How domains of the RNA polymerase I lobe binding subunits drive transcription elongation
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB86837 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR14711221.fastq.gz | Fastqsanger.gz | |||
| ERR14711222.fastq.gz | Fastqsanger.gz | |||
| ERR14711223.fastq.gz | Fastqsanger.gz | |||
| ERR14711224.fastq.gz | Fastqsanger.gz | |||
| ERR14711225.fastq.gz | Fastqsanger.gz |
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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
Project description:Recent studies reveal a striking phenomenon that RNA Polymerase II (Pol II) appears to travel on gene body in an accelerated fashion, but the mechanism has remained unknown. We performed synchronized transcription coupled with deep sequencing, observing an inverse relationship between initial rate and acceleration in different cell types. We directly tested several correlative events and detected a positive contribution of the splicing commitment factor SRSF2 to Pol II acceleration, suggesting a functional benefit of co-transcriptional pre-mRNA splicing in transcription elongation. Unexpectedly, we found that perturbation of Pol II Ser2 phosphorylation had little impact on Pol II elongation or acceleration. While H3K79me2 has been positively correlated with Pol II elongation, we showed that reduction of this histone modification event actually accelerated Pol II elongation. Together, these data suggest a combined effect of gradual gain-of-competence and gradual lost-of-epigenetic barriers as the mechanism for accelerated Pol II elongation. DRB time-course releasing assay under functional perturbation
2015-12-31 | E-GEOD-66021 | biostudies-arrayexpress
Project description:The rate of RNA polymerase II (pol II) elongation can influence splice site selection in nascent transcripts, yet the extent and physiological relevance of this kinetic coupling between transcription and alternative splicing is not well understood. We performed experiments to perturb pol II elongation and then globally compared alternative splicing patterns with genome-wide pol II occupancy. RNA binding and RNA processing functions were significantly enriched among the genes with pol II elongation inhibition-dependent changes in alternative splicing. Under conditions that interfere with pol II elongation, including cell stress, increased pol II occupancy was detected in the intronic regions flanking the alternative exons in these genes, and these exons generally became more included. A disproportionately high fraction of these exons introduced premature termination codons that elicited nonsense-mediated mRNA decay (NMD), thereby further reducing transcript levels. Our results provide evidence that kinetic coupling between transcription, alternative splicing and NMD affords a rapid mechanism by which cells can respond to changes in growth conditions, including cell stress, to coordinate the levels of RNA processing factors with mRNA levels. In order to identify alternative splicing events influenced by changes in pol II elongation, we performed quantitative alternative splicing microarray profiling (Pan et al., 2004 (PMID 15610736); Shai et al., 2006 (PMID 16403798)) of RNA isolated from stimulated Jurkat T lymphoma cells, cultured separately in the presence or absence of two different drugs that can inhibit pol II elongation: 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) and camptothecin.
2010-12-09 | E-GEOD-25287 | biostudies-arrayexpress
Project description:RNA polymerase II (Pol II) elongation is a critical step in gene expression. Here we find that NDF, which was identified as a bilaterian nucleosome-destabilizing factor, is also a Pol II transcription factor that stimulates elongation with plain DNA templates in the absence of nucleosomes. NDF binds directly to Pol II and enhances elongation by a different mechanism than does transcription factor TFIIS. Moreover, yeast Pdp3, which is related to NDF, binds to Pol II and stimulates elongation. Thus, NDF is a Pol II-binding transcription elongation factor that is localized over gene bodies and is conserved from yeast to humans.
2022-03-10 | GSE185464 | GEO
Project description:The rate of RNA polymerase II (pol II) elongation can influence splice site selection in nascent transcripts, yet the extent and physiological relevance of this kinetic coupling between transcription and alternative splicing is not well understood. We performed experiments to perturb pol II elongation and then globally compared alternative splicing patterns with genome-wide pol II occupancy. RNA binding and RNA processing functions were significantly enriched among the genes with pol II elongation inhibition-dependent changes in alternative splicing. Under conditions that interfere with pol II elongation, including cell stress, increased pol II occupancy was detected in the intronic regions flanking the alternative exons in these genes, and these exons generally became more included. A disproportionately high fraction of these exons introduced premature termination codons that elicited nonsense-mediated mRNA decay (NMD), thereby further reducing transcript levels. Our results provide evidence that kinetic coupling between transcription, alternative splicing and NMD affords a rapid mechanism by which cells can respond to changes in growth conditions, including cell stress, to coordinate the levels of RNA processing factors with mRNA levels. To monitor pol II distributions, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) was performed using an anti-pol II antibody (4H8) and cross-linked chromatin preparations from Jurkat cells, treated with or without pol II elongation inhibitor 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) at 10 and 25 ug/ml respectively prior to phorbol 12-myristate 13-acetate (PMA) stimulation, for 5000+ alternative splicing events.
2010-12-09 | E-GEOD-25494 | biostudies-arrayexpress
Project description:The Pol II elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, Spt6- and FACT-depleted cells exhibit processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6- depleted strains, but it is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes.
2024-07-08 | GSE262747 | GEO
Project description:Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter proximal regions and within the first 20-30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20-30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an "accelerator" over specific early gene body regions.
2019-06-28 | GSE116169 | GEO
Project description:Spt5 is a highly conserved RNA polymerase II (Pol II)-associated pausing and elongation factor. However, its impact on global elongation and Pol II processivity in mammalian cells has not been clarified. Here, we show that depleting Spt5 in mouse embryonic fibroblasts (MEFs) does not cause global elongation defects or decreased elongation rates. Instead, in the absence of Spt5, a fraction of Pol II molecules are dislodged during elongation thus decreasing the number of Pol II complexes that complete the transcription cycle. Most strikingly, this decrease is restricted to a narrow window between 15-20 kb from the promoter, a distance which coincides with the stage where accelerating Pol II attains maximum elongation speed and processivity. Consequently, long genes show a greater dependency on Spt5 for optimal elongation efficiency and overall gene expression than short genes. We propose that an important role of Spt5 in mammalian elongation is to promote the processivity of those Pol II complexes that are transitioning towards maximum elongation speed 15-20 kb from the promoter.
2018-03-06 | GSE106313 | GEO
Project description:We used Global Run-on and Sequencing (GRO-seq) to measure the rate of transcription elongation by RNA polymerase II (Pol II) following gene activation. We observed that Pol II elongation rates can vary as much as 4-fold at different genomic loci and in response to two distinct cellular signaling pathways (i.e., estrogen and TNFM-NM-1). Elongation rates are slowest near the promoter and increase during the first ~15 kb transcribed into the gene body. Gene body elongation rates correlate with the density of Pol II, consequently resulting in systematically higher rates of transcript production at genes with higher Pol II density. By monitoring Pol II dynamics following short inductions, we found that E2 stimulates gene expression by increasing Pol II initiation, whereas TNFM-NM-1 stimulates the release of Pol II from promoter proximal pause sites. Collectively, our results identify previously uncharacterized variation in the rate of Pol II elongation and highlight elongation as an important, variable, and regulated rate limiting step in the transcription cycle. Using GRO-seq over a time course (0, 10, 25, and 40 min) of estrogen signaling in ER-alpha positive MCF-7 human breast cancer cells.
2013-04-10 | E-GEOD-41323 | biostudies-arrayexpress
Project description:We used Global Run-on and Sequencing (GRO-seq) to measure the rate of transcription elongation by RNA polymerase II (Pol II) following gene activation. We observed that Pol II elongation rates can vary as much as 4-fold at different genomic loci and in response to two distinct cellular signaling pathways (i.e., estrogen and TNFM-NM-1). Elongation rates are slowest near the promoter and increase during the first ~15 kb transcribed into the gene body. Gene body elongation rates correlate with the density of Pol II, consequently resulting in systematically higher rates of transcript production at genes with higher Pol II density. By monitoring Pol II dynamics following short inductions, we found that E2 stimulates gene expression by increasing Pol II initiation, whereas TNFM-NM-1 stimulates the release of Pol II from promoter proximal pause sites. Collectively, our results identify previously uncharacterized variation in the rate of Pol II elongation and highlight elongation as an important, variable, and regulated rate limiting step in the transcription cycle. Using GRO-seq over a time course (0, 10, 25, and 40 min) of estrogen signaling in ER-alpha positive MCF-7 human breast cancer cells.
2013-04-10 | E-GEOD-41324 | biostudies-arrayexpress