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Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration [Mnase-seq]

ABSTRACT: mRNA homeostasis is favored by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5’ exhibited significant reductions in slope that were compatible with decreased elongation rates. This phenomenon was also observed soon after the nuclear pool of Xrn1 was depleted. Nucleosome mapping confirmed drastically altered chromatin dynamics. We also found an accumulation of arrested RNA pol II at the 3’ end, immediately upstream of polyadenylation sites of most genes, with particular incidence in those functionally related to regulatory processes. Lack of Xrn1 provoked alterations in RNA pol II CTD phosphorylation and increased recruitment of 3’ pre-mRNA processing factors. However, accumulation of RNA pol II at 3’ ends was rather related to the nucleosomal configuration of those regions.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE153035 | GEO | 2021/05/24

REPOSITORIES: GEO

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Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration [RNA-seq]

Project description:mRNA homeostasis is favored by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5’ exhibited significant reductions in slope that were compatible with decreased elongation rates. This phenomenon was also observed soon after the nuclear pool of Xrn1 was depleted. Nucleosome mapping confirmed drastically altered chromatin dynamics. We also found an accumulation of arrested RNA pol II at the 3’ end, immediately upstream of polyadenylation sites of most genes, with particular incidence in those functionally related to regulatory processes. Lack of Xrn1 provoked alterations in RNA pol II CTD phosphorylation and increased recruitment of 3’ pre-mRNA processing factors. However, accumulation of RNA pol II at 3’ ends was rather related to the nucleosomal configuration of those regions.

2021-05-24 | GSE153036 | GEO

The mRNA degradation factor Xrn1 regulates transcription elongation in parallel to Ccr4

Project description:During the last decade several examples of coordination between gene transcription and mRNA degradation have been reported. mRNA imprinting by Rpb4 and 7 subunits of RNA polymerase II (RNAPII) and by the Ccr4-Not complex allows controlling its fate during transcription. Transcription regulation by mRNA degradation factors like Xrn1 constitutes a feedback loop that contributes to mRNA homeostasis. Mechanistic details of these phenomena are unclear. Most studies involve measurement of mRNA decay rates, usually by stressing procedures such as transcriptional shut-off or incorporation of modified nucleotides that can lead to biased results. In this work we have used the easily repressible yeast GAL1 gene to perform a genetic analysis of mRNA synthesis and degradation under physiological conditions. We combined this experimental approach with computational multi-agent modelling, testing different possibilities of Xrn1 and Ccr4-Not action in gene transcription. This double strategy brought us to conclude that Xrn1 regulates RNAPII backtracking in a Ccr4-independent manner. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII molecules. We found that xrn1∆ and ccr4∆ exhibited very different patterns of TFIIS/RNPAII which confirmed their differential role in controlling transcription elongation.

2019-07-17 | GSE127179 | GEO

The Not4 ubiquitin ligase utilizes its conserved RNA binding domains to regulate global proteostasis and RNA polymerase II-dependent transcription

Project description:The conserved Ccr4-Not complex regulates all aspects of the RNA polymerase II (Pol II) gene expression pathway, while it also controls proteasome assembly and function. The Not4 RING domain ubiquitin ligase is a core subunit that also contains an RNA recognition motif (RRM) and C3H1 domain (collectively referred to as the RRM-C) whose function is unknown. Herein, we demonstrate that the Not4 RRM-C contributes to both Not4-dependent regulation of the proteasome and Pol II. Disruption of both Not4 ligase activity and the RRM-C domain simultaneously replicates the proteasome-dependent deubiquitylation and catalytic activity misregulation found in Not4-deficient cells. Transcriptome analysis reveals that the Not4 RRM-C affects a subset of Pol II-dependent genes involved in specific biological functions, including transcription elongation, cyclin-dependent kinase regulated nutrient responses, and ribosomal biogenesis. At these genes, the Not4 RRM-C negatively regulates Pol II binding. While Not4 RRM-C disruption modestly increases between Ccr4-Not and Pol II, a Not4 ligase mutant decreases Ccr4-Not association with Pol II, thus implicating Not4-dependent ubiquitylation in mediating Ccr4-Not interaction with Pol II. Collectively, our results suggests the cellular RNA environment may integrate control of global proteostasis and Pol II transcription through the Not4 ubiquitin ligase.

2017-08-03 | GSE102192 | GEO

Allosteric transcription stimulation by super elongation complex

Project description:The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and a subcomplex, ELL2-EAF1, which stimulates transcription elongation by RNA polymerase II (Pol II). Here we report the cryo-EM structure of ELL2-EAF1 bound to a Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the Pol II lobe, explaining how SEC delivers P-TEFb to Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that elongation stimulation requires the dimerization module and an ELL2 protein linker that tethers this module to the Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a further closed conformation of the Pol II active center cleft.

2021-07-15 | PXD024457 | Pride

Distribution of total RNA polymerase II along the 5'/3' regions in xrn1 mutants

Project description:Determination of 3' or 5' intragenic RNA pol II occupancy. Maintaining the proper mRNA levels is a key aspect in the regulation of gene expression. The balance between mRNA synthesis and decay determines these levels. Using a whole-genome analysis, we demonstrate that most yeast mRNAs are degraded by the 5'-to-3' pathway (the "decaysome"), as proposed previously. Unexpectedly, the level of these mRNAs is highly robust to perturbations in this major pathway, as defects in various decaysome components lead to transcription down-regulation. Moreover, these components shuttle between the cytoplasm and the nucleus, in a manner dependent on proper mRNA degradation. In the nucleus, they associate with chromatin - preferentially ~30bp upstream of transcription start-sites - and directly stimulate transcription initiation and elongation. Hence, the decaysome has a dual role in maintaining mRNA levels. Significantly, proper import of some decaysome components seems to play a key role in coupling the two functions. The gene expression process is therefore circular, whereby the hitherto first and last stages are interconnected. This study focuses on the distribution of RNA pol II in the 3' or 5' region of 377 S. cerevisiae ORFs. S. cerevisiae cells grown in YPD to exponential phase were subjected to chromatin immunoprecipitation (ChIP) with anti-Rpb3 antibody. Data were normalized using whole cell extract hybridized on the same array. Home-made macroarrays containing 300 bp from both the 5' or 3' ends of each ORF were used.

2013-05-22 | E-GEOD-43604 | biostudies-arrayexpress

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

Distribution of total RNA polymerase II along the 5'/3' regions in xrn1 mutants

2013-05-22 | GSE43604 | GEO

SPT6 functions in transcriptional pause/release via PAF1C recruitment

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute 5 protein-depletion approach, prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Mechanistically, loss of SPT6 results in loss of PAF1 complex (PAF1C) from RNA Pol II, leading to NELF-bound RNA Pol II release into the gene bodies. Furthermore, SPT6 and/or PAF1 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through the recruitment of PAF1C during the early elongation.

2022-08-09 | PXD033771 | Pride

Genomic Run-On (GRO): determination of the nascent transcriptional rate and mRNA amount in Xrn1 mutants

Project description:Maintaining the proper mRNA levels is a key aspect in the regulation of gene expression. The balance between mRNA synthesis and decay determines these levels. Using a whole-genome analysis, we demonstrate that most yeast mRNAs are degraded by the 5'-to-3' pathway (the "decaysome"), as proposed previously. Unexpectedly, the level of these mRNAs is highly robust to perturbations in this major pathway, as defects in various decaysome components lead to transcription down-regulation. Moreover, these components shuttle between the cytoplasm and the nucleus, in a manner dependent on proper mRNA degradation. In the nucleus, they associate with chromatin - preferentially ~30bp upstream of transcription start-sites - and directly stimulate transcription initiation and elongation. Hence, the decaysome has a dual role in maintaining mRNA levels. Significantly, proper import of some decaysome components seems to play a key role in coupling the two functions. The gene expression process is therefore circular, whereby the hitherto first and last stages are interconnected. This study focuses on the transcriptional activity of RNA pol II in 6035 S. cerevisiae ORFs. S. cerevisiae cells grown in YPD to exponential phase were subjected to Genomic Run-On. Data were normalized using gDNA hybridized on the same array and by the software ArrayStat. Home-made macroarrays containing 6035 ORFs were used.

2013-05-22 | E-GEOD-29519 | biostudies-arrayexpress

Structural basis of human transcription-DNA repair coupling

Project description:Transcription-coupled DNA repair (TCR) efficiently removes bulky DNA lesions from transcribed parts of the genome and constitutes a major defence against DNA damage by UV irradiation. TCR begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CsB, the E3 ubiquitin ligase complex CRL4CsA, and the UV-stimulated scaffold protein A (UVSSA). Here we provide five high-resolution structures of Pol II transcription complexes with bound TCR factors and complementary biochemical data. Together with published work, our results converge on a model for the molecular mechanism of transcription-DNA repair coupling. In this model, Pol II stalling triggers the formation of an alternative transcription elongation complex that we call ECact. In ECact, CsB has replaced the elongation factor DSIF, binds the PAF1 complex, and repositions upstream DNA such that it contacts the elongation factor SPT6. The CsB translocase now pulls on the upstream DNA template, thereby pushing Pol II forward. If the lesion cannot be bypassed, Pol II gets stably stalled. CRL4CsA spans over the Pol II clamp to reach the Pol II jaw and direct ubiquitination of RPB1 residue lysine-1268. This triggers the recruitment of TFIIH to UVSSA, which is located at downstream DNA, and enables nucleotide excision repair. Finally, large-scale conformational changes in CRL4CsA enable ubiquitination of CsB and the release of TCR factors, thereby allowing Pol II to resume elongation and continue transcription over repaired DNA.

2022-02-17 | PXD025328 | Pride

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