Control of 3' splice site selection in S. cerevisiae by a highly conserved amino acid within the Prp8 α-finger domain.

Precise recognition of the boundaries between exons and introns (splice sites [SS]) is essential for the fidelity of gene expression. In contrast with the 5'SS, the consensus 3'SS sequence in both Saccharomyces cerevisiae and humans is just three nucleotides long: YAG. How the correct 3'SS is chosen among many possible alternates by the spliceosome is often unclear but likely involves proofreading by the Prp22 ATPase. In cryo-EM structures of spliceosome product (P) complexes, glutamine 1594 in the highly conserved α-finger domain of the Prp8 protein interacts directly with the -3 pyrimidine of the 3'SS. To investigate the role of this interaction, we constructed a Prp8Q1594A mutant and studied the impact on splicing and 3'SS selection. Using splicing reporter assays and RNA-seq, we show that Prp8Q1594A enables use of nonconsensus 3'SS by relaxing sequence requirements at the -3 and -2 positions. Consequently, this can change how adjacent 3'SS compete with one another during mRNA formation. The ability of Prp8Q1594A to support splicing at non-YAG sites depends on the splicing factors Prp18 and Fyv6, and Prp8Q1594A has genetic interactions with Prp22 mutants. Together, these findings suggest that the Prp8 α-finger acts as a sensor of 3'SS accommodation within the spliceosome active site. We propose that conformational change of the α-finger either allows or inhibits binding of the Prp22 C-terminal tail. This may provide a mechanism for regulating Prp22 activity in response to 3'SS binding.