Senn, K.; Lipinski, K.; Zeps, N. J.; Griffin, A. F.; Wilkinson, M. E.; Hoskins, A.

Pre-mRNA splicing is catalyzed in two steps: 5\' splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (1st and 2nd step factors). We recently identified Fyv6 (FAM192A in humans) as a 2nd step factor in S. cerevisiae; however, we did not determine how widespread Fyv6\'s impact is on the transcriptome. To answer this question, we have used RNA-Seq to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3\' SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-EM structure of a yeast product complex spliceosome containing Fyv6 at 2.3 [A]. The structure reveals that Fyv6 is the only 2nd step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the 1st step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6{Delta} suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of Prp22-dependent, BP distal 3\' SS.