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RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit [CRAC]

ABSTRACT: Genome wide screens identified negative genetic interactions between several cofactors of the exosome nuclease complex and the Bre5-Ubp3 ubiquitin protease complex. RNA-binding was shown for Bre5 with enrichment for sites over exon 2 of spliced pre-mRNAs and close to poly(A) sites. An inducible splicing-reporter showed a requirement for Bre5 in efficient in vivo splicing and for normal RNAPII elongation, specifically on splicing-competent genes. A Bre5-Ubp3 sensitive site of RNAPII ubiquitination was mapped at Lys1246 at the entrance to the active site of the large subunit. Ubiquitinated RNAPII was depleted at the TSS but enriched at the 5’ end of exon 2 and upstream of poly(A) sites, similar to Bre5. Mutation of Lys1246 reduced RNAPII occupancy upstream of the poly(A) site, consistent with reduced pausing at a potential surveillance site, but increased RNAPII residence downstream of the poly(A) site. Strains expressing RNAPII with the Lys1246 mutation showed increased levels of unspliced but poly(A)+ RNA, indicating reduced cotranscriptional splicing efficiency. We propose that ubiquinitation of RNAPII is induced by RNA processing events and linked to transcriptional pausing, which is released by Bre5-Ubp3 associated with the nascent transcript.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE94941 | GEO | 2017/10/22

SECONDARY ACCESSION(S): PRJNA374885

REPOSITORIES: GEO

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RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit [RNA-Seq]

Project description:Genome wide screens identified negative genetic interactions between several cofactors of the exosome nuclease complex and the Bre5-Ubp3 ubiquitin protease complex. RNA-binding was shown for Bre5 with enrichment for sites over exon 2 of spliced pre-mRNAs and close to poly(A) sites. An inducible splicing-reporter showed a requirement for Bre5 in efficient in vivo splicing and for normal RNAPII elongation, specifically on splicing-competent genes. A Bre5-Ubp3 sensitive site of RNAPII ubiquitination was mapped at Lys1246 at the entrance to the active site of the large subunit. Ubiquitinated RNAPII was depleted at the TSS but enriched at the 5’ end of exon 2 and upstream of poly(A) sites, similar to Bre5. Mutation of Lys1246 reduced RNAPII occupancy upstream of the poly(A) site, consistent with reduced pausing at a potential surveillance site, but increased RNAPII residence downstream of the poly(A) site. Strains expressing RNAPII with the Lys1246 mutation showed increased levels of unspliced but poly(A)+ RNA, indicating reduced cotranscriptional splicing efficiency. We propose that ubiquinitation of RNAPII is induced by RNA processing events and linked to transcriptional pausing, which is released by Bre5-Ubp3 associated with the nascent transcript.

2017-10-22 | GSE94942 | GEO

Regulation of pre-mRNA splicing by m6A through the low-complexity protein hnRNPG and co-transcriptional pausing

Project description:Co-transcriptional processing of nascent transcripts is coupled with transcription through the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). The mRNA modification N6-methyladenosine (m6A) occurs co-transcriptionally and impacts pre-mRNA processing. How alternative splicing of pre-mRNA is co-transcriptionally regulated in an m6A-dependent manner is not well understood. Furthermore, splicing regulation through RNAPII pausing has been frequently suggested but the underlying control mechanism remains unclear. Here, we show that the m6A reader protein hnRNPG directly binds to the phosphorylated CTD of RNAPII using Arg-Gly-Gly (RGG) motifs in its low-complexity region. This RGG-phospho-CTD interaction and nascent RNA binding by hnRNPG enables its co-transcriptional association with RNAPII, resulting in transcriptome-wide regulation of alternative splicing and transcript abundance. m6A sites near exon splice sites in nascent mRNA further modulate hnRNPG binding and exon splicing. Exon inclusion is associated with RNAPII pausing downstream of the m6A site. Our results reveal an integrated nuclear mechanism of m6A-mediated gene regulation, in which an m6A reader protein regulates gene expression by using RGG motifs to co-transcriptionally interact with both RNAPII and m6A-modified nascent pre-mRNA, while the interplay between hnRNPG binding and RNAPII pausing modulates alternative splicing regulation.

2019-09-05 | GSE114311 | GEO

TCERG1 regulates alternative splicing of Bcl-x gene by modulating the rate of RNAPII transcription

Project description:Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNAPII may therefore have a fundamental impact in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-xs) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show evidence for in vitro and in vivo interaction of TCERG1 with the Bcl-x pre-mRNA. Transcription profile analysis reveals that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the pro-apoptotic Bcl-xS 5’ splice site. ChIP-Seq

2012-01-12 | E-GEOD-33716 | biostudies-arrayexpress

TCERG1 regulates alternative splicing of Bcl-x gene by modulating the rate of RNAPII transcription

2012-01-12 | GSE33716 | GEO

Effects of dynamic cytosine methylation on alternative pre-mRNA splicing in T lymphocytes

Project description:Intragenic 5-methylcytosine and CTCF mediate opposing affects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF binding sites were unclear. In this study, we identify the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF-mediated alternative splicing through conversion of 5-methylcytosine to its oxidation derivatives. Transcriptional profiling of human T-lymphocytes in the naÃ¯ve and activated states was performed by RNA-Seq. Methylation status (5mC and 5hmC) was assayed by medIP-Seq. CTCF binding sites were identified by ChIP-Seq.

2016-01-04 | E-GEOD-74850 | biostudies-arrayexpress

Transcription elongation factor ELL2 influences splicing versus poly(A) site choice in the Ig heavy chain gene

Project description:Processing of Immunoglobulin heavy chain (IgH) mRNA is a paradigm for competition between splicing and polyadenylation. In plasma cells pre-mRNA is polyadenylated mainly at the promoter-proximal secretory site while B-cells utilize a cryptic 5’ splice site in the last secretory-specific exon; these are mutually exclusive events for all IgH pre-mRNAs. Transcription elongation factor ELL2, more abundant in plasma cells relative to B-cells, was down-modulated by overexpression of heterogenous ribonucleoprotein F, a condition which reduced production of secretory IgH mRNA. Transfection of B-cells with ELL2 and the IgH reporter showed an accelerated use of the secretory poly(A) site, positioned in competition with the splice to M1; a small interfering RNA to ELL2 reduced expression of IgH secretory mRNA. Co-transcription factors ELL1 and PC4 were ineffective at driving secretory-poly(A) site use. ELL2 had little effect on poly(A) site choice with reporters containing tandem-linked poly(A) sites. Shorter forms of ELL2 protein result from both internal initiation at M186 and protein processing. An alternative splicing reporter driven by IgH or non-Ig promoters revealed that ELL2 and its M186 initiated form were able to accelerate exon skipping. Therefore, ELL2 influences IgH pre-mRNA processing through facilitating skipping of the alternative splice to the membrane form.

2008-06-01 | GSE11586 | GEO

Effects of dynamic cytosine methylation on alternative pre-mRNA splicing in T lymphocytes

2016-01-04 | GSE74850 | GEO

Transcription profiling of mouse B cells transfected with ELL2 vs. controls reveals transcription elongation factor ELL2 influences splicing versus poly(A) site choice in the Ig heavy chain gene

Project description:Processing of Immunoglobulin heavy chain (IgH) mRNA is a paradigm for competition between splicing and polyadenylation. In plasma cells pre-mRNA is polyadenylated mainly at the promoter-proximal secretory site while B-cells utilize a cryptic 5â?? splice site in the last secretory-specific exon; these are mutually exclusive events for all IgH pre-mRNAs. Transcription elongation factor ELL2, more abundant in plasma cells relative to B-cells, was down-modulated by overexpression of heterogenous ribonucleoprotein F, a condition which reduced production of secretory IgH mRNA. Transfection of B-cells with ELL2 and the IgH reporter showed an accelerated use of the secretory poly(A) site, positioned in competition with the splice to M1; a small interfering RNA to ELL2 reduced expression of IgH secretory mRNA. Co-transcription factors ELL1 and PC4 were ineffective at driving secretory-poly(A) site use. ELL2 had little effect on poly(A) site choice with reporters containing tandem-linked poly(A) sites. Shorter forms of ELL2 protein result from both internal initiation at M186 and protein processing. An alternative splicing reporter driven by IgH or non-Ig promoters revealed that ELL2 and its M186 initiated form were able to accelerate exon skipping. Therefore, ELL2 influences IgH pre-mRNA processing through facilitating skipping of the alternative splice to the membrane form. Experiment Overall Design: AxJ plasma cells were stably transfected to overexpress hnRNP-F, -H or empty vector. Clones showing high overexpression levels of F or H by western blot were selected. The IgH sec to mb ratios of these clones were determined. A global gene expression analysis was performed on mRNA from two clones from hnRNP-F, which demonstrated a lower sec:mb ratio, and one from each of the controls: overexpression of hnRNP-H, or transfection with empty vector, or A20 B-cells, using Affymetrix gene micro array technology.

2008-06-18 | E-GEOD-11586 | biostudies-arrayexpress

The chromatin remodeler Ino80 mediates alternative RNAPII pausing site determination

Project description:Promoter-proximal pausing of RNAPII is a critical step in early transcription elongation to precisely regulate gene expression. Here, we observed evidence of promoter-proximal pausing like distributions of RNAPII in S. cerevisiae. We find that Ino80p loss caused the transition of RNAPII pausing to the alternative pausing site at which is tightly associated with the +1 nucleosome. These Ino80p dependent genes showed better nucleosome positioning around the main pausing site which suggested the regulation of RNAPII pausing by Ino80p in a nucleosome context-dependent manner. Furthermore, we examined the similar INO80 dependent RNAPII pausing site determination in mESCs. Altogether, we hypothesize a highly conserved role of the chromatin remodeling Ino80 complex in controlling the early RNAPII elongation to establish intact pausing in various organisms, from budding yeast to mouse.

2021-08-20 | GSE158622 | GEO

Precise Maps of RNA Polymerase Reveal How Promoters Direct Initiation and Pausing

Project description:Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a Precision nuclear Run-On and sequencing assay (PRO-seq) to map the genome-wide distribution of transcriptionally-engaged Pol II at base-pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' poly-adenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing . We test this ‘Complex Interaction’ model with insertional mutagenesis of the Drosophila Hsp70 core promoter. Identification of RNA polymerase active sites in Drosophila S2 cell line using PRO-seq method. Identification of transcription initiation sites in Drosophila S2 cell line using PRO-cap method. Identification of changes in RNA polymerase active sites on transgenic Hsp70 promoters upon disruption of DNA sequence elements in 3 transgenic fly lines using PRO-seq method.

2013-02-01 | E-GEOD-42117 | biostudies-arrayexpress

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