Pre-mRNA Splicing is Facilitated by an Optimal RNA Polymerase II Elongation Rate
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: David Bentley
PROVIDER: E-GEOD-63375 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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).
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Project description:HEK293T cells were transfected with the Rbp1-amr or slow (R729H-amr) α-amanitin resistant subunit of RNA Pol II and selected with α-amanitin 24 hours after transfection for additional 24 hours. Total RNA was extracted and global changes in gene expression were determined using microarray chips. MiRNAs are transcribed by RNA pol II but the transcriptional features influencing their synthesis are poorly defined. Here we report that a TATA-box in miRNA and a subset of protein-coding genes is associated with increased sensitivity to a slow rate of transcription elongation. We also show that promoters driven by TATA-box or NF-κB elicit high transcription re-initiation rate, but paradoxically lower levels of miRNA. Interestingly, miRNA synthesis was converted to a more productive mode by decreasing initiation rate, but less productive when the re-initiation rate increased. This phenomenon was found to be associated with a delay in miR-146a induction by NF-κB. We also demonstrate that miRNAs are remarkably strong pause sites. Our findings suggest that lower efficiency of miRNA synthesis directed by the TATA-box or NF-κB is a consequence of frequent transcription initiation that lead to Pol II crowding at pause sites, thereby increasing the chance of collision and premature termination. These findings highlight the importance of the transcription initiation mechanism for miRNA synthesis, and have implications for TATA-box promoters in general. HEK293T cells were transfected with plasmids directing the expression of α-amanitin-resistant variants of Pol II (Rpb1-amr and R749H-amr). α-amanitin was added and RNA was prepared 24 and 48 h later, respectively. The data provided is from 3 Rpb1-amr vs 3 R749H-amr (6 samples).
2013-07-01 | E-GEOD-48163 | biostudies-arrayexpress
Project description:RNAP II is frequently paused near gene promoters in mammals and its transition to productive elongation requires active recruitment of P-TEFb, a cyclin-dependent kinase for RNAP II and other key transcription elongation factors. A fraction of P-TEFb is sequestered in an inhibitory complex containing the 7SK noncoding RNA, but it has been unclear how P-TEFb is switched from the 7SK complex to RNAP II during transcription activation. We report that SRSF2 (also known as SC35, an SR splicing factor) is part of the 7SK complex assembled at gene promoters and plays a direct role in transcription pause release. We demonstrate RNA-dependent, coordinated release of SRSF2 and P-TEFb from the 7SK complex and transcription activation via SRSF2 binding to promoter-associated nascent RNA. These findings reveal an unanticipated SR protein function, a role for promoter-proximal nascent RNA in gene activation, and an analogous mechanism to HIV Tat/TAR for activating cellular genes. SR ChIP-seq, Pol II ChIP-seq, and Gro-seq
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2014-06-09 | E-GEOD-49134 | biostudies-arrayexpress
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
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Project description:Posttranslational histone modifications play important roles in regulating chromatin structure and function. Histone H2B ubiquitination and deubiquitination have been implicated in transcriptional regulation, but the function of H2B deubiquitination is not well defined, particularly in higher eukaryotes. Here we report the purification of USP49 as a histone H2B specific deubiquitinase and demonstrate that H2B deubiquitination by USP49 is required for efficient co-transcriptional splicing of a large set of exons. USP49 forms a complex with RVB1 and SUG1, and specifically deubiquitinates histone H2B in vitro and in vivo. USP49 knockdown results in small changes in gene expression, but affects the abundance of over 9,000 isoforms. Exons down-regulated in USP49 knockdown cells show both elevated levels of alternative splicing and a general decrease in splicing efficiency. Importantly, USP49 is relatively enriched at this set of exons. USP49 knockdown increased uH2B levels at these exons as well as upstream 3’ and downstream 5’ intronic splicing elements. Change in H2B ubiquitination level, as modulated by USP49, regulates U1A and U2B association with chromatin and binding to nascent pre-mRNA. Although H3 levels are relatively stable after USP49 depletion, H2B levels at these exons are dramatically increased, suggesting that uH2B may enhance nucleosome stability. Therefore, this study identifies USP49 as a histone H2B specific deubiquitinase and uncovers a critical role for H2B deubiquitination in co-transcriptional pre-mRNA processing events. Examination of gene expression in wild type and USP49 knockdown cells [RNA-Seq]
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Project description:Posttranslational histone modifications play important roles in regulating chromatin structure and function. Histone H2B ubiquitination and deubiquitination have been implicated in transcriptional regulation, but the function of H2B deubiquitination is not well defined, particularly in higher eukaryotes. Here we report the purification of USP49 as a histone H2B specific deubiquitinase and demonstrate that H2B deubiquitination by USP49 is required for efficient co-transcriptional splicing of a large set of exons. USP49 forms a complex with RVB1 and SUG1, and specifically deubiquitinates histone H2B in vitro and in vivo. USP49 knockdown results in small changes in gene expression, but affects the abundance of over 9,000 isoforms. Exons down-regulated in USP49 knockdown cells show both elevated levels of alternative splicing and a general decrease in splicing efficiency. Importantly, USP49 is relatively enriched at this set of exons. USP49 knockdown increased uH2B levels at these exons as well as upstream 3’ and downstream 5’ intronic splicing elements. Change in H2B ubiquitination level, as modulated by USP49, regulates U1A and U2B association with chromatin and binding to nascent pre-mRNA. Although H3 levels are relatively stable after USP49 depletion, H2B levels at these exons are dramatically increased, suggesting that uH2B may enhance nucleosome stability. Therefore, this study identifies USP49 as a histone H2B specific deubiquitinase and uncovers a critical role for H2B deubiquitination in co-transcriptional pre-mRNA processing events. Examination of histone H2B ubiquitination in wild type and USP49 knockdown cells [ChIP-Seq]
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