine-γ-synthase, which catalyzes the first committed step of methionine biosynthesis. CGS1 gene expression is feedback regulated in response to S-adenosyl-l-methionine (AdoMet) by a coupled process of translation arrest and CGS1 mRNA degradation. In vitro translation studies using wheat germ extract revealed that AdoMet induces transient translation elongation arrest at Ser-94, located immediately downstream of the MTO1 region, which is the cis-element for translation arrest. Upon translation arrest, peptidyl-tRNA^Ser resides in the A-site and the ribosome is arrested at the pre-translocation step. The nascent peptide containing the MTO1 region adopts a compact conformation in the arrested ribosome, and the 28S rRNA of the ribosomal exit tunnel region may also undergo conformation changes. This pre-translocation step arrest feature makes the CGS1 system unique among the nascent peptide-mediated ribosome stalling systems.
• A halt in poly(A) shortening during S-adenosyl-L-methionine-induced translation arrest in CGS1 mRNA of Arabidopsis thaliarta

  Yui Yamashita, Ingrid Lambein, Soko Kobayashi, Hitoshi Onouchi, Yukako Chiba, Satoshi Naito

  GENES & GENETIC SYSTEMS 88 (4) 241 - 249 1341-7568 2013/08 [Refereed][Not invited]
   

  Cystathionine gamma-synthase (CGS) catalyzes the first committed step of methionine (Met) biosynthesis in plants. Expression of the Arabidopsis thaliana CGS1 gene is negatively feedback-regulated in response to the direct Met metabolite S-adenosyl-L-methionine (AdoMet). This regulation occurs at the step of mRNA stability during translation and is coupled with AdoMet-induced CGS1-specific translation arrest. In general, mRNA decay is initiated by a shortening of the poly(A) tail. However, this process has not been studied in detail in cases where regulatory events, such as programmed translation arrest, are involved. Here, we report that the poly(A) tail of the full-length CGS1 mRNA showed an apparent increase from 50-80 nucleotides (nt) to 140-150 nt after the induction of CGS1 mRNA degradation. This finding was unexpected because mRNAs that are destined for degradation harbored longer poly(A) tail than mRNAs that were not targeted for degradation. The results suggest that poly(A) shortening is inhibited or delayed during AdoMet-induced translation arrest of CGS1 mRNA. We propose an explanation for this phenomenon that remains consistent with the recent model of actively translating mRNA. We also found that CGS1 mRNA degradation intermediates, which are 51-truncated forms of CGS1 mRNA, had a short poly(A) tail of 10-30 nt. This suggests that poly(A) shortening occurs rapidly on the degradation intermediates. The present study highlights CGS1 mRNA degradation as a useful system to understand the dynamic features of poly(A) shortening.
• SS-mPMG and SS-GA: Tools for finding pathways and dynamic simulation of metabolic networks

  Tetsuo Katsuragi, Naoaki Ono, Keiichi Yasumoto, Md. Altaf-Ul-Amin, Masami Y. Hirai, Kansuporn Sriyudthsak, Yuji Sawada, Yui Yamashita, Yukako Chiba, Hitoshi Onouchi, Toru Fujiwara, Satoshi Naito, Fumihide Shiraishi, Shigehiko Kanaya

  Plant and Cell Physiology 54 (5) 728 - 739 0032-0781 2013/05 [Refereed][Not invited]
   

  Metabolomics analysis tools can provide quantitative information on the concentration of metabolites in an organism. In this paper, we propose the minimum pathway model generator tool for simulating the dynamics of metabolite concentrations (SS-mPMG) and a tool for parameter estimation by genetic algorithm (SS-GA). SS-mPMG can extract a subsystem of the metabolic network from the genome-scale pathway maps to reduce the complexity of the simulation model and automatically construct a dynamic simulator to evaluate the experimentally observed behavior of metabolites. Using this tool, we show that stochastic simulation can reproduce experimentally observed dynamics of amino acid biosynthesis in Arabidopsis thaliana. In this simulation, SS-mPMG extracts the metabolic network subsystem from published databases. The parameters needed for the simulation are determined using a genetic algorithm to fit the simulation results to the experimental data. We expect that SS-mPMG and SS-GA will help researchers to create relevant metabolic networks and carry out simulations of metabolic reactions derived from metabolomics data. © 2013 The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.
• Changes in mRNA stability associated with cold stress in arabidopsis cells

  Yukako Chiba, Katsuhiko Mineta, Masami Y. Hirai, Yuya Suzuki, Shigehiko Kanaya, Hiro Takahashi, Hitoshi Onouchi, Junji Yamaguchi, Satoshi Naito

  Plant and Cell Physiology 54 (2) 181 - 194 1471-9053 2013 [Refereed][Not invited]
   

  Control of mRNA half-life is a powerful strategy to adjust individual mRNA levels to various stress conditions, because the mRNA degradation rate controls not only the steady-state mRNA level but also the transition speed of mRNA levels. Here, we analyzed mRNA half-life changes in response to cold stress in Arabidopsis cells using genome-wide analysis, in which mRNA half-life measurements and transcriptome analysis were combined. Half-lives of average transcripts were determined to be elongated under cold conditions. Taking this general shift into account, we identified more than a thousand transcripts that were classified as relatively stabilized or relatively destabilized. The relatively stabilized class was predominantly observed in functional categories that included various regulators involved in transcriptional, post-transcriptional and post-translational processes. On the other hand, the relatively destabilized class was enriched in categories related to stress and hormonal response proteins, supporting the idea that rapid decay of mRNA is advantageous for swift responses to stress. In addition, pentatricopeptide repeat, cyclin-like F-box and Myb transcription factor protein families were significantly over-represented in the relatively destabilized class. The global analysis presented here demonstrates not only the importance of mRNA turnover control in the cold stress response but also several structural characteristics that might be important in the control of mRNA stability. © 2011 The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.
• BAIUCAS: a novel BLAST-based algorithm for the identification of upstream open reading frames with conserved amino acid sequences and its application to the Arabidopsis thaliana genome

  Hiro Takahashi, Anna Takahashi, Satoshi Naito, Hitoshi Onouchi

  BIOINFORMATICS 28 (17) 2231 - 2241 1367-4803 2012/09 [Refereed][Not invited]
   

  Motivation: Upstream open reading frames (uORFs) are often found in the 5'-untranslated regions of eukaryotic messenger RNAs. Some uORFs have been shown to encode functional peptides involved in the translational regulation of the downstream main ORFs. Comparative genomic approaches have been used in genomewide searches for uORFs encoding bioactive peptides, and by comparing uORF sequences between a few selected species or among a small group of species, uORFs with conserved amino acid sequences (UCASs) have been identified in plants, mammals and insects. Regulatory regions within uORF-encoded peptides that are involved in translational control are typically 10-20 amino acids long. Detection of homology between such short regions largely depends on the selection of species for comparison. To maximize the chances of identifying UCASs with short conserved regions, we devised a novel algorithm for homology search among a large number of species and the automatic selection of uORFs conserved in a wide range of species. Results: In this study, we developed the BAIUCAS (BLAST-based algorithm for identification of UCASs) method and identified 18 novel Arabidopsis uORFs whose amino acid sequences are conserved across diverse eudicot species, which include uORFs not found in previous comparative genomic studies due to low sequence conservation among species. Therefore, BAIUCAS is a powerful method for the identification of UCASs, and it is particularly useful for the detection of uORFs with a small number of conserved amino acid residues.
• Boron-Dependent Degradation of NIP5;1 mRNA for Acclimation to Excess Boron Conditions in Arabidopsis

  Mayuki Tanaka, Junpei Takano, Yukako Chiba, Fabien Lombardo, Yuki Ogasawara, Hitoshi Onouchi, Satoshi Naito, Toru Fujiwara

  PLANT CELL 23 (9) 3547 - 3559 1040-4651 2011/09 [Refereed][Not invited]
   

  Boron (B) is an essential plant micronutrient that is toxic at higher levels. NIP5;1 is a boric acid channel required for B uptake and growth under B deficiency. Accumulation of the NIP5;1 transcript is upregulated under B deficiency in Arabidopsis thaliana roots. To elucidate the mechanism of regulation, the 5' untranslated region (UTR) of NIP5;1 was tested for its ability to confer B-dependent regulation using beta-glucuronidase and green fluorescent protein as reporters. This analysis showed that the 5' UTR was involved in NIP5;1 transcript accumulation in response to B conditions. We also found that high-B conditions trigger NIP5;1 mRNA degradation and that the sequence from +182 to +200 bp in the 5' UTR is required for this mRNA destabilization. In the nip5;1-1 mutant background, a NIP5;1 complementation construct without the 5' UTR produced high levels of mRNA accumulation, increased B concentrations in tissues, and reduced growth under high-B conditions. These data suggest that the 5' UTR controls B-dependent NIP5;1 mRNA degradation and that NIP5;1 mRNA degradation is important for plant acclimation to high-B conditions.
• Arabidopsis cell-free extract, ACE, a new in vitro translation system derived from Arabidopsis callus cultures.

  Katsunori Murota, Yuka Hagiwara-Komoda, Keisuke Komoda, Hitoshi Onouchi, Masayuki Ishikawa, Satoshi Naito

  Plant & cell physiology 52 (8) 1443 - 53 0032-0781 2011/08 [Refereed][Not invited]
   

  The analysis of post-transcriptional regulatory mechanisms in plants has benefited greatly from the use of cell-free extract systems. Arabidopsis as a model system provides extensive genetic resources; however, to date a suitable cell-free translation system from Arabidopsis has not been available. In this study, we devised an Arabidopsis cell-free extract (ACE) to be used for in vitro translation studies. Protoplasts were prepared from callus cultures derived from Arabidopsis seedlings, and cell-free extracts were prepared after evacuolation of the protoplasts by Percoll gradient centrifugation. The new ACE system exhibits translation activity comparable with that of the wheat germ extract system. We demonstrated that ACE prepared from the 5'-3' exoribonuclease-deficient mutant of Arabidopsis, xrn4-5, exhibited increased stability of an uncapped mRNA as compared with that from wild-type Arabidopsis. We applied the ACE system to study post-transcriptional regulation of AtCGS1. AtCGS1 codes for cystathionine γ-synthase (CGS) that catalyzes the first committed step of methionine and S-adenosyl-l-methionine (AdoMet) biosynthesis in plants, and is feedback regulated by mRNA degradation coupled with translation elongation arrest. The ACE system was capable of reproducing translation elongation arrest and subsequent AtCGS1 mRNA degradation that are induced by AdoMet. The ACE system described here can be prepared in a month after seed sowing and will make it possible to study post-transcriptional regulation of plant genes while taking advantage of the genetics of Arabidopsis.
• S-Adenosyl-L-methionine Induces Compaction of Nascent Peptide Chain inside the Ribosomal Exit Tunnel upon Translation Arrest in the Arabidopsis CGS1 Gene

  Noriyuki Onoue, Yui Yamashita, Nobuhiro Nagao, Derek B. Goto, Hitoshi Onouchi, Satoshi Naito

  JOURNAL OF BIOLOGICAL CHEMISTRY 286 (17) 14903 - 14912 0021-9258 2011/04 [Refereed][Not invited]
   

  Expression of the Arabidopsis CGS1 gene, encoding the first committed enzyme of methionine biosynthesis, is feedback-regulated in response to S-adenosyl-L-methionine (AdoMet) at the mRNA level. This regulation is first preceded by temporal arrest of CGS1 translation elongation at the Ser-94 codon. AdoMet is specifically required for this translation arrest, although the mechanism by which AdoMet acts with the CGS1 nascent peptide remained elusive. We report here that the nascent peptide of CGS1 is induced to form a compact conformation within the exit tunnel of the arrested ribosome in an AdoMet-dependent manner. Cysteine residues introduced into CGS1 nascent peptide showed reduced ability to react with polyethyleneglycol maleimide in the presence of AdoMet, consistent with a shift into the ribosomal exit tunnel. Methylation protection and UV cross-link assays of 28 S rRNA revealed that induced compaction of nascent peptide is associated with specific changes in methylation protection and UV cross-link patterns in the exit tunnel wall. A 14-residue stretch of amino acid sequence, termed the MTO1 region, has been shown to act in cis for CGS1 translation arrest and mRNA degradation. This regulation is lost in the presence of mto1 mutations, which cause single amino acid alterations within MTO1. In this study, both the induced peptide compaction and exit tunnel change were found to be disrupted by mto1 mutations. These results suggest that the MTO1 region participates in the AdoMet-induced arrest of CGS1 translation by mediating changes of the nascent peptide and the exit tunnel wall.
• Analysis of boron-dependent regulatory mechanism of NIP5;1, transcript accumulation in Arabidopsis thaliana

  Tanaka Mayuki, Takano Junpei, Chiba Yukako, Onouchi Hitoshi, Naito Satoshi, Fujiwara Toru

  Plant and Cell Physiology Supplement 日本植物生理学会 2011 (0) 540 - 540 2011 

  NIP5;1 encodes s a boron (B) channel required for normal plant growth under low B conditions and its transcript accumulation is upregulated under low B conditions. At the annual meeting last year, we reported that at least +182-+200 region of 5'untranlated region (5'UTR) is important for NIP5;1 transcript accumulation in response to low B.
  To elucidate mechanisms of NIP5;1 regulation in response to B, the half-life of NIP5;1 mRNA was estimated by determining time course of mRNA decay after exposure to an RNA synthesis inhibitor under the high and low B conditions. We established constructs carrying either intact 5'UTR or 5'UTR without the sequence from +182 to +200 between the GUS reporter gene and the cauliflower mosaic virus 35S RNA promoter. In transgenic plants carrying the intact 5'UTR, the GUS mRNA level under the high B condition more rapidly decreased compared to the low B condition. In transgenic plants carrying the 5'UTR without +182-+200 construct, GUS mRNA level was not significantly different between B treatments. These results indicated that the sequence from +182 to +200 is required for B-dependent mRNA degradation.
• Comparative metabolomics charts the impact of genotype-dependent methionine accumulation in Arabidopsis thaliana

  Miyako Kusano, Atsushi Fukushima, Henning Redestig, Makoto Kobayashi, Hitomi Otsuki, Hitoshi Onouchi, Satoshi Naito, Masami Yokota Hirai, Kazuki Saito

  AMINO ACIDS 39 (4) 1013 - 1021 0939-4451 2010/10 [Refereed][Not invited]
   

  Methionine (Met) is an essential amino acid for all organisms. In plants, Met also functions as a precursor of plant hormones, polyamines, and defense metabolites. The regulatory mechanism of Met biosynthesis is highly complex and, despite its great importance, remains unclear. To investigate how accumulation of Met influences metabolism as a whole in Arabidopsis, three methionine over-accumulation (mto) mutants were examined using a gas chromatography-mass spectrometry-based metabolomics approach. Multivariate statistical analyses of the three mto mutants (mto1, mto2, and mto3) revealed distinct metabolomic phenotypes. Orthogonal projection to latent structures-discriminant analysis highlighted discriminative metabolites contributing to the separation of each mutant and the corresponding control samples. Though Met accumulation in mto1 had no dramatic effect on other metabolic pathways except for the aspartate family, metabolite profiles of mto2 and mto3 indicated that several extensive pathways were affected in addition to over-accumulation of Met. The pronounced changes in metabolic pathways in both mto2 and mto3 were associated with polyamines. The findings suggest that our metabolomics approach not only can reveal the impact of Met over-accumulation on metabolism, but also may provide clues to identify crucial pathways for regulation of metabolism in plants.
• Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways

  Junpei Takano, Mayuki Tanaka, Atsushi Toyoda, Kyoko Miwa, Koji Kasai, Kentaro Fuji, Hitoshi Onouchi, Satoshi Naito, Toru Fujiwara

  Proceedings of the National Academy of Sciences of the United States of America 107 (11) 5220 - 5225 0027-8424 2010/03/16 [Refereed][Not invited]
   

  Boron (B) is essential for plant growth but is toxic when present in excess. In the roots of Arabidopsis thaliana under B limitation, a boric acid channel, NIP5 1, and a boric acid/borate exporter, BOR1, are required for efficient B uptake and subsequent translocation into the xylem, respectively. However, under high-B conditions, BOR1 activity is repressed through endocytic degradation, presumably to avoid B toxicity. In this study, we investigated the localization of GFP-tagged NIP5 1 and BOR1 expressed under the control of their native promoters. Under B limitation, GFP-NIP5 1 and BOR1-GFP localized preferentially in outer (distal) and inner (proximal) plasma membrane domains, respectively, of various root cells. The polar localization of the boric acid channel and boric acid/borate exporter indicates the radial transport route of B toward the stele. Furthermore, mutational analysis revealed a requirement of tyrosine residues, in a probable cytoplasmic loop region of BOR1, for polar localization in various cells of the meristem and elongation zone. The same tyrosine residues were also required for vacuolar targeting upon high B supply. The present study of BOR1 and NIP5 1 demonstrates the importance of selective endocytic trafficking in polar localization and degradation of plant nutrient transporters for radial transport and homeostasis of plant mineral nutrients.
• Nascent peptide-mediated translation elongation arrest of Arabidopsis thaliana CGS1 mRNA occurs autonomously.

  Hitoshi Onouchi, Yuhi Haraguchi, Mari Nakamoto, Daisuke Kawasaki, Yoko Nagami-Yamashita, Katsunori Murota, Azusa Kezuka-Hosomi, Yukako Chiba, Satoshi Naito

  Plant & cell physiology 49 (4) 549 - 56 0032-0781 2008/04 [Refereed][Not invited]
   

  The Arabidopsis thaliana CGS1 gene encodes cystathionine gamma-synthase, the first committed enzyme of methionine biosynthesis in higher plants. Expression of CGS1 is feedback-regulated at the step of mRNA degradation in response to S-adenosyl-L-methionine (AdoMet). A short stretch of amino acid sequence, termed the MTO1 region, encoded within the first exon of CGS1 itself acts in cis in the regulation. In vitro analyses using wheat germ extract (WGE) revealed that AdoMet induces temporal translation arrest of CGS1 mRNA prior to mRNA degradation. This translational pausing occurs immediately downstream of the MTO1 region and is mediated by the nascent MTO1 peptide. In order to elucidate further the nature of this unique regulatory mechanism, we have examined whether a non-plant system also contains the post-transcriptional regulation activity. Despite the fact that mammals do not carry cystathionine gamma-synthase, AdoMet was able to induce the MTO1 sequence-dependent translation elongation arrest in rabbit reticulocyte lysate (RRL) in a similar manner to that observed in WGE. This result suggests that MTO1 peptide-mediated translation arrest does not require a plant-specific factor and rather most probably occurs via a direct interaction between the nascent MTO1 peptide and the ribosome that has translated it. In contrast, decay intermediates of CGS1 mRNA normally observed upon induction of CGS1 mRNA decay in plant systems were not detected in RRL, raising the possibility that CGS1 mRNA degradation involves a plant-specific mechanism.
• Ribosome stacking defines CGS1 mRNA degradation sites during nascent peptide-mediated translation arrest

  Yuhi Haraguchi, Yoshitomo Kadokura, Mari Nakamoto, Hitoshi Onouchi, Satoshi Naito

  PLANT AND CELL PHYSIOLOGY 49 (3) 314 - 323 0032-0781 2008/03 [Refereed][Not invited]
   

  Expression of the Arabidopsis CGS1 gene that codes for cystathionine -synthase is feedback-regulated at the step of mRNA degradation in response to S-adenosyl-L-methionine (AdoMet). This regulation occurs during translation and involves AdoMet-induced temporal translation arrest prior to the mRNA degradation. Here, we have identified multiple intermediates of CGS1 mRNA degradation with different 5 ends that are separated by approximately 30 nucleotides. Longer intermediates were found to be produced as the number of ribosomes loaded on mRNA was increased. Sucrose density gradient centrifugation experiments showed that the shortest mRNA degradation intermediate was associated with monosomes, whereas longer degradation intermediates were associated with multiple ribosomes. Immunoblot analyses revealed a ladder of premature polypeptides whose molecular weights corresponded to products of ribosomes in a stalled stack. An increase in smaller premature polypeptides was observed as the number of ribosomes loaded on mRNA increased. These results show that AdoMet induces the stacking of ribosomes on CGS1 mRNA and that multiple mRNA degradation sites probably correspond to each stacked ribosome.
• Promoter region of the beta subunit gene of beta-conglycinin responds to methionine and glutathione in transient assays using Arabidopsis protoplasts

  Naoko Ohkama-Ohtsu, Azusa Kezuka, Hitoshi Onouchi, Toru Fujiwara, Satoshi Naito

  SOIL SCIENCE AND PLANT NUTRITION 54 (1) 128 - 132 0038-0768 2008/02 [Refereed][Not invited]
   

  The effects of methionine and glutathione on the promoter activities of the beta subunit gene of beta-conglycinin, a seed storage protein of soybean, were studied in a transient assay system using protoplasts derived from Arabidopsis thaliana liquid callus cultures. The promoter activity of the beta subunit gene was repressed by methionine and glutathione in a manner similar to that observed in transgenic plants. This is the first report of a transient assay system to study the regulation of gene expression by methionine and glutathione.
• Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis

  H Onouchi, Y Nagami, Y Haraguchi, M Nakamoto, Y Nishimura, R Sakurai, N Nagao, D Kawasaki, Y Kadokura, S Naito

  GENES & DEVELOPMENT 19 (15) 1799 - 1810 0890-9369 2005/08 [Refereed][Not invited]
   

  Expression of the Arabidopsis CGS1 gene that codes for cystathionine gamma-synthase is feedback regulated at the step of mRNA stability in response to S-adenosyl-L-methionine (AdoMet). A short stretch of amino acid sequence, called the MTO1 region, encoded by the first exon of CGS1 itself is involved in this regulation. Here, we demonstrate, using a cell-free system, that AdoMet induces temporal translation elongation arrest at the Ser-94 codon located immediately downstream of the MTO1 region, by analyzing a translation intermediate and performing primer extension inhibition (toeprint) analysis. This translation arrest precedes the formation of a degradation intermediate of CGS1 mRNA, which has its 5' end points near the 5' edge of the stalled ribosome. The position of ribosome stalling also suggests that the MTO1 region in nascent peptide resides in the ribosomal exit tunnel when translation elongation is temporarily arrested. In addition to the MTO1 region amino acid sequence, downstream Trp-93 is also important for the AdoMet-induced translation arrest. This is the first example of nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in eukaryotes. Furthermore, our data suggest that the ribosome stalls at the step of translocation rather than at the step of peptidyl transfer.
• Mizoguchi T, Wright L, Fujiwara S, Cremer F, Lee K, Onouchi H, Mouradov A, Fowler S, Kamada H, Putterill J, Coupland G. : "Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in Arabidopsis" Plant Cell, 17:2255-2270 (2005)*

  2005 [Refereed][Not invited]
  
• Autoregulated MRNA stability of the gene for cystathionine gamma-synthase, the key-step enzyme of methionine biosynthesis in Arabidopsis

  Y Nagami, H Onouchi, Y Haraguchi, R Sakurai, M Nakamoto, N Nagao, S Naito

  Sulfur Transport and Assimilation in Plants in the Post Genomic Era 73 - 80 2005 [Not refereed][Invited]
  
• Dynamics of methionine biosynthesis

  Derek B. Goto, Hitoshi Onouchi, Satoshi Naito

  Plant Biotechnology Japanese Society for Plant Cell and Molecular Biology 22 (5) 379 - 388 1342-4580 2005 [Refereed][Invited]
   

  Methionine biosynthesis in plants provides the primary source of this essential amino acid. Early pioneering works characterised many of the steps involved in the methionine biosynthetic pathway and properties of the enzymes involved. They also showed that methionine biosynthesis was strictly controlled in higher plants and part of a larger, complex pathway that involves the biosynthesis of lysine, threonine and isoleucine. The adoption of the model plant Arabidopsis thaliana has enabled us to isolate mto mutants that over-accumulate soluble methionine. Through the analysis of these mto mutants, valuable insight has been gained into the regulation and in vivo dynamics of the methionine biosynthetic pathway in higher plants. Each of the three mutant mto loci identified to date (mto1, mto2, mto3) disrupt different processes within the pathway and display distinct phenotypic profiles. They have also revealed that, in addition to the common feedback controls, the pathway is subject to changing temporal and spatial regulation over the course of the plant life cycle.
• Autoregulation of the gene for cystathionine gamma-synthase in arabidopsis: post-transcriptional regulation induced by S-adenosylmethionine

  H Onouchi, Lambein, I, R Sakurai, A Suzuki, Y Chiba, S Naito

  BIOCHEMICAL SOCIETY TRANSACTIONS 32 597 - 600 0300-5127 2004/08 [Not refereed][Invited]
   

  Cystathionine gamma-synthase (CGS) catalyses the first committed step of methionine biosynthesis in higher plants. CGS is encoded by the CGS1 gene in Arabidopsis. Stability of CGS1 mRNA is down-regulated in response to methionine application and the exon 1-coding region of CGS1 itself is necessary and sufficient for this regulation. mto1 (for (m) under bare (t) under bar hionine (o) under bar veraccumulation) mutants of Arabidopsis, which carry single amino-acid sequence alterations within CGS1 exon 1, a re deficient in this regulation and overaccumulate methionine. Since CGS1 exon 1 acts in cis during this regulation, we have proposed a model that the regulation occurs during translation of CGS1 mRNA when the nascent polypeptide of CGS and its mRNA are in close proximity. in fact, application of the translation inhibitor cycloheximide abolished this regulation in vivo. This model predicts that the regulation can be reproduced in an in vitro translation system. Studies using the in vitro translation system of wheatgerm extract have indicated that S-adenosylmethionine, a direct metabolite of methionine, is the effector of this regulation. A 5'-truncated RNA species, which is a probable degradation intermediate of CGS1 mRNA in vivo, was also detected in vitro, suggesting that the wheatgerm in vitro translation system reflects the in vivo regulation.
• Decay kinetics of autogenously regulated CGS1 mRNA that codes for cystathionine gamma-synthase in Arabidopsis thaliana

  Lambein, I, Y Chiba, H Onouchi, S Naito

  PLANT AND CELL PHYSIOLOGY 44 (9) 893 - 900 0032-0781 2003/09 [Refereed][Not invited]
   

  Cystathionine gamma-synthase (CGS) catalyses the first committed step in methionine (Met) biosynthesis in higher plants. Stability of CGS1 mRNA encoding CGS in Arabidopsis thaliana is regulated by negative feedback in response to Met application and the amino acid sequence of CGS itself acts in cis in this regulation. It is proposed that the regulation occurs during translation when the nascent polypeptide of CGS and its mRNA are in close proximity. This model predicts that inhibition of translation abolishes the regulation. To test this, we analysed the effect of translation inhibitor cycloheximide on the CGS1 mRNA decay. The half-life of CGS1 mRNA after the addition of transcription inhibitor actinomycin D in the absence and presence of 1 mM Met was 154+/-11 min and 81+/-5 min, respectively. Simultaneous addition of actinomycin D and cycloheximide stabilized CGS1 mRNA both in the presence and absence of Met, as essentially no decrease of CGS1 mRNA was observed. Moreover, cycloheximide treatment inhibited production of the truncated CGS1 RNA species, a possible degradation intermediate. These results indicated that inhibition of translation abolishes the CGS1 mRNA-specific decay process. Kinetic analyses indicated that about half the CGS1 mRNA is destined to CGS1 mRNA-specific decay when 1 mM Met was applied.
• S-adenosyl-L-methionine is an effector in the posttranscriptional autoregulation of the cystathionine gamma-synthase gene in Arabidopsis

  Y Chiba, R Sakurai, M Yoshino, K Ominato, M Ishikawa, H Onouchi, S Naito

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100 (18) 10225 - 10230 0027-8424 2003/09 [Refereed][Not invited]
   

  Cystathionine gamma-synthase, the first committed enzyme of methionine biosynthesis in higher plants, is encoded by the CGS1 gene in Arabidopsis thaliana. We have shown previously that the stability of the CGS1 mRNA is negatively regulated in response to methionine application [Chiba, Y., Ishikawa, M., Kijima, F., Tyson, R. H., Kim, J., Yamamoto, A., Nambara, E., Leustek, T., Walisgrove, R. M. & Naito, S. (1999) Science 286, 1371-1374]. To determine whether methionine itself is the effector of the CGS1 exon 1-mediated posttranscriptional regulation, we carried out transfection experiments. The results suggested that, rather than methionine, S-adenosyl-L-methionine (AdoMet), or one of its metabolites, acts as the effector of this regulation. To further identify the actual effector, we exploited the wheat germ in vitro translation system. The effects of various metabolites and analogs of AdoMet were tested by using RNA carrying a CGS1 exon 1-reporter fusion. These tests identified AdoMet as the effector of this regulation. S-adenosyl-L-ethionine, an analog of AdoMet, also had effector activity. A. thaliana mto1 mutants, which are deficient in this regulation, showed a much reduced response to AdoMet in vitro, with a leaky allele showing a less reduced response. RNA translated in vitro in the presence of AdoMet contained a 5'-truncated RNA species, similar to the one that we previously suggested was an in vivo degradation intermediate of CGS1 mRNA. Together, the results show that the basic reactions of CGS1 exon 1-mediated posttranscriptional regulation occur in the wheat germ in vitro translation system, and that AdoMet acts as the effector.
• 細胞内シグナル伝達の機構 新生ペプチドによる遺伝子発現制御 (植物の形づくり--遺伝子からみた分子メカニズム) -- (細胞分化を促すシグナル伝達系)

  尾之内 均, 千葉 由佳子, 内藤 哲

  蛋白質核酸酵素 共立出版 47 (12) 1730 - 1734 0039-9450 2002/09 [Not refereed][Invited]
  
• Identification of a short highly conserved amino acid sequence as the functional region required for Posttranscriptional autoregulation of the cystathionine gamma-synthase gene in Arabidopsis

  K Ominato, H Akita, A Suzuki, F Kijima, T Yoshino, M Yoshino, Y Chiba, H Onouchi, S Naito

  JOURNAL OF BIOLOGICAL CHEMISTRY 277 (39) 36380 - 36386 0021-9258 2002/09 [Refereed][Not invited]
   

  Cystathionine gamma-synthase (CGS) catalyzes the first committed step of Met biosynthesis in plants. We have previously shown that expression of the gene for CGS is feedback-regulated at the level of mRNA stability, and that the amino acid sequence encoded by the first exon of the CGS gene itself is responsible for the regulation (Chiba, Y., Ishikawa, M., Kijima, F., Tyson, R. H., Kim, J., Yamamoto, A., Nambara, E., Leustek, T., Wallsgrove, R. M., and Naito, S. (1999) Science 286, 1371-1374). To identify the functional region within CGS exon 1, deletion analysis was performed. The results showed that the 41-amino acid region of exon 1 highly conserved among plants is necessary and sufficient for the regulation. Analyses of in vivo and in vitro generated mutations that abolish the regulation identified the functionally important amino acids as 11-13 residues within this conserved region. The importance of these residues was confirmed by deletion analysis within the conserved region. These studies identified the functional region of CGS exon 1 required for the posttranscriptional autoregulation of the CGS gene as (A)RRNCSNIGVAQ(I), with uncertainty of the first and last residues. This sequence is almost perfectly conserved among CGS sequences of higher plants but cannot be found elsewhere in the public databases.
• [Regulation of gene expression by nascent peptide].

  Onouchi H, Chiba Y, Naito S

  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 12 Suppl 47 (12 Suppl) 1730 - 1734 0039-9450 2002/09 [Not refereed][Invited]
  
• The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana, required for formation of a symmetric flat leaf lamina, encodes a member of a novel family of proteins characterized by cysteine repeats and a leucine zipper

  H Iwakawa, Y Ueno, E Semiarti, H Onouchi, S Kojima, H Tsukaya, M Hasebe, T Soma, M Ikezaki, C Machida, Y Machida

  PLANT AND CELL PHYSIOLOGY 43 (5) 467 - 478 0032-0781 2002/05 [Refereed][Not invited]
   

  The ASYMMETRIC LEAVES2 (AS2) gene of Arabidopsis thaliana is involved in the establishment of the leaf venation system, which includes the prominent mid-vein, as well as in the development of a symmetric lamina. The gene product also represses the expression of class 1 knox homeobox genes in leaves. We have characterized the AS2 gene, which appears to encode a novel protein with cysteine repeats (designated the C-motif) and a leucine-zipper-like sequence in the amino-terminal half of the primary sequence. The Arabidopsis genome contains 42 putative genes that potentially encode proteins with conserved amino acid sequences that include the C-motif and the leucine-zipper-like sequence in the amino-terminal half. Thus, the AS2 protein belongs to a novel family of proteins that we have designated the AS2 family. Members of this family except AS2 also have been designated ASLs (AS2-like proteins). Transcripts of AS2 were detected mainly in adaxial domains of cotyledonary primordia. Green fluorescent protein-fused AS2 was concentrated in plant cell nuclei. Overexpression of AS2 cDNA in transgenic Arabidopsis plants resulted in upwardly curled leaves, which differed markedly from the downwardly curled leaves generated by loss-of-function mutation of AS2. Our results suggest that AS2 functions in the transcription of a certain gene(s) in plant nuclei and thereby controls the formation of a symmetric flat leaf lamina and the establishment of a prominent mid-vein and other patterns of venation.
• A single-nucleotide mutation in a gene encoding S-adenosylmethionine synthetase is associated with methionine over-accumulation phenotype in Arabidopsis thaliana

  DB Goto, M Ogi, F Kijima, T Kumagai, F van Werven, H Onouchi, S Naito

  GENES & GENETIC SYSTEMS 77 (2) 89 - 95 1341-7568 2002/04 [Refereed][Not invited]
   

  Met-overaccumulating mutants provide a powerful genetic tool for examining both the regulation of the Met biosynthetic pathway and an vivo developmental responses of gene expression to altered Met levels. We have previously reported the identification of two Arabidopsis thaliana Met over-accumulation (mto) mutants, mto1-1 and mto2-1, that carry mutations in the genes encoding cystathionine gamma-synthase (CGS) and threonine synthase (TS), respectively. A third mutant, mto3-1, has recently been reported to carry a mutation in the gene encoding S-adenosylmethionine synthetase 3 (SAMS3). Here, we report the isolation of a new ethionine-resistant A. thaliana mutant that over-accumulates soluble Met approximately 20-fold in young rosettes. The causal mutation was determined to be a single, recessive mutation that was mapped to chromosome 3. Sequence analysis identified a single nucleotide change in the gene encoding SAMS3 that was distinct from the mto3-1 mutation and altered the amino acid sequence of the enzyme active site. This mutation was therefore referred to as mto3-2. Although Met over-accumulation in the mto3-2 mutant was similar to that in the mto2-1 mutant, CGS mRNA levels did not respond to the mto3-2 mutation and were similar to that in equivalent wild-type plants.
• The first exon coding region of cystathionine gamma-synthase gene is necessary and sufficient for downregulation of its own mRNA accumulation in transgenic Arabidopsis thaliana

  A Suzuki, Y Shirata, H Ishida, Y Chiba, H Onouchi, S Naito

  PLANT AND CELL PHYSIOLOGY 42 (10) 1174 - 1180 0032-0781 2001/10 [Refereed][Not invited]
   

  Expression of the gene for cystathionine gamma -synthase (CGS), which catalyzes the key step of methionine biosynthesis, is feedback regulated at the level of mRNA stability. The first exon polypeptide of CGS is suggested to be involved in this regulation and amino acid sequence alterations caused by mto1 mutations in that region lead to an overaccumulation of CGS mRNA [Chiba et al. (1999) Science 286: 1371-1374]. Transgenic Arabidopsis thaliana harboring chimeric constructs in which wild-type or mto1 mutant CGS exon 1 are fused in-frame to reporter genes and driven by the cauliflower mosaic virus 35S RNA promoter were constructed. Studies with these transgenic lines demonstrated that the coding region of CGS exon 1 is necessary and sufficient for downregulation of its own mRNA accumulation in response to methionine application and that this region acts in cis in this process.
• The transposition pattern of the Ac element and its use for targeted transposition in Arabidopsis thaliana

  Machida Chiyoko, Onouchi Hitoshi, Tanaka Hirokazu, Hamada Susumu, Ishikawa Takaaki, Semiarti Endang, Iwakawa Hidekazu, Nomura Kiyohito, Machida Yasunori

  Annual report of Research Institute for Biological Function 中部大学 1 (0) 9 - 16 1346-4205 2001/03/31 [Not refereed][Invited]
   

  In order to evaluate feasibility of the gene tagging by the maize transposable element Ac in heterologous plant systems, we have investigated physical distances and directions of transposition of the element in Arabidopsis thaliana and tobacco cultured cell line BY-2. We prepared a T-DNA construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-SceI (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. A number of transgenic Arabidopsis plants were generated, each of which had a single copy ...
• Suarez-Lopez P, Wheatley K, Robson F, Onouchi H, Valverde F and Coupland G.:"CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis", Nature, 410(6832):1116-1120 (2001).*

  2001 [Refereed][Not invited]
  
• A subtilisin-like serine protease is required for epidermal surface formation in Arabidopsis embryos and juvenile plants.

  Tanaka, H, Onouchi, H, Kondo, M, Hara-Nishimura, I, Nishimura, M, Machida, C, Machida, Y

  Development 128 4681 - 4689 2001 [Refereed][Not invited]
  
• The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates formation of a symmetric lamina, establishment of venation and repression of meristem-related homeobox genes in leaves.

  Semiarti, E, Onouchi, H, Torikai, S, Ishikawa, T, Machida, Y, Machida, C

  Development 128 1771 - 1783 2001 [Refereed][Not invited]
  
• The transposition pattern of the Ac element in tobacco cultured cells.

  Endang Semiarti, Hitoshi Onouchi, Satomi Torikai, Takaaki Ishikawa, Yasunori Machida, Chiyoko Machida

  Genes Genet. Syst. The Genetics Society of Japan 76 (2) 131 - 139 1341-7568 2001 [Refereed][Not invited]
   

  We investigated physical distances and directions of transposition of the maize transposable element Ac in tobacco cultured cells. We introduced a T-DNA construct that carried a non-autonomous derivative of Ac (designated dAc-I-RS) that included sites for cleavage by restriction endonuclease MluI. Another cleavage site was also introduced into the T-DNA region outside of the dAc-I-RS transposable element. The tobacco cultured cell line BY-2 was transformed with the T-DNA and several transformed lines that had a single copy of the T-DNA at a different chromosomal location were isolated. These lines were co-cultured with Agrobacterium tumefaciens cells that carried a cDNA for the Ac transposase gene under the control of various promoters. Sublines of cultured cells in which dAc-I-RS had been transposed, were isolated. The genomic DNAs of these sublines were isolated and digested with MluI. Sizes of DNA segments generated by digestion were determined by pulse-field gel electrophoresis. Our results showed that 20 to 70% of transposition events had occurred within several hundreds kilo-base pairs (kb) on the same chromosome. These results demonstrate that the Ac-Ds element preferentially transposed to regions near the original site in a tobacco chromosome. In addition, the present results are an example of asymmetric transposition as demonstrated by the distance of transposition on the chromosome.
• Mutations in the WUSCHEL gene of Arabidopsis thaliana result in the development of shoots without juvenile leaves

  Susumu Hamada, Hitoshi Onouchi, Hirokazu Tanaka, Mitsuko Kudo, Yao‐Guang Liu, Daisuke Shibata, Chiyoko Machida, Yasunori Machida

  The Plant Journal 24 (1) 91 - 101 0960-7412 2000/10 

  Summary The vegetative growth of Arabidopsis thaliana can be divided into two phases. The transition from the juvenile (early) phase to the adult (later) phase is associated with changes in several morphological features of leaves, such as the shape of leaf blades, the number of trichomes and patterns of venation. In a screening of mutants with altered morphological identities of leaves, we found one which we named juvenile leafless and misshapen shoot apical meristem (jam). The mutation represented a new allele of the WUSCHEL (WUS) gene, and, in its presence, plants produced no juvenile leaves. Analysis of the morphology of mutant plants revealed that all the rosette leaves had characteristics of adult leaves. The formation of the first rosette leaf in the wus(jam) mutant was markedly delayed, and occurred at the almost same time as formation of the third or fourth leaf in wild‐type plants. In the wild‐type, these leaves correspond to the first adult leaves. Analysis by RT–PCR showed that transcripts of WUS accumulated in shoot apices and roots, but not in cotyledons and leaves. The present results suggest that the WUS gene controls the morphological traits of rosette leaves either directly or indirectly. In view of the predicted function of the WUS gene, namely maintenance of stem cells within the shoot apical meristem, we suggest that the lack of juvenile leaves in the mutant might have been caused by interruption of leaf initiation during the juvenile phase or by halting of an entire process of formation of juvenile leaves.
• Mutagenesis of plants overexpressing CONSTANS demonstrates novel interactions among Arabidopsis flowering-time genes

  H Onouchi, MI Igeno, C Perilleux, K Graves, G Coupland

  PLANT CELL 12 (6) 885 - 900 1040-4651 2000/06 [Refereed][Not invited]
   

  CONSTANS (CO) promotes flowering of Arabidopsis in response to long photoperiods. Transgenic plants carrying CO fused with the cauliflower mosaic virus 35S promoter (35S::CO) flowered earlier than did the wild type and were almost completely insensitive to length of day. Genes required for CO to promote flowering were identified by screening for mutations that suppress the effect of 35S::CO. Four mutations were identified that partially suppressed the early-flowering phenotype caused by 35S::CO. One of these mutations, suppressor of overexpression of CO 1 (soc1), defines a new locus, demonstrating that the mutagenesis approach is effective in identifying novel flowering-time mutations. The other three suppressor mutations are allelic with previously described mutations that cause late flowering. Two of them are alleles of ft, indicating that FT is required for CO to promote early flowering and most likely acts after CO in the hierarchy of flowering-time genes. The fourth suppressor mutation is an allele of fwa, and fwa soc1 35S::CO plants flowered at approximately the same time as co mutants, suggesting that a combination of fwa and soc1 abolishes the promotion of flowering by CO. Besides delaying flowering, fwa acted synergistically with 35S::CO to repress floral development after bolting. The latter phenotype was not shown by any of the progenitors and was most probably caused by a reduction in the function of LEAFY. These genetic interactions suggest models for how CO, FWA, FT, and SOC1 interact during the transition to flowering.
• Hamada S, Onouchi H, Tanaka H, Kudo M, Liu Y, Shibata D, Machida C and Machida M.: "Mutations in the WUSCHEL gene of Arabidopsis thaliana result in the development of shoots without juvenile leaves", Plant J., 24:1-13(2000)*

  2000 [Refereed][Not invited]
  
• Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z, Yanofsky MF, Coupland G.: "Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis", Science, 288:1613-1616 (2000)*

  2000 [Refereed][Not invited]
  
• MOLECULAR-GENETIC ANALYSIS OF THE AS2 GENE ON THE MECHANISM OF SYMMETRICAL LEAF DEVEL OPMENT IN Arabidopsis thaliana

  SEMIARTI Endang, IWAKAWA Hidekazu, TSUKAYA Hirokazu, ONOUCHI Hitoshi, MACHIDA Chiyoko, MACHIDA Yasunori

  Plant and cell physiology 日本植物生理学会 41 (0) s212  0032-0781 2000 [Refereed][Not invited]
  
• GIGANTEA : a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains

  FOWLER S.

  EMBO J. 18 4679 - 4688 1999 [Refereed][Not invited]
   

  Fowler S, Lee K, Onouchi H, Samach A, Richardson K, Morris B, Coupland G, Putterill J. "GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains", EMBO J. , 18, 4679-88(1999)*
• Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91(phox))

  MA Torres, H Onouchi, S Hamada, C Machida, KE Hammond-Kosack, JDG Jones

  PLANT JOURNAL 14 (3) 365 - 370 0960-7412 1998/05 [Refereed][Not invited]
   

  An NADPH oxidase analogous to that in mammalian phagocytes has been hypothesized to produce reactive oxygen species (ROS) in the plant defence response. A. thaliana contains at least six gp91(phox) homologues, designated AtrbohA-F (A. thaliana Respiratory Burst Oxidase Homologues), which map to different positions. Transcripts of three of these genes can be detected in healthy plants by RNA gel blot analyses. The Atrboh gene products are closely related to gp91(phox) and the intron locations suggest a common evolutionary origin. A putative EF-hand Ca2+-binding motif in the extended N-terminal region of the Atrboh proteins suggests a direct regulatory effect of Ca2+ on the activity of the NADPH oxidase in plants.
• Torres MA, Onouchi H, Hamada S, Machida C, Hammond-Kosack KE, Jones JD. :"Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox)". Plant J., 14(3):365-370 (1998)*

  1998 [Refereed][Not invited]
  
• Characterization of the transposition pattern of the Ac element in Arabidopsis thaliana using endonuclease I-SceI

  C Machida, H Onouchi, J Koizumi, S Hamada, E Semiarti, S Torikai, Y Machida

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 94 (16) 8675 - 8680 0027-8424 1997/08 [Refereed][Not invited]
   

  We have investigated physical distances and directions of transposition of the maize transposable element Ac in Arabidopsis thaliana, We prepared a transferred DNA (T-DNA) construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-SceI (designated dAc-I-RS element), Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS, Three transgenic Arabidopsis plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location, These transgenic plants were crossed with the Arabidopsis that carried the gene for Ac transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with endonuclease I-SceI, sizes of segment of DNA were determined by pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements, Our results showed that 50% of all transposition events had occurred within 1,700 kb on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability, The data thus indicate that the Ac-Ds system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of Ac in Arabidopsis, In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites.
• トランスポソンタギング

  町田 千代子, 尾之内 均, 浜田 進

  化学と生物 学会出版センタ- 34 (9) 617 - 623 0453-073X 1996/09/25 [Not refereed][Invited]
  
• A new method of manipulation of chromosomes in higher plant

  MACHIDA Chiyoko, ONOUCHI Hitoshi

  Bioscience & industry バイオインダストリ-協会 54 (8) 16 - 22 0914-8981 1996/08/01 [Not refereed][Invited]
  
• Manipulation of plant chromosomes using the Ac transposale element and R-RS site-specific recombination system from Z. rouxii.

  MACHIDA C, ONOUCHI H, KOIZUMI J, KUDO M, MACHIDA Y

  育種学最近の進歩 37 (0) 13 - 16 1995/11 [Not refereed][Invited]
  
• VISUALIZATION OF SITE-SPECIFIC RECOMBINATION CATALYZED BY A RECOMBINASE FROM ZYGOSACCHAROMYCES-ROUXII IN ARABIDOPSIS-THALIANA

  H ONOUCHI, R NISHIHAMA, M KUDO, Y MACHIDA, C MACHIDA

  MOLECULAR & GENERAL GENETICS 247 (6) 653 - 660 0026-8925 1995 [Refereed][Not invited]
   

  Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic beta-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F-1 and F-2 progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F-1 progeny and most of the F-2 progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F-2 individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F-1 generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.
• Analysis of Plant Genes Using T-DNA Transfer from Agrobacterium.

  MACHIDA Chiyoko, ONOUCHI Hitoshi, MACHIDA Yasunori

  Nippon Nōgeikagaku Kaishi Japan Society for Bioscience, Biotechnology, and Agrochemistry 67 (4) 711 - 715 0002-1407 1993
• Onouchi H, Yokoi K, Machida C, Matsuzaki H, Oshima Y, Matsuoka K, Nakamura K, Machida Y. "Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells." Nucleic Acids Res., 19(23):6373-6378(1991)*

  1991 [Refereed][Not invited]
  

Books etc

• 植物ゲノム科学辞典

  尾之内 均 (Contributor)
  朝倉書店 2009
• RNA実験ノート

  (Contributor)
  羊土社 2008
• 生化学辞典（第４版）

  尾之内 均 (Contributor)
  東京化学同人 2007

Conference Activities & Talks

• 維管束形成を司るLONESOME HIGHWAY遺伝子の上流ORFが介する翻訳制御とmRNA分解制御  [Not invited]

  梅原俊一, 木俣薫織, 戸田智美, 遠洞弥生, 大角有里沙, 蝦名績, 内藤 哲, 尾之内均

  第59回日本植物生理学会年会  2018/03
• 核小体ストレスに応答して翻訳を制御するシロイヌナズナANAC082遺伝子の上流ORF  [Not invited]

  佐々木 駿, 工藤 凛, 渡部 俊, 大林 祝, 杉山 宗隆, 刑部 祐里子, 刑部 敬史, 内藤 哲, 尾之内 均

  第59回日本植物生理学会年会  2018/03
• リボソームの停滞を引き起こす新規被子植物uORFの同定  [Not invited]

  林憲哉, 佐々木駿, Zhihang Feng, 藤原徹, 内藤哲, 尾之内均

  第58回日本植物生理学会年会  2017/03
• ペプチド配列依存的に遺伝子発現を制御するシロイヌナズナの上流ORFの探索と制御機構の解析  [Invited]

  尾之内均, 林憲哉, 梅原俊一, 木俣薫織, 高橋広夫, 内藤哲

  日本遺伝学会第88回大会ワークショップ  2016/09
• 維管束形成を制御するLONESOME HIGHWAY遺伝子のuORFペプチドが介する翻訳制御機構  [Not invited]

  大角有里沙, 木俣薫織, 梅原俊一, 戸田智美, 遠洞弥生, 蝦名績, 内藤哲, 尾之内均

  第57回植物生理学会年会  2016/03
• リボソームアレストを引き起こす被子植物uORFの探索  [Not invited]

  林憲哉, 高橋広夫, 内藤哲, 尾之内均

  第57回植物生理学会年会  2016/03
• Bioinformatic identification method of evolutional ranges for potential functional non-coding molecules on genomes  [Not invited]

  Takahashi H, Takahashi A, Hayashi N, Satoshi N, Onouchi H

  International Meeting "Non-coding DNA and Chromosomal Integrity -toward the finding of Intermeres-"  2015/08
• リボソームアレストを引き起こす被子植物のCPuORFの探索と機能解析  [Not invited]

  林憲哉, 高橋広夫, 内藤哲, 尾之内均

  第17回RNA学会年会  2015/07
• 翻訳制御に関わる種間保存性uORFペプチドの網羅的探索・進化保存性評価手法の開発と動植物ゲノムへの応用  [Not invited]

  髙橋広夫, 髙橋アンナ, 林憲哉, 内藤哲, 尾之内均

  第17回RNA学会年会  2015/07
• 制御ペプチドをコードする二つのuORFが関与する翻訳制御機構  [Not invited]

  工藤凜, 小山博彰, 大谷美紗都, 高橋広夫, 内藤哲, 尾之内均

  第17回RNA学会年会  2015/07
• Identification of novel Arabidopsis upstream open reading frames that control translation of the main coding sequences in a peptide sequence-dependent manner  [Invited]

  ONOUCHI Hitoshi

  Frontiers in Plant RNA Research  2012/10
• 植物の uORF にコードされるペプチドが関与する翻訳制御機構の多様性  [Not invited]

  尾之内 均

  第14回日本RNA学会年会  2012/07
• uORFがコードするペプチドにより発現が制御されるシロイヌナズナ遺伝子の探索  [Not invited]

  尾之内 均

  第53回日本植物生理学会年会  2012/03
• Nascent peptide-mediated control of gene expression in Arabidopsis: Feedback regulation of methionine biosynthesis and beyond  [Invited]

  ONOUCHI Hitoshi

  12th International Congress on Amino Acids, Peptides and Protein  2011/08

MISC

• 真核生物における新生ペプチドに依存したリボソームの停止にかかる出口トンネル狭窄部位の関与:逆遺伝学と生化学によるアプローチ

  高松世大, 大橋悠文, 尾上典之, 田島陽子, 今道朋哉, 森本恭子, 米澤進哉, 尾之内均, 山下由衣, 内藤哲  日本分子生物学会年会プログラム・要旨集(Web)  42nd-  2019
• シロイヌナズナリボソームタンパク質uL4は翻訳途上の新生ポリペプチド鎖と相互作用してリボソームの翻訳を制御する

  高松世大, 大橋悠文, 尾上典之, 尾之内均, 山下由衣, 内藤哲  日本植物細胞分子生物学会大会・シンポジウム講演要旨集  36th-  2018
• シロイヌナズナリボソームタンパク質uL4は翻訳途上の新生ポリペプチド鎖と相互作用したリボソームの翻訳を制御する

  高松世大, 大橋悠文, 尾上典之, 尾之内均, 山下由衣, 内藤哲  日本植物生理学会年会(Web)  59th-  2018
• シロイヌナズナCGS1遺伝子におけるS-アデノシルメチオニンに応答した翻訳停止

  高松世大, 今道朋哉, 米澤進哉, 楠本奈央, 大橋悠文, 尾上典之, 尾之内均, 山下由衣, 内藤哲  日本分子生物学会年会プログラム・要旨集(Web)  41st-  2018
• シロイヌナズナTTM3遺伝子の上流ORFは後期促進複合体の構成因子をコードする

  垣内俊哉, 伊藤正樹, 高橋広夫, 刑部祐里子, 刑部敬史, 内藤哲, 尾之内均  日本植物生理学会年会(Web)  59th-  ROMBUNNO.P.120  2018  [Not refereed][Not invited]
  
• Identification of Arabidopsis upstream open reading frames that control gene expression in a peptide sequence-dependent manner and analysis of their regulatory mechanisms

  Hitoshi Onouchi, Noriya Hayashi, Shunichi Umehara, Kaori Kimata, Hiro Takahashi, Satoshi Naito  GENES & GENETIC SYSTEMS  91-  (6)  329  -329  2016/12  [Not refereed][Not invited]
  
• Development of identification method based on cross-sectional analysis of transcriptome database for conserved peptide uORFs and its application to animal and plant genomes

  Hiro Takahashi, Noriya Hayashi, Hitoshi Onouchi  GENES & GENETIC SYSTEMS  91-  (6)  329  -329  2016/12  [Not refereed][Not invited]
  
• Boron-dependent degradation of NIP5;1 mRNA through AUGUAA sequences, in the 5 ' UTR in Arabidopsis thaliana

  Mayuki Tanaka, Naoyuki Sotta, Kyoko Miwa, Yukako Chiba, Hitoshi Onouchi, Satoshi Naito, Toru Fujiwara  GENES & GENETIC SYSTEMS  91-  (6)  329  -329  2016/12  [Not refereed][Not invited]
  
• 翻訳とその制御

  尾之内均, 山下由衣  植物学の百科事典  620  -621  2016  [Not refereed][Not invited]
  
• P4-2-3 シロイヌナズナのホウ素輸送体、NIP5;1のホウ素に応答したmRNA蓄積の制御機構の解析(ポスター,4-2 植物の微量栄養素,2015年度京都大会)

  田中 真幸, 反田 直之, 千葉 由佳子, 尾之内 均, 内藤 哲, 藤原 徹  日本土壌肥料学会講演要旨集  (61)  70  -70  2015/09/09
• Feedback regulation of methionine biosynthesis mediated by ribosome stalling in Arabidopsis

  Yui Yamashita, Yubun Ohashi, Seidai Takamatsu, Noriyuki Onoue, Kyoko Morimoto, Yoko Tajima, Shiro Aono, Hitoshi Onouchi, Satoshi Naito  AMINO ACIDS  47-  (8)  1657  -1657  2015/08  [Not refereed][Not invited]
  
• シロイヌナズナCGS1 mRNAの翻訳停止におけるリボソーム出口トンネルの関与

  大橋悠文, 高松世大, 尾上典之, 山下由衣, 尾之内均, 内藤哲  日本植物細胞分子生物学会大会・シンポジウム講演要旨集  33rd-  2015
• シロイヌナズナCGS1遺伝子の翻訳アレストにおけるリボソーム出口トンネルの役割

  高松世大, 大橋悠文, 尾上典之, 山下由衣, 尾之内均, 内藤哲  日本RNA学会年会要旨集  17th-  2015
• シロイヌナズナCGS1mRNAの新生ペプチドによる翻訳アレストへのリボソーム出口トンネルの関与

  高松世大, 大橋悠文, 尾上典之, 山下由衣, 尾之内均, 内藤哲  日本生化学会大会(Web)  88th-  2015
• 2P-208 ESUCA : evolutionary search for upstream open reading frames with conserved amino acid sequences and its application to plant and animal genomes

  Takahashi Hiro, Takahashi Anna, Takemoto Mariko, Ebina Isao, Kimata Kaori, Igarashi Takuya, Kudo Rin, Osumi Arisa, Naito Satoshi, Onouchi Hitoshi  日本生物工学会大会講演要旨集  66-  158  -158  2014
• どう活かす他人のデータ : バイオインフォマティクス活用法(続・生物工学基礎講座-バイオよもやま話-)

  高橋 広夫, 岩川 秀和, 尾之内 均, 小島 晶子, 町田 千代子  生物工学会誌 : seibutsu-kogaku kaishi  91-  (9)  520  -525  2013
• 2P-184 Development of evolutionary search for upstream open reading frames with conserved amino acid sequences

  Takahashi Hiro, Takahashi Anna, Takemoto Mariko, Watanabe Shun, Ebina Isao, Endo Yayoi, Koyama Hiroaki, Toda Satomi, Seto Ryuta, Naito Satoshi, Onouchi Hitoshi  日本生物工学会大会講演要旨集  65-  150  -150  2013
• シロイヌナズナCGS1mRNAの翻訳停止におけるリボソーム出口トンネルの解析

  大橋悠文, 尾上典之, 尾之内均, 内藤哲  日本RNA学会年会要旨集  15th-  2013
• シロイヌナズナの低温ストレス応答に関連したmRNA制御システム

  千葉由佳子, 千葉由佳子, 峯田克彦, 平井優美, 鈴木悠也, 金谷重彦, 高橋広夫, 尾之内均, 山口淳二, 内藤哲  日本RNA学会年会要旨集  14th-  131  2012/07/18  [Not refereed][Not invited]
  
• 低温ストレス応答に関連したmRNA分解制御

  千葉由佳子, 千葉由佳子, 峯田克彦, 平井優美, 鈴木悠也, 高橋広夫, 尾之内均, 山口淳二, 内藤哲  日本植物生理学会年会要旨集  53rd-  214  2012/03/09  [Not refereed][Not invited]
  
• シロイヌナズナ細胞抽出液を用いたシロイヌナズナのシスタチオニンγ‐シンターゼmRNAの分解中間体の解析

  室田勝功, 薦田(萩原)優香, 薦田圭介, 尾之内均, 石川雅之, 内藤哲  日本植物生理学会年会要旨集  53rd-  214  2012/03/09  [Not refereed][Not invited]
  
• Arabidopsis Cell-free Extract, ACE, a New In Vitro Translation System Derived from Arabidopsis Callus Cultures (vol 52, pg 1443, 2011)

  Katsunori Murota, Yuka Hagiwara-Komoda, Keisuke Komoda, Hitoshi Onouchi, Masayuki Ishikawa, Satoshi Naito  PLANT AND CELL PHYSIOLOGY  53-  (3)  602  -602  2012/03  [Not refereed][Not invited]
  
• シロイヌナズナCGS1mRNAにおける複数のリボソームの停滞:ピューロマイシンを用いた翻訳伸長停止段階の特定

  山下由衣, 門倉嘉知, 佐竹暁子, 尾之内均, 尾之内均, 内藤哲  日本RNA学会年会要旨集  14th-  2012
• 4Fp19 Development of the BLAST-based algorithm for identification of upstream open reading frames with conserved amino acid sequences

  Takahashi Hiro, Takahashi Anna, Takemoto Mariko, Watanabe Shun, Ebina Isao, Endo Yayoi, Koyama Hiroaki, Toda tomomi, Seto Ryuta, Naito Satoshi, Onouchi Hitoshi  日本生物工学会大会講演要旨集  64-  217  -217  2012
• Nascent peptide-mediated control of gene expression in Arabidopsis: feedback regulation of methionine biosynthesis and beyond

  Hitoshi Onouchi, Isao Ebina, Mariko Takemoto, Shun Watanabe, Noriyuki Onoue, Yui Yamashita, Yayoi Endo, Hiroaki Koyama, Yoko Yamashita-Nagami, Hiro Takahashi, Satoshi Naito  AMINO ACIDS  41-  S68  -S69  2011/07  [Not refereed][Not invited]
  
• S-アデノシルメチオニンに応答したシロイヌナズナCGS1mRNAにおける翻訳伸長の一時停止とリボソーム内新生ペプチドの収縮構造

  山下由衣, 尾上典之, 尾之内均, 尾之内均, 内藤哲  日本植物細胞分子生物学会大会・シンポジウム講演要旨集  29th-  2011
• シロイヌナズナの試験管内翻訳系を用いた5’‐3’エキソヌクレアーゼAtXRN4による5’キャップ構造を持たないmRNAの分解に関する解析

  室田勝功, 薦田(萩原)優香, 薦田圭介, 尾之内均, 石川雅之, 内藤哲  日本分子生物学会年会プログラム・要旨集(Web)  34th-  4T8A-4 (WEB ONLY)  2011  [Not refereed][Not invited]
  
• CGS1遺伝子発現制御における翻訳伸長停止時のリボソーム出口トンネル内部の変化と新生ペプチドの二次構造形成

  尾上典之, 山下由衣, 尾之内均, 内藤哲  日本RNA学会年会要旨集  12th-  2010
• シロイヌナズナCGS1 mRNAの翻訳停止における新生ペプチドのフォールディングの解析

  山下由衣, 尾上典之, 尾之内均, 内藤哲  日本蛋白質科学会年会プログラム・要旨集  10th-  2010
• 新生ペプチドによる翻訳伸長の停止と共役したmRNA分解

  原口雄飛, 室田勝功, 尾上典之, 長谷川傑, 川崎大輔, 小松陽平, 永見陽子, 門倉嘉知, 平田健, 中嶋一恵, 中本真理, 尾之内均, 尾之内均, 内藤哲  日本植物生理学会年会要旨集  50th-  2009
• リボソームの翻訳制御:シロイヌナズナCGS1遺伝子の新生ペプチドとS-アデノシルメチオニンによる翻訳伸長停止機構の解析

  尾上典之, 古家由理, 竹原好美, 尾之内均, 尾之内均, 内藤哲  日本RNA学会年会要旨集  11th-  2009
• CGS1遺伝子の新生ペプチドとS-アデノシルメチオニンによるリボソームの翻訳アレスト機構

  尾上典之, 長谷川傑, 中嶋一恵, 平松俊行, 細谷孝充, 鈴木正昭, 大野敏, 横川隆志, 西川一八, 尾之内均, 尾之内均, 内藤哲  日本植物生理学会年会要旨集  50th-  2009
• 新生ペプチドのフォールディングがシロイヌナズナCGS1 mRNAの翻訳停止に与える影響

  山下由衣, 尾上典之, 尾之内均, 内藤哲  日本分子生物学会年会講演要旨集  32nd-  (Vol.3)  2009
• AtCGS1遺伝子の新生ペプチドとS-アデノシルメチオニンによるリボソームの翻訳アレスト機構

  尾上典之, 本間翔, 森本恭子, 竹原好美, 平松俊行, 細谷孝充, 鈴木正昭, 大野敏, 横川隆志, 西川一八, 尾之内均, 内藤哲  日本RNA学会年会要旨集  10th-  2008
• リボソームから見たシスタチオニンγシンターゼ遺伝子の転写後制御機構

  長谷川傑, 中嶋一恵, 室田勝功, 尾上典之, 尾之内均, 内藤哲  生化学  2008
• シロイヌナズナCGS1遺伝子の新生ペプチドによるリボソームの翻訳アレスト-リボソームexit tunnelの関与-

  尾上典之, 中嶋一恵, 室田勝功, 長谷川傑, 尾之内均, 内藤哲  生化学  2007
• Translation elongation arrest and coupled mRNA decay mechanisms for regulation of the arabidopsis CGS1 gene that encodes a key enzyme in methionine biosynthesis

  Hitoshi Onouchi, Yuhi Haraguchi, Kazue Nakajima, Katsunori Murota, Yoshitomo Kadokura, Daisuke Kawasaki, Ken Hirata, Yoko Nagami, Satoshi Naito  PLANT AND CELL PHYSIOLOGY  48-  S11  -S11  2007  [Not refereed][Not invited]
  
• Post-transcriptional autoregulation of CGS gene expression: mRNA cleavages coupled with translation elongation arrest in the CGS1 gene of Arabidopsis thaliana

  Yuhi Haraguchi, Ryoko Sakurai, Yoshitomo Kadokura, Hitoshi Onouchi, Satoshi Naito  PLANT AND CELL PHYSIOLOGY  48-  S36  -S36  2007  [Not refereed][Not invited]
  
• Post-transcriptional autoregulation of CGS gene expression: Conservation among plant species

  Noriyuki Onoue, Nobuhiro Naga, Daisuke Kawasaki, Hitoshi Onouchi, Satoshi Naito  PLANT AND CELL PHYSIOLOGY  48-  S213  -S213  2007  [Not refereed][Not invited]
  
• Post-transcriptional autoregulation of CGS gene expression: Analysis of stalled ribosomes

  Yoshitomo Kadokura, Yuhi Haraguchi, Yoko Nagarui, Hitoshi Onouchi, Satoshi Naito  PLANT AND CELL PHYSIOLOGY  48-  S214  -S214  2007  [Not refereed][Not invited]
  
• シロイヌナズナ細胞抽出液を用いたシロイヌナズナのシスタチオニンγ‐シンターゼにおける転写後制御機構の解析

  室田勝功, 薦田圭介, 尾之内均, 石川雅之, 内藤哲  日本植物生理学会年会要旨集  47th-  193  2006/03  [Not refereed][Not invited]
  
• シスタチオニンγ-シンターゼ遺伝子の転写後制御:ヒメツリガネゴケの遺伝子配列を用いた解析

  川崎大輔, 尾上典之, 尾之内均, 内藤哲  日本植物生理学会年会要旨集  47th-  2006
• Posttranscriptional regulation of Arabidopsis cystathionine gamma-synthase

  R Sakurai, N Nagao, S Homma, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  47-  S114  -S114  2006  [Not refereed][Not invited]
  
• mRNA degradation coupled with ribosorne stalling ir the CGS1 gene of Arabidopsis thaliana

  Y Haraguchi, Y Kadokura, M Nakamoto, Y Nagami, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  47-  S114  -S114  2006  [Not refereed][Not invited]
  
• Posttranscriptional regulation of' cystathionine y-synthase gene of the moss Physcomitrella patens

  D Kawasaki, N Onoue, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  47-  S113  -S113  2006  [Not refereed][Not invited]
  
• Analysis of posttranscriptional regulation of Arabidopsis cystathionine gamma-synthase in Arabidopsis cell extract

  K Murota, K Komoda, H Onouchi, M Ishikawa, S Naito  PLANT AND CELL PHYSIOLOGY  47-  S113  -S113  2006  [Not refereed][Not invited]
  
• シロイヌナズナのシスタチオニンγ‐シンターゼにおける転写後制御:シロイヌナズナ細胞抽出液を用いた試験管内翻訳系の確立

  室田勝功, 薦田圭介, 尾之内均, 石川雅之, 内藤哲  日本植物生理学会年会要旨集  46th-  223  2005/03  [Not refereed][Not invited]
  
• Posttranscriptional regulation of Arabidopsis cystathionine gamma-synthase: Establishment of in vitro translation system derived from Arabidopsis cell extract

  K Murota, K Komoda, H Onouchi, M Ishikawa, S Naito  PLANT AND CELL PHYSIOLOGY  46-  S142  -S142  2005  [Not refereed][Not invited]
  
• Post-transcriptional regulation of Arabidopsis cystathionine gamma-synthase: Analysis of mutant involved in the regulation

  K Terasawa, A Suzuki, Y Kawai, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  46-  S243  -S243  2005  [Not refereed][Not invited]
  
• Posttranscriptional regulation of Arabidopsis cystathionine gamma-synthase: In vitro analysis of translation elongation arrest in response to S-adenosylmethionine

  Y Nagami, Y Haraguchi, M Nakamoto, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  46-  S243  -S243  2005  [Not refereed][Not invited]
  
• Posttranscriptional regulation of Arabidopsis cystathionine gamma-synthase: Analysis of mRNA degradation mechanism coupled with translational pausing in vitro

  Y Haraguchi, R Sakurai, M Nakamoto, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  46-  S243  -S243  2005  [Not refereed][Not invited]
  
• Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in Arabidopsis.

  Tsuyoshi Mizoguchi, Louisa Wright, Sumire Fujiwara, Frédéric Cremer, Karen Lee, Hitoshi Onouchi, Aidyn Mouradov, Sarah Fowler, Hiroshi Kamada, Joanna Putterill, George Coupland  The Plant cell  17-  (8)  2255  -2270  2005  [Not refereed][Not invited]
   

  The circadian clock acts as the timekeeping mechanism in photoperiodism. In Arabidopsis thaliana, a circadian clock-controlled flowering pathway comprising the genes GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) promotes flowering specifically under long days. Within this pathway, GI regulates circadian rhythms and flowering and acts earlier in the hierarchy than CO and FT, suggesting that GI might regulate flowering indirectly by affecting the control of circadian rhythms. We studied the relationship between the roles of GI in flowering and the circadian clock using late elongated hypocotyl circadian clock associated1 double mutants, which are impaired in circadian clock function, plants overexpressing GI (35S:GI), and gi mutants. These experiments demonstrated that GI acts between the circadian oscillator and CO to promote flowering by increasing CO and FT mRNA abundance. In addition, circadian rhythms in expression of genes that do not control flowering are altered in 35S:GI and gi mutant plants under continuous light and continuous darkness, and the phase of expression of these genes is changed under diurnal cycles. Therefore, GI plays a general role in controlling circadian rhythms, and this is different from its effect on the amplitude of expression of CO and FT. Functional GI:green fluorescent protein is localized to the nucleus in transgenic Arabidopsis plants, supporting the idea that GI regulates flowering in the nucleus. We propose that the effect of GI on flowering is not an indirect effect of its role in circadian clock regulation, but rather that GI also acts in the nucleus to more directly promote the expression of flowering-time genes.
• Identification of the region required for translocation of cystathionine gamma-synthase to chloroplast

  T Sugiyama, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  45-  S222  -S222  2004  [Not refereed][Not invited]
  
• Genetic identification of factors that are involved in the post-transcriptional regulation of the cystathionine gamma-synthase gene expression

  A Suzuki, K Terasawa, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  45-  S224  -S224  2004  [Not refereed][Not invited]
  
• In vitro analysis of the necessary region for S-adenosylmethionine-induced translation arrest of Arabidopsis cystathionine gamma-synthase mRNA

  Y Nagami, M Nakamoto, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  45-  S176  -S176  2004  [Not refereed][Not invited]
  
• Autoregulation of mRNA stability of Arabidopsis cystathionine Gamma-synthase: Translation arrest induced by S-adenosylmethionine

  H Onouchi, M Nakamoto, Y Nagami, Y Chiba, S Naito  PLANT AND CELL PHYSIOLOGY  45-  S123  -S123  2004  [Not refereed][Not invited]
  
• Post-transcriptional regulation of Arabidopsis thaliana cystathionine gamma-synthase: Analysis of the mRNA degradation in the in vitro translation system

  R Sakurai, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  45-  S123  -S123  2004  [Not refereed][Not invited]
  
• Autoregulation of mRNA Stability of Arabidopsis Cystathionine gamma-Synthase : Analysis of the mRNA degradation in the in vitro translation system

  R Sakurai, Y Nishimura, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  44-  S52  -S52  2003  [Not refereed][Not invited]
  
• Isolation of Arabidopsis mutants involved in the posttranscriptional regulation of the cystathionine gamma-synthase gene

  A Suzuki, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  44-  S52  -S52  2003  [Not refereed][Not invited]
  
• The autoregulation of cystathionine gamma-synthase mRNA stability : Analysis of intermediates of translation products using the in vitro translation system

  M Nakamoto, Y Nishimura, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  44-  S52  -S52  2003  [Not refereed][Not invited]
  
• シロイヌナズナのシスタチオニンγ‐シンターゼ遺伝子におけるmRNA安定性の自己制御

  尾之内均, 千葉由佳子, 吉野道子, 内藤哲  生化学  74-  (8)  698  2002/08/25  [Not refereed][Not invited]
  
• Autoregulation of mRNA stability of Arabidopsis cystathionine gamma-synthase

  H Onouchi, Y Chiba, M Yoshino, R Sakurai, Lambein, I, S Naito  PLANT AND CELL PHYSIOLOGY  43-  S48  -S48  2002  [Not refereed][Not invited]
  
• Genetic identification of factors involved in cystathionine gamma-synthase mRNA stability

  A Suzuki, Lambein, I, Y Shirata, Y Chiba, H Onouchi, S Naito  PLANT AND CELL PHYSIOLOGY  43-  S47  -S47  2002  [Not refereed][Not invited]
  
• S-ADENOSYLMETHIONINE IS A LIKELY EFFECTOR FOR THE FEEDBACK REGULATION OF mRNA STABILITY OF CYSTATHIONINE γ-SYNTHASE :

  ONOUCHI Hitoshi, SAKURAI Ryoko, ISHIKAWA Mari, OMINATO Kimihiro, CHIBA Yukako, NAITO Satoshi  Plant and cell physiology  42-  s191  2001
• Study of cystathionine γ-synthase mRNA stability in Arabidopsis callus and tobacco BY2 suspension cultures :

  Lambein Ingrid, Chiba Yukako, Kanno Tatsuo, Onouchi Hitoshi, Naito Satoshi  Plant and cell physiology  42-  s191  2001
• Analysis of autoregulation of cystathionine γ-synthase mRNA stability in transgenic Arabidopsis thaliana :

  Suzuki Akinori, Shirata Yukie, Chiba Yukako, Onouchi Hitoshi, Naito Satoshi  Plant and cell physiology  42-  s191  2001
• Effect of the WUS (JAM) gene on the regulation of leaf identity in Arabidopsis thaliana :

  Hamada Susumu, Kudo Mitsuko, Onouchi Hitoshi, Liu Yao Guang, Shibata Daisuke, Machida Chiyoko, Machida Yasunori  Plant and cell physiology  42-  s54  2001
• SUPPRESSOR MUTAGENESIS OF OVER-EXPRESSION OF THE CONSTANS GENE IN ARABIDOPSIS :

  ONOUCHI Hitoshi, SAMACH Alon, IGENO Isabel, PERILLEUX Clair, GOLD Scott E., YANOFSKY Martin F., COUPLAND George  Plant and cell physiology  41-  s127  2000
• The ALE1 gene is involved in endosperm development and epidermal differentiation in arabidopsis :

  TANAKA Hirokazu, ONOUCHI Hitoshi, WATANABE Daisuke, TSUKAYA Hirokazu, MACHIDA Chiyoko, MACHIDA Yasunori  Plant and cell physiology  41-  s206  2000
• The transposition pattern of the Ac element and its use for targeted transposition in Arabidopsis thaliana

  Machida Y, Onouchi H, Tanaka H, Hamada S, Ishikawa T, Semiarti E, Iwakawa H, Nomura K, Machida C  The 13th Symposium on Plant Biotechnology  13-  11  -15  1999  [Not refereed][Not invited]
  
• The ABNORMAL LEAF-SHAPE genes regulate epidermal differentiation in Arbidopsis thaliana

  TANAKA H., ONOUCHI H., WATANABE D., TSUKAYA H., MACHIDA C., MACHIDA Y.  日本分子生物学会年会プログラム・講演要旨集  21-  635  -635  1998/12/01
• The role of the ASYMMETRIC LEAVES 2(AS2)gene of Arabidopsis thaliana in leaf development

  SEMIARTI Endang, ONOUCHI Hitoshi, TSUKAYA Hirokazu, MACHIDA Chiyoko, MACHIDA Yasunori  日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan  62-  97  -97  1998/09/01
• シロイヌナズナの葉の表皮細胞分化の分子遺伝学的解析

  田中 博和, 尾之内 均, 渡辺 大輔, 塚谷 裕一, 町田 千代子, 町田 泰則  日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan  62-  97  -97  1998/09/01
• Isolation and Characterization of the JUVENILE-LEAF-LESS AND MISSHAPEN-APICAL-MERISTEM (JAM) Gene of Arabidopsis thaliana

  HAMADA Susumu, KUDO Mitsuko, ONOUCHI Hitoshi, MACHIDA Chiyoko, MACHIDA Yasunori  Plant and cell physiology  39-  S60  -S60  1998/05
• Acトランスポゾンタギングによる葉の形態異常変異株aleの単離と解析

  田中 博和, 尾之内 均, 町田 千代子, 町田 泰則  日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan  61-  288  -288  1997/09
• 幼若葉が欠損するシロイヌナズナjam変異体の単離と解析

  浜田 進, 工藤 光子, 尾之内 均, 町田 千代子, 町田 泰則  日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan  61-  289  -289  1997/09  [Not refereed][Not invited]
  
• A transposon insertion mutant of the NADPH oxidase gp91-phox gene homolog in Arabidopsis

  ONOUCHI Hitoshi, MACHIDA Chiyoko, MACHIDA Yasunori  Plant and cell physiology  38-  s70  1997/03
• シロイヌナズナのNADPHオキシダーゼ系gp91-phox相同遺伝子欠損変異株の解析

  尾之内 均, 町田 千代子, 町田 泰則  日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan  60-  247  -247  1996/10
• トランスポソンAcの転移マップを利用した遺伝子クローニング

  SEMIARTI Endang, 尾之内 均, 浜田 進, 小泉 順, 町田 千代子, 町田 泰則  日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan  60-  246  -246  1996/10  [Not refereed][Not invited]
  
• シロイヌナズナのNADPHオキシダーゼ系gp91-phox相同遺伝子欠損変異株の解析

  尾之内 均, 町田 千代子, 町田 泰則  日本分子生物学会年会プログラム・講演要旨集  19-  305  -305  1996/08/01
• 部位特異的組換えを利用したシロイヌナズナ染色体操作法の開発

  工藤 光子, 小泉 順, 尾之内 均, 町田 千代子, 町田 泰則  日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan  59-  302  -302  1995/09/24
• トランスポゾンタギング法により分離されたシロイヌナズナ形態異常変異株の解析

  尾之内 均, 小泉 順, 工藤 光子, 町田 千代子, 町田 泰則  日本植物学会大会研究発表記録 = Proceedings of the annual meeting of the Botanical Society of Japan  59-  303  -303  1995/09/24
• ISOLATION OF ARABIDOPSIS MUTANTS USINGTHE Ac TRANSPOSABLE EREMENTS AND THE SITE-SPECIFIC RECOMBINATIONSYSTEM

  ONOUCHI Hitoshi, KOIZUMI Jun, KUDO Mitsuko, MACHIDA Chiyoko, MACHIDA Yasunori  Plant and cell physiology  36-  S62  1995/03
• 酵母でみられる部位特異的組換え反応のシロイヌナズナ植物体での発現

  尾之内均, 西浜竜一, 町田千代子, 町田泰則  日本分子生物学会年会プログラム・講演要旨集  16th-  320  1993/11  [Not refereed][Not invited]
  
• Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells.

  ONOUCHI H.  Nucl. Acids Res.  19-  6373  -6378  1991

Research Grants & Projects

• 上流ORFが関与する細胞環境感知機構の解明とゲノム育種への応用

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2019/04 -2023/03 

  Author : 尾之内 均

   

  研究代表者のグループはこれまでに、進化的に保存された配列を持つシロイヌナズナのuORFの中から、下流の主要ORFの翻訳に影響を与えるものを同定した。その中には、AUG以外の開始コドンで始まるuORF（非AUG開始型uORF）が４つ含まれる。それらの非AUG開始型uORFの１つを持つ遺伝子はストレスに応答して転写が促進されるが、その遺伝子の発現がポリアミンに応答して翻訳段階で抑制され、その翻訳制御に非AUG開始型uORFが関与することを本年度の研究において見出した。さらに、非AUG開始型uORFの開始コドンの下流に存在するRNA二次構造がこの翻訳制御に必要であることを明らかにした。この翻訳制御は、植物細胞内でポリアミンが過剰に蓄積した場合に、ポリアミン濃度を適切な範囲内に維持する役割を担っていると考えられる。 他の３つの非AUG開始型uORFのうちの２つでは、uORFのペプチド配列が植物で広く保存されている。これらの２つのuORFのペプチド配列の中で翻訳制御に重要なアミノ酸残基の同定を行い、それぞれC末端側の8アミノ酸および15アミノ酸の領域が重要であることを見出した。 また、これまでに生理学的役割を明らかにしたAUG開始型uORF及び非AUG開始型uORFについて、それらが介する翻訳制御に関与するトランス因子を同定するために、トランス因子として予想される候補タンパク質が欠損したシロイヌナズナ変異体をゲノム編集技術を用いて作出した。
• ゲノム編集による5´非翻訳領域の改変によりタンパク質発現量を増加させる技術の開発

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2019/06 -2022/03 

  Author : 尾之内 均

   

  近年、ゲノム編集技術が作物の品種改良に活用されるようになってきている。しかし、遺伝子組換えにあたらない範囲でのゲノム編集技術の作物育種への利用はほとんど遺伝子破壊に限られており、転写や翻訳を抑制する配列が同定されている一部の遺伝子以外は、外来導入DNAを残さずに特定のタンパク質の発現量を増加させることは現状では困難である。そこで本研究では、より多くの作物遺伝子において遺伝子組換えに該当せずにタンパク質の発現量を増加させることを可能にするために、mRNAの翻訳効率に影響を与える5´非翻訳領域配列をゲノム編集を用いて改変することでタンパク質発現量を増加させる方法の開発を目指す。 2019年度の研究において、CRISPR/Cas9システムを用いてイネの遺伝子の5´非翻訳領域に変異を導入し、5´非翻訳領域に様々な変異が生じた変異体を分離した。2020年度の研究では、それらの変異がタンパク質発現量にどのような影響を与えるかを調べるために、変異が生じた5´非翻訳領域をクローニングしてルシフェラーゼ遺伝子につなぎ、一過的発現解析により変異の影響を検討した。その結果、5´非翻訳領域に生じた変異によりルシフェラーゼの発現量が様々なレベルに変化し、それらの変異の中でルシフェラーゼの発現量を増加させるものも見つかった。さらに、それらの変異のヘテロ接合体の次世代の個体の中から、ゲノム編集用コンストラクトが除かれた個体や変異をホモ接合体として持つ個体をそれぞれ分離した。
• Elucidation of translation-arresting sequences in genomes using computer-based intersectional analyses of life science databases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018 -2020 

  Author : 高橋 広夫, 伊藤 素行, 尾之内 均, 佐々木 博己, 町田 泰則

   

  Upstream Open Reading Frame (uORF)は、AUG以外の開始コドンから翻訳が始まる非AUG開始型uORFが存在する。そのような非AUG開始型uORFの中で翻訳アレストを起こすものを同定するために、ESUCAパイプラインを改変して進化的に保存された配列を持つ非AUG開始型uORFを動植物のゲノムから探索した。その結果、そのような非AUG開始型uORFが14個同定された。それらの中から、主要ORFの翻訳制御に関与するものを一過的発現解析により4個同定した。また、胃がん発症機構解明のため、本年度は、高度遺伝子増幅領域内の遺伝子発現がプロモーターのメチル化と関連しているか、メチロームデータとの3層オミクス統合解析を行い、遺伝子の発現量とメチル化の間に、強い相関があることを見出した。遺伝子増幅に伴って過剰発現する遺伝子にはGATA6のような新たながん遺伝子が多く、発現が低下している遺伝子にはGSDMAのようながん抑制遺伝子が含まれていた。これらにより、3層オミクス統合解析によって、腹膜転移性胃がんの増幅遺伝子の全体像が明らかになった。
• uORFペプチドによる翻訳制御の新たな役割と機構の解明

  文部科学省：科学研究費補助金（基盤研究(C)）

  Date (from‐to) : 2016/04 -2018/03 

  Author : 尾之内 均
• uORFにコードされるペプチドによる翻訳制御の植物における多様な役割の解明

  文部科学省：科学研究費補助金（基盤研究(C)）

  Date (from‐to) : 2013/04 -2015/04 

  Author : 尾之内 均
• 植物アミノ酸代謝のオミクス統合解析による解明

  科学技術振興事業団：戦略的基礎研究推進事業 (CREST)

  Date (from‐to) : 2007/09 -2013/03 

  Author : 平井優美
• 植物における翻訳アレストにより誘導されるmRNA分解機構

  文部科学省：科学研究費補助金(基盤研究(C))

  Date (from‐to) : 2009 -2011 

  Author : 尾之内 均

   

  植物において翻訳アレストとmRNA分解の共役の普遍的性について検討する目的で、新生ペプチドによって翻訳アレストが引き起こされる可能性が考えられるuORFの系を用いて解析を行った。前年度に新たに同定したペプチド配列依存的に翻訳抑制を起こす2つのuORFについて、試験管内翻訳系において翻訳アレストが起こるかの検討を行ったが、通常の翻訳条件では翻訳アレストは観察されなかった。細胞内に存在する何らかの代謝産物が翻訳アレストを誘導するために必要である可能性も考えられるため、今後、どのような条件で翻訳抑制が誘導されるかについての詳細な検討を行う。また、それらのuORFにおいて翻訳抑制と共役したmRNA分解の促進が見られるかを検討するために、各遺伝子の翻訳抑制に必要な野生型配列と翻訳抑制が起こらない変異型配列をそれぞれ導入した形質転換植物を作出した。次年度に、これらの植物を用いて、このuORFによる翻訳抑制と共役してmRNA分解が誘導されるかを検討する予定である。今年度に新たに同定したペプチド配列依存的に翻訳抑制を起こすuORFの中に、翻訳阻害剤であるシクロヘキシミド処理によってmRNA蓄積量が増加するものを見いだした。このuORFでは、翻訳と共役してmRNA分解が起こる可能性が考えられる。このuORFについて、アミノ酸置換解析を行うことによって、uORFにコードされるペプチド中の翻訳抑制...
• 植物の発生分化におけるuORFによる翻訳制御機構

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2009 -2010 

  Author : 尾之内 均, 内藤 哲

   

  これまでに同定したシロイヌナズナのuORFペプチドが関与する発現制御の中で、複数のuORFが関与するものを2つ見いだした。それらの系では、ペプチド配列依存的に翻訳を抑制するuORFのさらに上流に別のuORFが存在し、上流側のuORFを削除した場合に主要なORFの発現が強く抑制され、その効果は下流側のuORFのアミノ酸配列に依存することが示された。これらのことから、上流側のuORFは下流のuORFの翻訳効率を調節する働きがあることが示唆された。また、シロイヌナズナにおいてアミノ酸配列依存的な発現制御がみられなかったuORFの中に、アブラナ科植物では他の科の植物と比べてアミノ酸配列の保存性が低いものがいくつか見いだされた。そのうちの1つの遺伝子について、アミノ酸配列がより高度に保存されているポプラのオルソログのuORFを用いて、ペプチド配列依存的な発現制御がみられるかを検討した。その結果、ポプラのオルソログのuORFはアミノ酸配列依存的に主要なORFの発現を抑制することを見いだした。この遺伝子の5'UTRにも複数のuORFが存在し、ポプラにおいてペプチド配列依存的な発現制御がみられた下流側のuORFだけでなく、上流側のuORFのアミノ酸配列も双子葉植物間で高度に保存されている。上流側のuORFの役割について解析したところ、シロイヌナズナとポプラのいずれにおいても上流側のuORFは...
• 新生ペプチドによる翻訳アレストと連携したRNA制御機構

  文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))

  Date (from‐to) : 2009 -2010 

  Author : 尾之内 均

   

  前年度にバイオインフォマティクス解析によって同定した植物間で保存されたアミノ酸配列を持つ14個のuORFの中から、uORFにコードされるペプチドが実際に翻訳制御に関与するものを同定する目的で、フレームシフト変異やアミノ酸置換によってアミノ酸配列を変化させ場合の下流のレポーター遺伝子の発現への影響を検討した。シロイヌナズナ培養細胞MM2dから調製したプロトプラストを用いた一過的発現解析の結果、アミノ酸配列依存的に下流ORFの発現に影響を与えるuORFを新たに5つ同定した。新たに同定したuORFについて、発現制御に重要な領域を同定するために、アミノ酸置換解析を行った。制御に重要な領域のアミノ酸配列に特に共通性はみられなかったが、C末端の15-20残基が発現制御に重要であるという傾向がみられた。昨年度の解析により、LONESOME HIGHWAY(LHW)遺伝子のuORFペプチドによる発現制御にはuORFの終止コドンが必要であるのに対し、ANAC082遺伝子のuORFペプチドによる発現制御は終止コドンに依存しないことが示された。今年度、新たに同定したuORFについても発現制御の終止コドン依存性を検討したところ、そのうちの一つのuORFでは発現制御が部分的に終止コドンに依存することが示された。これらの結果から、遺伝子によってuORFペプチドによる発現制御のメカニズムに違いがあることが...
• 葉の形態形成におけるリボソームによる制御

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2008 -2008 

  Author : 尾之内 均, 内藤 哲

   

  シロイヌナズナのリボソームタンパク質RRL17の変異が葉の形態形成に影響を与える原因を明らかにするために、まずrp117変異の影響によってリボソームの翻訳活性が低下した可能性を検討した。そのために、いくつかの遺伝子についてポリソームプロファイル解析を行った。その結果、いずれの遺伝子についても野生型株とrp117変異株の間で顕著な差はみられず、rp117変異はリボソームの翻訳活性に大きな影響を与えないことが示唆された。また、リボソームタンパク質の変異の新生ペプチドによる制御への影響と葉の形態異常との関連について調べる目的で、rp117b変異株と類似の葉の形態異常を示すrp14d変異株において、CGS1遺伝子の転写後制御への影響を解析した。その結果、rp117b変異とは異なり、rp14d変異はCG51遺伝子の転写後制御に影響を与えないことが示された。このことから、リボソームタンパク質変異株の葉の形態異常は、新生ペプチドによる制御への影響によるのではなく、リボソームの変異による他の影響によるものであることが示唆された。また、形態形成に関わる遺伝子のuORF配列による下流ORFの翻訳への影響を検討し、いくつかの遺伝子においてuORFの配列が下流ORFの翻訳制御に関与することを見いだした。そのうちの一つの遺伝子は、uORFペプチドによる翻訳抑制と選択的スプライシングの組み合わせにより制...
• Molecular genetic analysis and screen for nascent-peptide mediated regulation in Arabidopsis

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))

  Date (from‐to) : 2006 -2007 

  Author : Hitoshi NOUCHI

   

  In the course of study on the posttranscriptional regulation of the Arabidopsis CGS1 gene, which encodes the key enzyme of methionine biosynthesis, we have found that the nascent CGS1 peptide mediates its own translation elongation arrest in response to S-adenosylmethionine, a metabolite of methionine. Although several examples of nascent peptide-mediated ribosomal regulations have been reported in eukaryotes, all other regulatory nascent peptides are encoded by upstream ORFs (uORF) located in the 5'UTR, except for the CGS1 gene. In these systems, uORF-encoded peptides cause ribosome stalli...
• Molecular mechanism of translation arrest and mRNA degradation in cystathionine gamma-synthase, the key-step enzyme of methionine biosynthesis.

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2004 -2006 

  Author : Satoshi NAITO, 尾之内 均

   

  Cystathionine gamma-synthase (CGS) of Arabidopsis catalyzes the key step in methionine biosynthesis. Unlike many of the key-step enzymes in biosynthetic pathways, CGS is not an allosteric enzyme. Expression of CGS is feedback-regulated at the step of mRNA degradation and S-adenosylmethionine (SAM) acts as the effector. Upon mRNA degradation, 5'-truncated RNA species are formed as degradation intermediates. A short stretch of amino acids, termed the MTO1 region, located within the first exon-coding region of CGS itself, functions as the cis element in this regulation. CGS regulation occurs d...
• 翻訳伸長段階の制御を網羅的に検索する方法の開発

  文部科学省：科学研究費補助金(若手研究(B))

  Date (from‐to) : 2004 -2005 

  Author : 尾之内 均

   

  翻訳伸長段階の制御を検索するための方法として、翻訳の伸長が途中で停止することによってリボソームが5'側に偏在するようなmRNAを検索する方法の開発を試みた。そのために、ポリソームをマイクロコッカルヌクレアーゼで部分消化し、そのあとショ糖密度勾配遠心分離を行うことによってリボソームのmRNA上の偏在を検出することを計画した。しかし、翻訳伸長停止によってリボソームが偏在することが示されているシロイヌナズナのシスタチオニンγ-シンターゼ(CGS)mRNAにおいても、上述のような方法で顕著なリボソームの偏在は検出されなかった。したがって、この方法で翻訳伸長が途中で停止したmRNAを検索することは困難であると考えられたため、別の方法として翻訳停止したリボソームと翻訳伸長中のリボソームの翻訳阻害剤に対する感受性の違いを利用する方法を考えた。そのための予備実験として、CGSmRNA上で翻訳停止したリボソームの様々な翻訳伸長阻害剤に対する感受性を調べた。その結果、通常ではピューロマイシン処理後5分以内に大部分のポリペプチドがリボソームから解離するのに対し、CGS mRNA上で翻訳停止したリボソームではピューロマイシン処理30分後も半分近いポリペプチドが解離せずに残った。このようなピューロマイシンに対する感受性の違いを利用して、翻訳伸長停止を起こしたmRNAの検索を進めている。また、原核生物で...
• RNaseによるRNA分解制御

  文部科学省：科学研究費補助金(特定領域研究)

  Date (from‐to) : 2002 -2004 

  Author : 正木 春彦, 尾之内 均

   

  <代表者>1.コリシンDのtRNase高活性ドメインをC末端103残基(D-CRD595)に特定し、D-CRD595単独およびImmDとの複合体の構造をX線結晶解析で決定し、先にImmDとの複合体構造を決定していたC末端94残基(D-CRD604)と構造と活性を比較した。D-CRD595に比べD-CRD604はN末端9残基を欠き、活性を大きく損なうが、ImmD複合体構造はほとんど同一なので、9残基の特異的重要性が示唆された。ImmDはD-CRD595の活性中心に結合し、またImmDの結合前後でD-CRD595は構造をほとんど変えていなかった。2.D-CRD595領域のさまざまな点変異体を作製して各残基の寄与を評価した。予想に反しN末端9アミノ酸は個別には重要でなかった。Hisと二つのLysが触媒中心と考えられ、先のコリシンE5とも異なる新しいRNase反応機構が推定できた。3.D-CRD遺伝子を出芽酵母に導入し、E5-CRDの場合と同様に、CRDの誘導発現に伴ってコロニー形成が停止した。しかし、大腸菌と異なり、tRNA感受性は同等なのに殺菌的でなく静菌的に見え、細胞毒としての作用が細菌と真核細胞とで異なる可能性が生じた。<分担者>1.植物のメチオニン生合成系の鍵酵素であるシスタチオニンγ-シンターゼ(CGS)の遺伝子発現制御では、S-アデノシルメチオニンに応答して翻訳伸長が停...
• Molecular genetic studies of control of mRNA stability in the gene for the key enzyme of methionine biosynthesis

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2001 -2003 

  Author : Satoshi NAITO, 尾之内 均

   

  Expression of the gene for cystathionine gamma-synthase (CGS), the key enzyme of methionine biosynthesis in higher plants is feedback-regulated at the step of mRNA stability and the amino acid sequence in the exon 1 of CGS itself is involved in this regulation. In order to identity the factors that are involved in this regulation, a chimeric gene carrying green fluorescent protein gene (GFP) fused to the exon 1 of CGS gene and placed under the control of a cauliflower mosaic virus 35S promoter was constructed and introduced into wild-type Arabidopsis. The transgenic line termed GFPc4 was us...
• 分裂組織からの器官分化過程におけるシスタチオニンγ-シンターゼmRNA安定性制御

  文部科学省：科学研究費補助金(特定領域研究(A))

  Date (from‐to) : 2001 -2001 

  Author : 尾之内 均, 内藤 哲

   

  シスタチオニンγ-シンターゼ(CGS) mRNAの安定性制御機構を解明する目的で、この制御のエフェクター分子の同定を行った。小麦胚芽由来のin vitro翻訳系を用いたアッセイによって、メチオニンの代謝産物についてエフェクターとしての効果を調べたところ、S-アデノシルメチオニン(SAM)のみが効果を示し、SAMがCGS mRNA安定性制御のエフェクター分子であることが強く示唆された。また、SAMを基質とするメチル基転位酵素の競争阻害剤であるS-アデノシルホモシステインは、SAMの効果を阻害しなかった。このことから、SAMの効果はメチル基転位反応によるものではないことが示唆された。また、SAMのアナログについても、同じin vitroアッセイを用いて、CGS mRNA安定性制御のエフェクターとしての効果を調べた。その結果、SAMのメチル基をエチル基に置換したS-アデノシルエチオニン(SAE)も、SAMと同様の効果を示した。一方、SAMからメチル基を除いたS-アデノシルホモシステインは効果を示さなかった。したがって、SAMとSAEに共通する構造的特徴あるいは硫黄原子にアルキル基が付加することによって生じる正電荷が重要である可能性が考えられる。また、in vitro翻訳系を用いて、翻訳反応後のRNA分解中間体の解析を行った。これまでにin vivoの実験系でみられたのと同様の5'側...
• 植物における遺伝子発現の転写後制御
• Post-transcriptional regulation of gene expression in plant

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