Multi-step recognition of potential 5' splice sites by the Saccharomyces cerevisiae U1 snRNP
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.
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
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.
- Jonathan P Staley, University of Chicago, United States
- Preprint posted: May 18, 2021 (view preprint)
- Received: May 19, 2021
- Accepted: August 11, 2022
- Accepted Manuscript published: August 12, 2022 (version 1)
- Version of Record published: September 1, 2022 (version 2)
© 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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