A unified allosteric/torpedo mechanism for transcriptional termination on human protein-coding genes
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ABSTRACT:
PROVIDER: PRJNA566400 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10148479.fastq.gz | Fastqsanger.gz | |||
| SRR10148480.fastq.gz | Fastqsanger.gz | |||
| SRR10148481.fastq.gz | Fastqsanger.gz | |||
| SRR10148482.fastq.gz | Fastqsanger.gz | |||
| SRR10148483.fastq.gz | Fastqsanger.gz |
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Project description:The so-called allosteric and torpedo models have been used for the past thirty years to explain how transcription terminates on protein-coding genes. The former invokes conformational changes in the transcription complex and the latter involves degradation of the downstream product of poly(A) signal (PAS) processing. Here, we describe a single mechanism incorporating features of both models. We show that CPSF73 is indispensable for transcriptional termination on protein-coding and its loss causes profound read-through genome-wide. CPSF73 functions upstream of allosteric modifications to the elongation complex that cause Pol II to slow down after the PAS. This state is enriched by rapid depletion of XRN2 and promoted by protein phosphatase 1 (PP1), the inhibition of which confers runaway read-through in the absence of XRN2. These allosteric changes facilitate XRN2-dependent termination, by aiding its capture of Pol II, rather than constituting a termination pathway in themselves. Our experiments unify the long-standing allosteric and torpedo models for transcriptional termination.
2020-01-21 | GSE137727 | GEO
Project description:Termination of protein-coding gene transcription by RNA polymerase II (Pol II) depends on endonucleolytic cleavage at the poly(A) site and the activity of a 5’->3’ “torpedo” exoribonuclease. Other Pol II transcripts also undergo endonucleolytic cleavage suggesting common themes for its termination. Nevertheless, many RNA polymerases employ intrinsic/allosteric termination that directly defines transcript 3’ ends. We have analysed termination of snRNA transcription in humans, which utilises the endoribonucleolytic integrator complex. Integrator is involved, but termination continues after its depletion. This implicates additional mechanisms and shows that endo- and 5’->3’ exonuclease activities are not necessarily a basis for all Pol II termination. Although the alternative process acts as a failsafe in the absence of integrator it contributes significantly to snRNA termination in the natural situation. Long-read sequencing reveals its products to have stochastic 3’ ends that are terminated before integrator cleavage, ruling out 5’->3’ exonuclease contribution(s) and supporting an allosteric/intrinsic mechanism. Termination of some snRNA transcription occurs at T-runs further indicating common principles between this mechanism and those used by other RNA polymerases.
2020-11-04 | GSE150238 | GEO
Project description:An allosteric/intrinsic mechanism supports termination of snRNA transcription.
| PRJNA631616 | ENA
Project description:The allosteric and torpedo models have been used for 30 yr to explain how transcription terminates on protein-coding genes. The former invokes termination via conformational changes in the transcription complex and the latter proposes that degradation of the downstream product of poly(A) signal (PAS) processing is important. Here, we describe a single mechanism incorporating features of both models. We show that termination is completely abolished by rapid elimination of CPSF73, which causes very extensive transcriptional readthrough genome-wide. This is because CPSF73 functions upstream of modifications to the elongation complex and provides an entry site for the XRN2 torpedo. Rapid depletion of XRN2 enriches these events that we show are underpinned by protein phosphatase 1 (PP1) activity, the inhibition of which extends readthrough in the absence of XRN2. Our results suggest a combined allosteric/torpedo mechanism, in which PP1-dependent slowing down of polymerases over termination regions facilitates their pursuit/capture by XRN2 following PAS processing.
| S-EPMC6938672 | biostudies-literature
Project description:At the end of protein-coding genes, RNA polymerase (Pol) II undergoes a concerted transition that involves 3’-processing of the pre-mRNA and transcription termination. Here we conduct a genome-wide analysis of this 3’-transition in yeast. We find that the 3’-transition globally requires the Pol II elongation factor Spt5 and factors involved in the recognition of the poly-adenylation (pA) site and in endonucleolytic RNA cleavage. Pol II release from DNA occurs in a narrow termination window downstream of the pA site and generally requires the ‘torpedo’ exonuclease Rat1 (human XRN2). The Rat1-interacting factor Rai1 contributes to RNA degradation downstream of the pA site. Defects in the 3’-transition result in transcription interference at downstream genes, which is attenuated by a back-up termination mechanism.
2017-03-15 | GSE79222 | GEO
Project description:Termination of RNA polymerase II (RNAPII) transcription is a fundamental step of gene expression that is critical for determining the borders between genes. In budding yeast, termination at protein-coding genes is initiated by the cleavage/polyadenylation machinery, whereas termination of most noncoding RNA (ncRNA) genes occurs via the Nrd1-Nab3-Sen1 (NNS) pathway. Unexpectedly, we show here that NNS-like transcription termination is not conserved in fission yeast. Instead, genome-wide location analyses show global recruitment of mRNA 3’ end processing factors at the end of ncRNA genes, including snoRNAs and snRNAs, and that this recruitment coincides with high levels of Ser2 and Tyr1 phosphorylation on the RNAPII C-terminal domain. We further show that termination of mRNA and ncRNA transcription requires the conserved Ysh1/CPSF-73 and Dhp1/XRN2 nucleases, supporting widespread cleavage-dependent transcription termination in fission yeast. Our findings thus reveal that a common mode of transcription termination can produce functionally and structurally distinct types of polyadenylated and non-polyadenylated RNAs.
2018-08-10 | GSE115595 | GEO
Project description:RNA polymerase II (RNAPII) transcription involves initiation from promoters, transcript elongation through the gene body, and cessation of transcription in the downstream terminator regions. In contrast to bacteria, where terminators often contain specific DNA elements to direct RNAP dissociation1, termination by RNAPII is thought to be driven entirely by protein co-factors1-3. Here we use biochemical reconstitution to shed new light on RNAPII termination. Unexpectedly, transcription through a terminator region by pure RNAPII results in a significant amount of intrinsic polymerase dissociation at specific sequences containing T-tracts. A combination of biochemistry and single molecule analysis indicates that such intrinsic termination involves pausing without backtracking prior to spontaneous RNAPII dissociation from the DNA template. Importantly, while the ‘torpedo’ Rat1-Rai1 RNA exonuclease (XRN2 in humans) works inefficiently on paused or stopped polymerases, it greatly stimulates intrinsic termination. By contrast, elongation factor Spt4-Spt5 (DSIF in humans) suppresses such termination. Genome-wide analysis in yeast using 3’-end sequencing further supports the idea that transcriptional termination occurs by transcript cleavage at the polyA site exposing a new RNA-end that allows loading of the Rat1-Rai1 torpedo, which then catches up with a destabilised RNAPII at intrinsic termination sites containing T-tracts to terminate transcription.
2023-09-11 | GSE218927 | GEO
Project description:RNA polymerase II (RNAPII) transcription involves initiation from promoters, transcript elongation through the gene body, and cessation of transcription in the downstream terminator regions. In contrast to bacteria, where terminators often contain specific DNA elements to direct RNAP dissociation1, termination by RNAPII is thought to be driven entirely by protein co-factors1-3. Here we use biochemical reconstitution to shed new light on RNAPII termination. Unexpectedly, transcription through a terminator region by pure RNAPII results in a significant amount of intrinsic polymerase dissociation at specific sequences containing T-tracts. A combination of biochemistry and single molecule analysis indicates that such intrinsic termination involves pausing without backtracking prior to spontaneous RNAPII dissociation from the DNA template. Importantly, while the ‘torpedo’ Rat1-Rai1 RNA exonuclease (XRN2 in humans) works inefficiently on paused or stopped polymerases, it greatly stimulates intrinsic termination. By contrast, elongation factor Spt4-Spt5 (DSIF in humans) suppresses such termination. Genome-wide analysis in yeast using 3’-end sequencing further supports the idea that transcriptional termination occurs by transcript cleavage at the polyA site exposing a new RNA-end that allows loading of the Rat1-Rai1 torpedo, which then catches up with a destabilised RNAPII at intrinsic termination sites containing T-tracts to terminate transcription.
2023-09-11 | GSE218924 | GEO
Project description:We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a and Dcp2 and the termination factor TTF2 co-immunoprecipitate with Xrn2, the nuclear 5'-3' exonuclease torpedo that facilitates transcription termination at the 3' ends of genes. Dcp1a, Xrn2 and TTF2 localize near transcription start sites (TSSs) by ChIP-Seq. At genes with 5' peaks of paused pol II, knockdown of decapping or termination factors, Xrn2 and TTF2, shifted polymerase away from the TSS toward upstream and downstream distal positions. This re-distribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the torpedo mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated co-transcriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation. RNA pol II (GSE30895: GSM766171), Xrn2, TTF2 and Dcp1a were localized by ChIP-Seq in HeLa cells. RNA pol II was localized in control HEK293 cells and cells infected with lentiviruses expressing a scrambled control shRNA (scr), and shRNAs targeting the following proteins: Xrn2, TTF2, Xrn2+TTF2, Edc3, Dcp1a, and Dcp2.
2012-04-04 | E-GEOD-36185 | biostudies-arrayexpress
Project description:We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a and Dcp2 and the termination factor TTF2 co-immunoprecipitate with Xrn2, the nuclear 5'-3' exonuclease torpedo that facilitates transcription termination at the 3' ends of genes. Dcp1a, Xrn2 and TTF2 localize near transcription start sites (TSSs) by ChIP-Seq. At genes with 5' peaks of paused pol II, knockdown of decapping or termination factors, Xrn2 and TTF2, shifted polymerase away from the TSS toward upstream and downstream distal positions. This re-distribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the torpedo mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated co-transcriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation.
2012-04-05 | GSE36185 | GEO