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 (1 st and 2 nd step factors). We recently identified Fyv6 (FAM192A in humans) as a 2 nd 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 Å. The structure reveals that Fyv6 is the only 2 nd step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the 1 st step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6Δ 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.