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| Status |
Public on Oct 22, 2017 |
| Title |
RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit [CRAC] |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
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| Summary |
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.
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| Overall design |
RNA binding by Bre5 and Rpo21 was analysed by CRAC in S. cerevisiae. Bre5 full length protein carried a C-terminal HTP tag, in the wild-type background, in the absence of Ubp3 (ubp3∆), or in an rsp5-3 temperature sensitive strain. Bre5-HTP was analysed using a standard CRAC procedure (Granneman et al., 2009). Rpo21 full length protein also carried a C-terminal HTP tag, in the wild-type background or in the absence of Bre5 (bre5∆). To map the locations of RNAPII containing ubiquitinated Rpo21 we applied modification CRAC (mCRAC), including an affinity purification step to specifically enrich for ubiquitinated proteins using a MultiDsk construct (MD), comprised of five UBA domains from the yeast ubiquitin-binding protein Dsk2 fused to GST (Wilson et al., 2012). This procedure led to three samples: “GST-FT” represents the total Rpo21, “MD” represents the ubiquitinated Rpo21, “GST” represent the non-specific binding to a GST column. Rpo21-K1246-HTP was analysed using a standard CRAC procedure. All protein genes were tagged at their endogenous locus and were expressed under the control of their endogenous promoter. We have included duplicates samples and an untagged BY4741 negative control.
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| Contributor(s) |
Milligan L, Sayou C, Tuck A, Reid JE, Alexander R, de Lima Alves F, Rappsilber J, Beggs JD, Kudla G, Tollervey D |
| Citation(s) |
29027900 |
| Submission date |
Feb 15, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Camille Sayou |
| E-mail(s) |
camille.sayou@gmail.com
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| Organization name |
Wellcome Trust Centre for Cell Biology
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| Lab |
Tollervey lab
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| Street address |
Michael Swann Building, Max Born Crescent
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| City |
Edinburgh |
| ZIP/Postal code |
EH9 3BF |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL13821 |
Illumina HiSeq 2000 (Saccharomyces cerevisiae) |
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| Samples (22)
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| This SubSeries is part of SuperSeries: |
| GSE94944 |
RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit |
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| Relations |
| BioProject |
PRJNA374885 |
| SRA |
SRP099866 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE94941_RAW.tar |
158.2 Mb |
(http)(custom) |
TAR (of BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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