RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit
Abstract
Numerous links exist between co-transcriptional RNA processing and the transcribing RNAPII. In particular, pre-mRNA splicing was reported to be associated with slowed RNAPII elongation. Here we identify a site of ubiquitination (K1246) in the catalytic subunit of RNAPII close to the DNA entry path. Ubiquitination was increased in the absence of the Bre5-Ubp3 ubiquitin protease complex. Bre5 binds RNA in vivo, with a preference for exon 2 regions of intron-containing pre-mRNAs and poly(A) proximal sites. Ubiquitinated RNAPII showed similar enrichment. The absence of Bre5 led to impaired splicing and defects in RNAPII elongation in vivo on a splicing reporter construct. Strains expressing RNAPII with a K1246R mutation showed reduced cotranscriptional splicing. 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.
Data availability
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RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunitPublicly available at the NCBI Gene Expression Omnibus. Accession no:GSE94944, subseries GSE94942.
Article and author information
Author details
Funding
Wellcome (77248)
- David Tollervey
Medical Research Council
- Grzegorz Kudla
Wellcome (104648)
- Jean D Beggs
Wellcome (108504)
- Juri Rappsilber
Wellcome (93853)
- Jane E A Reid
Wellcome (97383)
- Grzegorz Kudla
Wellcome (92076)
- David Tollervey
Wellcome (200885)
- Robin Allshire
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Nick J Proudfoot, University of Oxford, United Kingdom
Version history
- Received: March 22, 2017
- Accepted: October 12, 2017
- Accepted Manuscript published: October 13, 2017 (version 1)
- Version of Record published: November 6, 2017 (version 2)
Copyright
© 2017, Milligan et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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- Chromosomes and Gene Expression
Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.
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- Chromosomes and Gene Expression
- Genetics and Genomics
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