Landscape of Transcription Termination in Arabidopsis Revealed by Single-molecule Nascent RNA Sequencing [BS-seq]
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PROVIDER: PRJNA763183 | ENA |
REPOSITORIES: ENA
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Project description:The dynamic process of transcription termination produces transient RNA intermediates, such as the full-length readthrough, 5’ and 3’ cleavage products, and polyadenylated nascent RNAs, which are difficult to distinguish from each other via short-read sequencing methods. Here, we used single-molecule nascent RNA sequencing to characterize these various forms of transient RNAs during termination at genome-wide scale in Arabidopsis. Our data revealed a wide range of termination windows (defined as the median readthrough distance) among genes, ranging from ~50 nt to over 1000 nt. The ends of termination windows precisely match the peak of Pol II occupancy and co-localize with termination factors. We also discovered a termination mechanism shaped by the downstream tRNA genes, suggesting that elongating Pol II can be expelled by Pol III activity. Mutation of the exonuclease AtXRN3 leads to a drastic accumulation of 3’ cleaved readthrough RNAs. Moreover, readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Our results revealed the diverse termination patterns in Arabidopsis, and our method is applicable to study the global termination landscape in a broad range of species.
2024-09-22 | GSE184099 | GEO
Project description:The dynamic process of transcription termination produces transient RNA intermediates, such as the full-length readthrough, 5’ and 3’ cleavage products, and polyadenylated nascent RNAs, which are difficult to distinguish from each other via short-read sequencing methods. Here, we used single-molecule nascent RNA sequencing to characterize these various forms of transient RNAs during termination at genome-wide scale in Arabidopsis. Our data revealed a wide range of termination windows (defined as the median readthrough distance) among genes, ranging from ~50 nt to over 1000 nt. The ends of termination windows precisely match the peak of Pol II occupancy and co-localize with termination factors. We also discovered a termination mechanism shaped by the downstream tRNA genes, suggesting that elongating Pol II can be expelled by Pol III activity. Mutation of the exonuclease AtXRN3 leads to a drastic accumulation of 3’ cleaved readthrough RNAs. Moreover, readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Our results revealed the diverse termination patterns in Arabidopsis, and our method is applicable to study the global termination landscape in a broad range of species.
2024-09-22 | GSE178980 | GEO
Project description:Landscape of Transcription Termination in Arabidopsis Revealed by Single-molecule Nascent RNA Sequencing
| PRJNA763181 | ENA
Project description:Landscape of Transcription Termination in Arabidopsis Revealed by Single-molecule Nascent RNA Sequencing [FLEP-seq]
| PRJNA741784 | ENA
Project description:BackgroundThe dynamic process of transcription termination produces transient RNA intermediates that are difficult to distinguish from each other via short-read sequencing methods.ResultsHere, we use single-molecule nascent RNA sequencing to characterize the various forms of transient RNAs during termination at genome-wide scale in wildtype Arabidopsis and in atxrn3, fpa, and met1 mutants. Our data reveal a wide range of termination windows among genes, ranging from ~ 50 nt to over 1000 nt. We also observe efficient termination before downstream tRNA genes, suggesting that chromatin structure around the promoter region of tRNA genes may block pol II elongation. 5' Cleaved readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Consistent with previous reports, we also observe long chimeric transcripts with cryptic splicing in fpa mutant; but loss of CG DNA methylation has no obvious impact on termination in the met1 mutant.ConclusionsOur method is applicable to establish a comprehensive termination landscape in a broad range of species.
| S-EPMC8613925 | biostudies-literature
Project description:CoPRO adapts PRO-cap for paired end sequencing, and includes a total of three different libraries that were enriched for either capped nascent RNAs only, uncapped nascent RNAs only, or both. With paired end sequencing, each read tells us where an RNA polymerase molecule initiated, and then where its active site is located. Comparison of the libraries for different capping states allows us identify the precise location of pausing, and of where nascent RNAs become capped across the tens of thousands of initiation sites that we detect. The paired nature of the data enabled identification of sites of transcription initiation with unprecedented precision: we could use the pattern of pausing and elongation as a sensitive way of calling real initiation sites, and filter out termination by comparing capped and uncapped treatments. Because CoPRO maps nascent RNA, it is not affected by the post-transcriptional stability of the initiation and pausing events detected. Thus, it puts non-coding transcription (such as eRNAs and upstream divergent RNAs) on an equal footing with longer lived RNAs like mRNA and lncRNAs. With our comprehensive maps of transcription initiation, we are able to compare the architecture of initiation sites with other features of the genome. For this purpose, we chose human K562 cells for direct comparison with dozens of publically available genome-wide datasets.
2018-07-02 | GSE116472 | GEO
Project description:The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit Pol II termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. ZC3H4, in turn, directly connects to the ZCCHC8 component of the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay.
2023-06-19 | GSE212208 | GEO
Project description:Termination of Pol II transcription is an important step in the transcription cycle and is responsible for dislodgement of polymerase from DNA which leads to the release of a functional transcript. Recent studies have identified key players important for termination and showed a conserved domain that interacts with the phosphorylated C-terminus of Pol II (CTD-Interacting-Domain, CID) to constitute a common feature of these proteins. However, the mechanism by which transcription termination is achieved, is not understood. Using genome-wide methods, we demonstrate that the fission yeast CID protein Seb1 is essential for termination of protein-coding and non-coding genes through interacting with S2-phosphorylated Pol II and nascent RNA. Furthermore, we present the crystal structures of the Seb1 CTD- and RNA-binding modules. Unexpectedly, the latter reveals a novel intertwined two-domain arrangement of a canonical RRM and a second domain. These results provide important insights into the mechanism underlying eukaryotic transcription termination.
2017-02-05 | GSE89914 | GEO
Project description:The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit Pol II termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. ZC3H4, in turn, directly connects to the ZCCHC8 component of the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay.
2023-06-19 | GSE212383 | GEO
Project description:The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit Pol II termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. ZC3H4, in turn, directly connects to the ZCCHC8 component of the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay.
2023-06-19 | GSE228383 | GEO