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MRNA decapping factors and the exonuclease Xrn2 function in widespread premature termination of RNA polymerase II transcription

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE36185 | GEO | 2012/04/05

SECONDARY ACCESSION(S): PRJNA151657

REPOSITORIES: GEO

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mRNA decapping factors and the exonuclease Xrn2 function in widespread premature termination of RNA polymerase II transcription

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

Effects of transcription elongation rate and Xrn2 exonuclease activity on RNA polymerase II termination suggest widespread kinetic competition

Project description:Transcription termination was analyzed by anti RNA pol II ChIP-seq in isogenic human HEK293 cell lines that inducibly express a-amanitin resistant mutants of the RNA polymerase II large subunit with slow and fast elongation rates and in lines that inducbily over-express WT or an active site mutant of the RNA exonuclease "torpedo" Xrn2. Transcription termination zones were mapped by anti-pol II ChIP-seq under conditions where transcription elongation rate was increased or decreased by point mutations in the large subunit of the enzyme. Termination was also assayed under conditions where Xrn2 exonuclease activity was inhibited by over-expression of an active site mutant (D235A).

2015-11-19 | E-GEOD-72800 | biostudies-arrayexpress

Xrn2 substrate mapping identifies torpedo loading sites and extensive premature termination of RNA pol II transcription

Project description:The exonuclease torpedo Xrn2 loads onto nascent RNA 5’-PO4 ends and chases down pol II to promote termination downstream of polyA sites. We report that Xrn2 is recruited to pre-initiation complexes and “travels” to 3’ ends of genes. Mapping of 5’-PO4 ends in nascent RNA identified Xrn2 loading sites stabilized by an active site mutant, Xrn2(D235A). Xrn2 loading sites are ~2-20 bases downstream of where CPSF73 cleaves at polyA sites and histone 3’ ends. We propose that processing of all mRNA 3’ ends comprises cleavage and limited 5’-3’ trimming by CPSF73 followed by hand-off to Xrn2. A similar hand-off occurs at tRNA 3’ ends where co-transcriptional RNAseZ cleavage generates novel Xrn2 substrates. Exonuclease-dead Xrn2 increased transcription in 3’ flanking regions by inhibiting polyA site-dependent termination. Surprisingly, the mutant Xrn2 also rescued transcription in promoter-proximal regions to the same extent as in 3’ flanking regions. eNET-seq revealed Xrn2-mediated degradation of sense and anti-sense nascent RNA within a few bases of the TSS where 5’-PO4 ends may be generated by decapping or endonucleolytic cleavage. These results suggest that a major fraction of pol II complexes terminate prematurely close to the start site under normal conditions by an Xrn2-mediated torpedo mechanism.

2022-11-17 | GSE217033 | GEO

A unified allosteric/torpedo mechanism for transcriptional termination on human protein-coding genes

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

Effects of transcription elongation rate and Xrn2 exonuclease activity on RNA polymerase II termination suggest widespread kinetic competition

2015-11-19 | GSE72800 | GEO

Control of RNA pol II speed by PNUTS-PP1 and Spt5 dephosphorylation facilitates termination by a “sitting duck torpedo” mechanism

Project description:Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA pol II elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, pol II decelerates from >2kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a new model for termination, the “sitting duck torpedo” mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homologue NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.

2019-10-30 | GSE134198 | GEO

Genome-wide analysis of RNA polymerase II termination at protein-coding genes

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

Characterization of the SF3B1 interactome

Project description:CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II). As part of P-TEFb, CDK9 phosphorylates the carboxyl-terminal domain (CTD) of pol II and elongation factors, including SPT5, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein-coding genes. Our phosphoproteomic analyses after CDK9 inhibition, using either DRB or an analog-sensitive CDK9 cell line, confirm the splicing factor SF3B1 as an additional key target of this kinase. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC. As part of this project, we characterized the interactome of SF3B1 in HeLa cells in duplicate.

2022-08-22 | PXD033694 | Pride

Transcription profiling by array of a stably RNAi-silenced nuclear 5’-3’ exonuclease XRN3 mutant line in Arabidopsis thaliana after transcriptional inhibition with cordycepin

Project description:In Arabidopsis thaliana two nuclear 5'-3' exonucleases, XRN2 & 3, are involved in the degradation and processing of several classes of nuclear RNAs and in transcription termination of RNA polymerase II. The role of XRN3 in Pol II transcription termination is investigated by combining two different types of RNA-seq data for Col-0 background WT and xrn3-8 mutant A. thaliana seedlings. The data for the experiment comprises single molecule Direct RNA Sequencing of unamplified RNA (3x WT, 4x xrn3-8 mutant) and high-depth Illumina strand-specific short read sequencing of PCR amplified ribosomal-RNA depleted RNA (Illumina TruSeq stranded ribozero Plant kit, 3x WT, 3x xrm3-8 mutant). In the xrn3 mutant, these data reveal a widespread accumulation of non-coding intergenic transcripts (xrn3-associated transcripts - XATs) generated by Pol II read-through transcription that are usually polyadenylated and lack the 5' cap structure. This data highlights the important role of exoribonucleases in the torpedo mechanism of Pol II transcription termination and show that a global disturbance in this process significantly impacts both gene expression and transcriptome integrity.

2019-09-02 | E-MTAB-5137 | biostudies-arrayexpress

Direct RNA Sequencing of polyadenylated RNA & high-depth Illumina strand-specific, short read RNA-seq of a stably RNAi-silenced nuclear 5'-3' exonuclease XRN3 mutant line in Arabidopsis thaliana

2019-09-02 | E-MTAB-5132 | biostudies-arrayexpress

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