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 S. 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 Prp8^Q1594A mutant and studied the impact on splicing and 3'SS selection. Using splicing reporter assays and RNA-seq, we show that Prp8^Q1594A enables use of non-consensus 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 for Prp8^Q1594A to support splicing at non-YAG sites depends on the splicing factors Prp18 and Fyv6, and Prp8^Q1594A 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 domain. This may provide a mechanism for regulating Prp22 activity in response to 3'SS binding.