九州大学-研究者情報 [野島孝之 (准教授) 生体防御医学研究所附属システム免疫学統合研究センター]

1.	Chihiro Nakayama, Yasukazu Daigaku, Yuki Aoi, Qi Fang, Hiroshi Kimura, Ali Shilatifard, Michael Tellier, Takayuki Nojima , NELF coordinates Pol II transcription termination and DNA replication initiation, BioRxiv [Preprint], https://doi.org/10.1101/2024.01.31.578294 , 2024.01.
2.	Nojima, Takayuki; Proudfoot, Nick J., Mechanisms of lncRNA biogenesis as revealed by nascent transcriptomics, NATURE REVIEWS MOLECULAR CELL BIOLOGY, 10.1038/s41580-021-00447-6, 23, 6, 389-406, 2022.06.
3.	Iannone Camilla, Kainov Yaroslav; Zhuravskaya Anna, Hamid Fursham, Nojima, Takayuki, Makeyev Eugene V, PTBP1-activated co-transcriptional splicing controls epigenetic status of pluripotent stem cells, MOLECULAR CELL, 10.1016/j.molcel.2022.12.014, 83, 2, 203-+, 2023.01.
4.	Tellier, Michael; Zaborowska, Justyna; Neve, Jonathan; Nojima, Takayuki; Hester, Svenja; Fournier, Marjorie; Furger, Andre; Murphy, Shona, CDK9 and PP2A regulate RNA polymerase II transcription termination and coupled RNA maturation, EMBO REPORTS, 10.15252/embr.202154520, 23, 10, 2022.10.
5.	Rui Sousa-Luís, Gwendal Dujardin, Inna Zukher, Hiroshi Kimura, Carika Weldon, Maria Carmo-Fonseca, Nick J.Proudfoot, and Takayuki Nojima, POINT technology illuminates the processing of polymerase-associated intact nascent transcripts, Molecular Cell, https://doi.org/10.1101/2020.11.09.374108, 2021.05, [URL], Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly because of technical limitations in purifying authentic nascent transcripts. We present a new approach to characterize nascent RNA, called polymerase intact nascent transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5' ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano), and profiles whole transcription units (POINT-seq). In particular, we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5' P-RNA at polyadenylation sites. Furthermore, POINT-nano reveals that co-transcriptional splicing either occurs immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing..

主要総説, 論評, 解説, 書評, 報告書等

全てを見る→

Takayuki Nojima, Nicholas J Proudfoot, Mechanism of lncRNA biogenesis as revealed by nascent transcriptomics, Nature Reviews Molecular Cell Biology, https://doi.org/10.1038/s41580-021-00447-6, 2022.03, [URL], Mammalian genomes express two principal gene categories through RNA polymerase II-mediated transcription: protein-coding transcription units and non-coding RNA transcription units. Non-coding RNAs are further divided into relatively abundant structural RNAs, such as small nuclear RNAs, and into a myriad of long non-coding RNAs (lncRNAs) of often low abundance and low stability. Although at least some lncRNA synthesis may reflect transcriptional ‘noise’, recent studies define unique functions for either specific lncRNAs or for the process of lncRNA synthesis. Notably, the transcription, processing and metabolism of lncRNAs are regulated differently from protein-coding genes. In this Review, we provide insight into the regulation of lncRNA transcription and processing gleaned from the application of recently devised nascent transcriptomics technology. We first compare and contrast different methodologies for studying nascent transcription. We then discuss the molecular mechanisms regulating lncRNA transcription, especially transcription initiation and termination, which emphasize fundamental differences in their expression as compared with protein-coding genes. When perturbed, lncRNA misregulation leads to genomic stress such as transcription–replication conflict and R-loop-mediated DNA damage. We discuss many unresolved but important questions about the synthesis and potential functions of lncRNAs..

主要学会発表等

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1.	野島孝之, The end of RNA synthesis, RNAフロンティアミーティング2022, 2023.10, [URL].
2.	Takayuki Nojima, POINTing towards transcription termination, 第30回 Hot Spring Harbor Symposium、クロマチン潜在能　合同国際シンポジウム , 2022.01, [URL], Transcription is terminated in appropriate region to prevent genome stresses such as transcription-replication conflict and RNA-DNA hybrid caused in extragenic region of eukaryote cells. Biochemical and genetic approaches identified several trans-factors and cis-elements involved in transcription termination. However, the mechanism and the rule remain largely unclear due to technical limitation and complexity created by other co-transcriptional events. In order to investigate precise mechanisms of transcription, we developed a nascent RNA technology named mammalian native elongating transcript-sequencing (mNET-seq) method that reveals Pol II pausing and RNA processing intermediates with the phosphorylation states at single nucleotide resolution. In mNET-seq analysis, RNA polymerase II (Pol II) machinery was specifically detected at termination region of protein coding genes with the CTD phosphorylation at threonine 4 position (T4P). Our group substantially extended mNET-seq method to dissect intact nascent RNA associated with elongating Pol II machinery. This new method was termed Polymerase Intact Nascent Transcript (POINT) technology. The POINT technology is applied to a template switching based 5’RACE method, resulting in detection of nascent transcript 5’ends at single nucleotide resolution (POINT-5) in illumina platform. Therefore POINT-5 method precisely profiles transcription start sites and co-transcriptional RNA cleavage sites. Notably rapid depletion of nuclear 5’-3’ exonuclease Xrn2 significantly induced a termination defect with increased RNA cleavage peaks only on polyadenylation sites of pre-mRNA genes, but not on other cleavage sites. This analysis revealed specificity of Xrn2-dependent RNA turnover. In conclusion, mNET and POINT technology variants will prove invaluable to dissect the termination mechanism not only for pre-mRNA gene, but also other gene category such as noncoding genes..
3.	Takayuki Nojima, Mechanism of Co-transcriptional RNA splicing, 第16回生命医科学研究所ネットワーク国際シンポジウム＆ KEY FORUM 2021, 2021.11, [URL], In mammalian cells, transcripts synthesised by RNA polymerase II (Pol II) are largely co-transcriptionally (co-T) spliced. The exact timing of intron excision after transcription may influence splice site usage, thus shaping the alternatively spliced transcriptome. However, the underlying splicing kinetics is still poorly understood due to technical limitations in the purification of authentic newly synthesized transcripts (nascent RNA). To overcome this issue, our group have developed mammalian native elongating transcript-sequencing (mNET-seq) technology to analyse nascent RNAs with Pol II phosphorylation states (Nojima et al., Cell 2015). mNET-seq method revealed Pol II pausing on exon and phosphorylated Pol II specific spliceosome assembly (Nojima et al., Mol Cell 2018). Furthermore, we recently developed a new technology to dissect intact nascent RNAs directly purified from all Pol II transcribing states, called Polymerase Intact Nascent Transcript (POINT) (Sousa-Luis et al., Mol Cell 2021). By combining Illumina and Nanopore technologies, we took advantage of the high coverage of the former and the long read lengths of the latter. POINT single-molecule long-read data in human cells revealed the presence of different classes of splicing kinetics. ~40% of analysed introns are immediately excised as soon as the downstream exon emerges from Pol II, whereas many other introns progressively undergo delayed co-T splicing while Pol II transcribes the next intron. Moreover, we found that splicing dynamics relate to the distance between the intron and the 3’ end of the gene. Using engineered cell-lines with auxin-inducible degradation of the endogenous cleavage factor CPSF73 gene, we detected higher levels of splicing in cleavage-deficient cells. Most likely, this is because the transcriptional read-through increased the time available for delayed co-T splicing. Taken together, our novel POINT technology is able to show variation in co-T processing splicing dynamics, suggesting the presence of both immediate and delayed co-T splicing mechanisms..
4.	Takayuki Nojima, End of RNA synthesis, 日本生化学会, 2021.10, Transcription is terminated in appropriate region to prevent genome stresses such as transcription-replication conflict and RNA-DNA hybrid caused in extragenic region of eukaryote cells. Biochemical and genetic approaches identified several trans-factors and cis-elements involved in transcription termination. However, the mechanism and the rule remain largely unclear due to technical limitation and complexity created by other co-transcriptional events. In order to investigate precise mechanisms of transcription, we developed a nascent RNA technology named mammalian native elongating transcript-sequencing (mNET-seq) method that reveals Pol II pausing and RNA processing intermediates with the phosphorylation states at single nucleotide resolution. In mNET-seq analysis, RNA polymerase II (Pol II) machinery was specifically detected at termination region of protein coding genes with the CTD phosphorylation at threonine 4 position (T4P). On the other hand, the T4P CTD mark is distributed throughout long noncoding RNA (lncRNA) gene units, suggesting transcription termination signals are embedded in lncRNA genes to fine tune lncRNA expression. We substantially extended mNET-seq method to dissect intact nascent RNA associated with elongating Pol II machinery. This new method was termed Polymerase Intact Nascent Transcript (POINT) technology. The POINT technology is applied to a template switching based 5’RACE method, resulting in detection of nascent transcript 5’ends at single nucleotide resolution (POINT-5) in illumina platform. Therefore POINT-5 method precisely profiles transcription start sites and co-transcriptional RNA cleavage sites. Notably rapid depletion of nuclear 5’-3’ exonuclease Xrn2 significantly induced a termination defect with increased RNA cleavage peaks only on polyadenylation sites of pre-mRNA genes, but not on other cleavage sites. This analysis revealed specificity of Xrn2-dependent RNA turnover. In conclusion, mNET and POINT technology variants will prove invaluable to dissect the termination mechanism not only for pre-mRNA gene, but also other gene category such as noncoding genes..

学会活動

所属学会名

日本RNA学会

日本生化学会

学会大会・会議・シンポジウム等における役割

2023.11.01～2023.11.01, 日本生化学会, 世話人.

2023.10.18～2023.10.23, RNAフロンティアミーティング2023, 世話人.

その他の研究活動

海外渡航状況, 海外での教育研究歴

University of Oxford, UnitedKingdom, 2010.09～2021.01.

外国人研究者等の受入れ状況

2022.06～2023.03, 1ヶ月以上, 九州大学, Canada, 学内資金.

受賞

令和５年度科学技術分野の文部科学大臣表彰科学技術賞（科学技術振興部門）, 文部科学省, 2023.04.

海外留学リサーチフェローシップ, 上原記念生命科学財団, 2010.09.

Exceptional Achievement Award, University of Oxford, 2014.09.

海外リサーチアワード, かなえ医薬振興財団, 2013.09.

ベストポスター賞, RNA society UK, 2018.01.

研究資金

科学研究費補助金の採択状況(文部科学省、日本学術振興会)

2008年度～2009年度, 特定領域研究, 分担, ヘルペスウイルスRNAの核外輸送制御機構の解明.

2008年度～2009年度, 新学術領域研究（研究領域提案型）, 代表, 悪性腫瘍特異的なRNA選択的スプライシングを制御する抗がん剤の開発.

2008年度～2009年度, 若手研究(B), 代表, ヘルペスウイルス感染による宿主選択的スプライシング制御と免疫回避機構.

2007年度～2008年度, 基盤研究(B), 分担, イントロンレスRNAの核外輸送の分子メカニズム.

2024年度～2025年度, 挑戦的研究（萌芽）, 分担, DNA損傷により惹起されるゲノム変異非依存的な新規ネオアンチゲン発生機構の解明.

2024年度～2026年度, 基盤研究(B), 分担, DNA複製・RNA転写コンフリクトのゲノム科学的解析.

2024年度～2026年度, 基盤研究(B), 代表, 細胞ストレスよる転写終結制御破綻メカニズムの解明.

2021年度～2023年度, 帰国発展研究, 代表, ＲＮＡプロセシングと共役するゲノム転写とその破綻機構の解明.

競争的資金(受託研究を含む)の採択状況

2022年度～2024年度, 金沢大学がん進展制御研究所　共同研究助成, 代表, がんクロマチン環境における非コードRNA産生機構の解明.

2021年度～2027年度, JST, 代表, 新生RNAライフサイクルを制御する転写終結機構の解明.

寄附金の受入状況

2023年度, 公益財団法人内藤記念科学振興財団, がん機能性⾮コードRNAの発現を左右する転写終結機構の分⼦解剖と医学的応⽤.

2022年度, 公益財団法人　ノバルティス科学技術振興財団
, 研究助成/非コードRNAを創出するがん特異的な転写終結機構の解明.

2022年度, 公益財団法人　新日本先進医療研究財団
, 研究助成/スプライシング阻害が引き起こす未成熟転写終結機構とそれ由来非コードRNA代謝物の細胞機能の解明.

2022年度, 公益財団法人　上原記念生命科学財団
, 研究助成/非コードRNA異常産生を防ぐ転写終結機構の解明.

2022年度, 公益財団法人　アステラス病態代謝研究会
, 研究助成/転写終結機構の解明とそれ由来代謝物の医学的応用.

2022年度, 公益財団法人
住友財団, 基礎科学研究助成/遺伝子代謝産物の長さを決定する未成熟転写終結機構の解明.

2022年度, 公益財団法人　金原一郎医学医療振興財団, 基礎医学医療研究助成金/未成熟転写終結を介する非コードRNA代謝物の産生機構と生物学的機能の解明.

2022年度, 公益財団法人第一三共生命科学研究振興財団, PIセットアップ研究助成/非コードRNA発現をON/OFFにする転写終結機構の解明とその医学的応用.

2022年度, 公益財団法人武田科学振興財団, 生命科学研究助成/非コードRNA機能を調節する転写終結機構の解明.

2022年度, 公益財団法人三菱財団, 自然研究科学助成/非コードRNA産生を制御する転写終結機構の解明.

2021年度, 公益財団法人高松宮妃癌研究基金, 研究助成/抗がん化合物で誘導される“未成熟”転写終結制御機構とそれ由来長鎖非コードRNAの生理学的機能解析.

2021年度, 公益財団法人持田記念医学薬学振興財団, 研究助成/ゲノム転写終結が制御する非コードRNA産生機構の解明とその医学的応用.

学内資金・基金等への採択状況

2021年度～2022年度, QRプログラム　（わかばチャレンジ）, 代表, クロマチンコンパクション機構の解明.

九大関連コンテンツ

pure2017年10月2日から、「九州大学研究者情報」を補完するデータベースとして、Elsevier社の「Pure」による研究業績の公開を開始しました。

QIR　九州大学学術情報リポジトリシステム情報科学研究院

生体防御医学研究所


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