Multi-step recognition of potential 5' splice sites by the Saccharomyces cerevisiae U1 snRNP

  1. Sarah R Hansen
  2. David S White
  3. Mark Scalf
  4. Ivan R Corrêa Jr
  5. Lloyd M Smith
  6. Aaron A Hoskins  Is a corresponding author
  1. University of Wisconsin-Madison, United States
  2. New England Biolabs, United States

Abstract

In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5' splice site (5' SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5' SS recognition have largely been done in vivo or in cellular extracts where the U1/5' SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used co-localization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5' SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5' SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5' SS and preferentially accumulate at these sequences rather than on close cognates.

Data availability

Source data files have been provided for Figure 1-Supplemental Figure 2. Due its large size, the source data for the single molecule microscopy experiments will be hosted by a campus web server and freely available for public download using Globus at the weblink belowhttps://app.globus.org/file-manager?origin_id=2b62cfc8-0c02-42ca-bb75-1a257d7b4284&origin_path=%2FWe have included this link in the manuscript text with the materials and methods section describing single molecule data collection.

Article and author information

Author details

  1. Sarah R Hansen

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    No competing interests declared.
  2. David S White

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0164-0125
  3. Mark Scalf

    Department of Chemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    No competing interests declared.
  4. Ivan R Corrêa Jr

    New England Biolabs, Ipswich, United States
    Competing interests
    Ivan R Corrêa, is employed by New England Biolabs.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3169-6878
  5. Lloyd M Smith

    Department of Chemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6652-8639
  6. Aaron A Hoskins

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    For correspondence
    ahoskins@wisc.edu
    Competing interests
    Aaron A Hoskins, is conducting sponsored research with and a scientific advisor for Remix Therapeutics, Inc..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9777-519X

Funding

National Institutes of Health (R01 GM122735)

  • Aaron A Hoskins

National Institutes of Health (R35 GM136261)

  • Aaron A Hoskins

National Institutes of Health (R35 GM126914)

  • Lloyd M Smith

National Institutes of Health (T32 GM008505)

  • Sarah R Hansen

National Institutes of Health (F32 GM143780)

  • David S White

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

Publication history

  1. Preprint posted: May 18, 2021 (view preprint)
  2. Received: May 19, 2021
  3. Accepted: August 11, 2022
  4. Accepted Manuscript published: August 12, 2022 (version 1)
  5. Version of Record published: September 1, 2022 (version 2)

Copyright

© 2022, Hansen 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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  1. Sarah R Hansen
  2. David S White
  3. Mark Scalf
  4. Ivan R Corrêa Jr
  5. Lloyd M Smith
  6. Aaron A Hoskins
(2022)
Multi-step recognition of potential 5' splice sites by the Saccharomyces cerevisiae U1 snRNP
eLife 11:e70534.
https://doi.org/10.7554/eLife.70534

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