1. Chromosomes and Gene Expression
Download icon

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

  1. Laura Milligan
  2. Camille Sayou
  3. Alex Tuck  Is a corresponding author
  4. Tatsiana Auchynnikava
  5. Jane E A Reid
  6. Ross Alexander
  7. Flavia de Lima Alves
  8. Robin Allshire
  9. Christos Spanos
  10. Juri Rappsilber
  11. Jean D Beggs
  12. Grzegorz Kudla
  13. David Tollervey  Is a corresponding author
  1. University of Edinburgh, United Kingdom
  2. Friedrich Miescher Institute for Biomedical Research, Switzerland
Research Article
  • Cited 9
  • Views 2,576
  • Annotations
Cite this article as: eLife 2017;6:e27082 doi: 10.7554/eLife.27082

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

The following data sets were generated

Article and author information

Author details

  1. Laura Milligan

    Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Camille Sayou

    Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8226-7272
  3. Alex Tuck

    Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
    For correspondence
    alex.tuck@fmi.ch
    Competing interests
    The authors declare that no competing interests exist.
  4. Tatsiana Auchynnikava

    Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Jane E A Reid

    Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Ross Alexander

    Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Flavia de Lima Alves

    Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  8. Robin Allshire

    Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Christos Spanos

    Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  10. Juri Rappsilber

    Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  11. Jean D Beggs

    Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  12. Grzegorz Kudla

    MRC Human Genetics Unit, IGMM, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  13. David Tollervey

    Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
    For correspondence
    d.tollervey@ed.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2894-2772

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

  1. Nick J Proudfoot, University of Oxford, United Kingdom

Publication history

  1. Received: March 22, 2017
  2. Accepted: October 12, 2017
  3. Accepted Manuscript published: October 13, 2017 (version 1)
  4. 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.

Metrics

  • 2,576
    Page views
  • 561
    Downloads
  • 9
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, PubMed Central, Crossref.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Chromosomes and Gene Expression
    2. Microbiology and Infectious Disease
    Michele Felletti et al.
    Research Article

    The ability to regulate DNA replication initiation in response to changing nutrient conditions is an important feature of most cell types. In bacteria, DNA replication is triggered by the initiator protein DnaA, which has long been suggested to respond to nutritional changes; nevertheless, the underlying mechanisms remain poorly understood. Here, we report a novel mechanism that adjusts DnaA synthesis in response to nutrient availability in Caulobacter crescentus. By performing a detailed biochemical and genetic analysis of the dnaA mRNA, we identified a sequence downstream of the dnaA start codon that inhibits DnaA translation elongation upon carbon exhaustion. Our data show that the corresponding peptide sequence, but not the mRNA secondary structure or the codon choice, is critical for this response, suggesting that specific amino acids in the growing DnaA nascent chain tune translational efficiency. Our study provides new insights into DnaA regulation and highlights the importance of translation elongation as a regulatory target. We propose that translation regulation by nascent chain sequences, like the one described, might constitute a general strategy for modulating the synthesis rate of specific proteins under changing conditions.

    1. Chromosomes and Gene Expression
    2. Developmental Biology
    Benoit Roch et al.
    Research Article

    We developed a Xrcc4M61R separation of function mouse line to overcome the embryonic lethality of Xrcc4 deficient mice. XRCC4M61R protein does not interact with Xlf, thus obliterating XRCC4-Xlf filament formation while preserving the ability to stabilize DNA Ligase IV. X4M61R mice, which are DNA repair deficient, phenocopy the Nhej1-/- (known as Xlf -/-) setting with a minor impact on the development of the adaptive immune system. The core NHEJ DNA repair factor XRCC4 is therefore not mandatory for V(D)J recombination aside from its role in stabilizing DNA ligase IV. In contrast, Xrcc4M61R mice crossed on Paxx-/-, Nhej1-/-, or Atm-/- backgrounds are severely immunocompromised, owing to aborted V(D)J recombination as in Xlf-Paxx and Xlf-Atm double KO settings. Furthermore, massive apoptosis of post-mitotic neurons causes embryonic lethality of Xrcc4M61R -Nhej1-/- double mutants. These in vivo results reveal new functional interplays between XRCC4 and PAXX, ATM and Xlf in mouse development and provide new insights in the understanding of the clinical manifestations of human XRCC4 deficient condition, in particular its absence of immune deficiency.