Country：United Kingdom

『超高解像度新生RNA 解析法の開発とゲノム転写制御の研究』

Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1101/2024.01.31.578294

Other Link： https://www.biorxiv.org/content/10.1101/2024.01.31.578294v1

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Nature Reviews Molecular Cell Biology  

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.

DOI： 10.1038/s41580-021-00447-6

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： https://doi.org/10.1101/2020.11.09.374108

Other Link： https://www.sciencedirect.com/science/article/pii/S1097276521001441?via%3Dihub

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.molcel.2018.10.011

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.molcel.2018.09.004

Event date： 2023.10

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：大阪大学　銀杏会館   Country：Japan  

Event date： 2022.1

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：zoom   Country：Japan  

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.

Other Link： http://hshis30.umin.ne.jp/

Event date： 2021.11

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：zoom   Country：Japan  

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.

Other Link： https://www.keyforum2021.org/

Event date： 2021.10

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：zoom   Country：Japan  

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.

Event date： 2023.7

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：国立遺伝学研究所   Country：Japan  

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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.

DOI： https://doi.org/10.1038/s41580-021-00447-6

Other Link： https://www.nature.com/articles/s41580-021-00447-6

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

DOI： https://doi.org/10.1093/nar/gkae1240

Language：Japanese   Publisher：(株)羊土社  

近年,転写されたばかりのRNA(新生RNA)を解析する技術が次々に開発されたことで,ヒトゲノムのほとんどの領域で転写が起きていることがわかってきた.われわれのゲノムは,タンパク質をコードする領域とそうでない領域(非コードDNA)の2つに分類される.それら2つのゲノム領域から転写されるRNAの質は同じであろうか.本稿では,単なる転写のノイズであると考えられてきた多くの長鎖非コードRNA(lncRNA)の発現解析方法,それによって明らかになったlncRNA転写制御と安定性制御機構について紹介する.(著者抄録)

Language：English   Publisher：Nature Reviews Molecular Cell Biology  

In the version of this article initially published, the technique fastGRO was presented in Fig. 2 and the main text as a chromatin- based run- on technique when in fact it is a nuclear run- on technique. Accordingly, in Fig. 2, right- hand “Techniques” section, the third balloon has been edited to include “fastGRO” and the seventh balloon edited to remove “fastGRO,” and the eighth sentence of the Fig. 2 legend has been amended to include fastGRO, as follows: “Nucleus: transcription run- on (TRO) analyses are performed in biochemically isolated nuclei using 5-bromouridine 5′-triphosphate (BrUTP) in GRO-seq37, 4- thiouridine (4sU) in fastGRO40 or biotin- labelled NTPs (biotin- NTP) in PRO- seq38.” Further, the penultimate sentence of the “In vitro RNA labelling” section has been amended to read “Finally, a variation on genomic TRO called fastGRO also employs 4- thiouridine (4sU) in the NRO reaction” (from the original “Finally, a variation on genomic TRO called fastGRO also employs the use of chromatin fractions as starting material but the nascent transcript is tagged with 4- thiouridine (4sU)….”). The changes are reflected in the HTML and PDF versions of the article.

DOI： 10.1038/s41580-022-00551-1

Scopus

PubMed

Type：Competition, symposium, etc. 

Number of participants：50

Type：Competition, symposium, etc. 

Number of participants：50

転写研究者のネットワーク作り

Fukuoka RNA commons は、生物現象を支える最も重要な分子の一つである 「RNA」の研究者を日本国内外からお招きし、九州大学学内のみならず、福岡エリアを中心に RNA研究の普及、さらには研究者・学生たちのネットワーク作りへの貢献を目指しています。