PT  - JOURNAL ARTICLE
AU  - Sarah R. Hansen
AU  - Mark Scalf
AU  - Ivan R. Corrêa, Jr
AU  - Lloyd M. Smith
AU  - Aaron A. Hoskins
TI  - Multi-Factor Authentication of Potential 5’ Splice Sites by the <em>Saccharomyces cerevisiae</em> U1 snRNP
AID  - 10.1101/2021.05.18.443434
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.05.18.443434
4099  - http://biorxiv.org/content/early/2021/05/18/2021.05.18.443434.short
4100  - http://biorxiv.org/content/early/2021/05/18/2021.05.18.443434.full
AB  - 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.IMPACT STATEMENT The yeast U1 snRNP recognizes multiple features of target RNAs to reversibly identify splicing-competent 5’ splice sites.Competing Interest StatementAAH is a consultant and carrying out sponsored research for Remix Therapeutics