1. Chromosomes and Gene Expression
  2. Genetics and Genomics

m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site

  1. Matthew T Parker
  2. Beth K Soanes
  3. Jelena Kusakina
  4. Antoine Larrieu
  5. Katarzyna Knop
  6. Nisha Joy
  7. Friedrich Breidenbach
  8. Anna V Sherwood
  9. Geoffrey J Barton
  10. Sebastian M Fica
  11. Brendan H Davies  Is a corresponding author
  12. Gordon Grant Simpson  Is a corresponding author
  1. University of Dundee, United Kingdom
  2. University of Leeds, United Kingdom
  3. University of Oxford, United Kingdom
https://doi.org/10.7554/eLife.78808
Share this article
Cite this article
  1. Matthew T Parker
  2. Beth K Soanes
  3. Jelena Kusakina
  4. Antoine Larrieu
  5. Katarzyna Knop
  6. Nisha Joy
  7. Friedrich Breidenbach
  8. Anna V Sherwood
  9. Geoffrey J Barton
  10. Sebastian M Fica
  11. Brendan H Davies
  12. Gordon Grant Simpson
(2022)
m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site
eLife 11:e78808.
https://doi.org/10.7554/eLife.78808
  2. Download BibTeX
  3. Download .RIS

Abstract

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5' splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N6 methylation, but the role of this m6A modification is poorly understood. Here we show that m6A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIO1, is required for Arabidopsis U6 snRNA m6A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5' splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m6A influences 3' splice site usage. We generalise these findings to reveal two major classes of 5' splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m6A modification contributes to the selection of degenerate 5' splice sites crucial to alternative splicing.

Data availability

Illumina sequencing data from the genetic screen that identified fio1-4 is available from ENA accession PRJEB51468. Col-0, fip37-4 and fio1-1 nanopore DRS data is available from ENA accession PRJEB51364. Col-0 and fio1-3 Illumina RNA-Seq data is available from ENA accession PRJEB51363.

The following data sets were generated

Article and author information

Author details

  1. Matthew T Parker

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0891-8495

  2. Beth K Soanes

    School of Biology, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  3. Jelena Kusakina

    School of Biology, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  4. Antoine Larrieu

    School of Biology, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  5. Katarzyna Knop

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2636-9450

  6. Nisha Joy

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  7. Friedrich Breidenbach

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9610-1927

  8. Anna V Sherwood

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  9. Geoffrey J Barton

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9014-5355

  10. Sebastian M Fica

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  11. Brendan H Davies

    School of Biology, University of Leeds, Leeds, United Kingdom
    For correspondence
    b.h.davies@leeds.ac.uk
    Competing interests
    The authors declare that no competing interests exist.

  12. Gordon Grant Simpson

    School of Life Sciences, University of Dundee, Dundee, United Kingdom
    For correspondence
    g.g.simpson@dundee.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6744-5889

Funding

Biotechnology and Biological Sciences Research Council (BB/W002302/1)

  • Geoffrey J Barton
  • Gordon Grant Simpson

Wellcome Trust (220212/Z/20/Z)

  • Sebastian M Fica

Global Challenges Research Fund (University of Dundee Global Challenges Research Fund)

  • Geoffrey J Barton
  • Gordon Grant Simpson

Biotechnology and Biological Sciences Research Council (BB/M010066/1)

  • Geoffrey J Barton
  • Gordon Grant Simpson

Biotechnology and Biological Sciences Research Council (BB/M004155/1)

  • Geoffrey J Barton
  • Gordon Grant Simpson

Biotechnology and Biological Sciences Research Council (BB/W007673/1)

  • Geoffrey J Barton
  • Gordon Grant Simpson

Biotechnology and Biological Sciences Research Council (BB/M000338/1)

  • Brendan H Davies

Biotechnology and Biological Sciences Research Council (BB/W007967/1)

  • Brendan H Davies

Biotechnology and Biological Sciences Research Council (BB/T007222/1)

  • Beth K Soanes
  • Brendan H Davies

HORIZON EUROPE Marie Sklodowska-Curie Actions (799300)

  • Katarzyna Knop

HORIZON EUROPE Marie Sklodowska-Curie Actions (896598)

  • Nisha Joy

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Jonathan P Staley, University of Chicago, United States

Version history

  1. Received: March 21, 2022
  2. Preprint posted: April 5, 2022 (view preprint)
  3. Accepted: November 20, 2022
  4. Accepted Manuscript published: November 21, 2022 (version 1)
  5. Version of Record published: December 30, 2022 (version 2)

Copyright

© 2022, Parker 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

  • 2,342
    views
  • 316
    downloads
  • 16
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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)

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

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

  1. Matthew T Parker
  2. Beth K Soanes
  3. Jelena Kusakina
  4. Antoine Larrieu
  5. Katarzyna Knop
  6. Nisha Joy
  7. Friedrich Breidenbach
  8. Anna V Sherwood
  9. Geoffrey J Barton
  10. Sebastian M Fica
  11. Brendan H Davies
  12. Gordon Grant Simpson
(2022)
m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site
eLife 11:e78808.
https://doi.org/10.7554/eLife.78808

Share this article

https://doi.org/10.7554/eLife.78808

Categories and tags

Research organisms

Further reading

    1. Chromosomes and Gene Expression
    2. Developmental Biology

    Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind

    F Javier DeHaro-Arbona, Charalambos Roussos ... Sarah Bray
    Research Article

    Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation.

    1. Chromosomes and Gene Expression

    The role of RNA in the maintenance of chromatin domains as revealed by antibody mediated proximity labelling coupled to mass spectrometry

    Rupam Choudhury, Anuroop Venkateswaran Venkatasubramani ... Axel Imhof
    Research Article

    Eukaryotic chromatin is organized into functional domains, that are characterized by distinct proteomic compositions and specific nuclear positions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To gain molecular insight into these domains and explore their composition, we developed an antibody-based proximity-biotinylation method targeting the RNA and proteins constituents. The method that we termed Antibody-Mediated-Proximity-Labelling-coupled to Mass Spectrometry (AMPL-MS) does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize the composition of chromatin domains based on specific signature proteins or histone modifications. To demonstrate the utility of our approach we used AMPL-MS to characterize the molecular features of the chromocenter as well as the chromosome territory containing the hyperactive X-chromosome in Drosophila. This analysis identified a number of known RNA binding proteins in proximity of the hyperactive X and the centromere, supporting the accuracy of our method. In addition, it enabled us to characterize the role of RNA in the formation of these nuclear bodies. Furthermore, our method identified a new set of RNA molecules associated with the Drosophila centromere. Characterization of these novel molecules suggested the formation of R-loops in centromeres, which we validated using a novel probe for R-loops in Drosophila. Taken together, AMPL-MS improves the selectivity and specificity of proximity ligation allowing for novel discoveries of weak protein-RNA interactions in biologically diverse domains.

    1. Cancer Biology
    2. Chromosomes and Gene Expression

    Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition

    Gregory Caleb Howard, Jing Wang ... William P Tansey
    Research Article

    The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the ‘WIN’ site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.