TY - JOUR TI - Multi-step recognition of potential 5' splice sites by the Saccharomyces cerevisiae U1 snRNP AU - Hansen, Sarah R AU - White, David S AU - Scalf, Mark AU - CorrĂȘa, Ivan R AU - Smith, Lloyd M AU - Hoskins, Aaron A A2 - Staley, Jonathan P A2 - Struhl, Kevin A2 - Staley, Jonathan P A2 - Walter, Nils VL - 11 PY - 2022 DA - 2022/08/12 SP - e70534 C1 - eLife 2022;11:e70534 DO - 10.7554/eLife.70534 UR - https://doi.org/10.7554/eLife.70534 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 colocalization 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. KW - splicing KW - RNA KW - snRNP KW - single-molecule fluorescence KW - CoSMoS KW - spliceosome JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -