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bioRxiv · 10.1101/2025.06.16.659908

Control of 3' Splice Site Selection in S. cerevisiae by a Highly Conserved Amino Acid within the Prp8 α-finger Domain

Liu, Y.·Paulson, J. C.·Hoskins, A. A.
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Abstract

Precise recognition of the boundaries between exons and introns (splice sites, SS) is essential for the fidelity of gene expression. In contrast with the 5SS, the consensus 3SS sequence in both S. cerevisiae and humans is just three nucleotides long: YAG. How the correct 3SS 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 -finger domain of the Prp8 protein interacts directly with the -3 pyrimidine of the 3SS. To investigate the role of this interaction, we constructed a Prp8Q1594A mutant and studied the impact on splicing and 3SS selection. Using splicing reporter assays and RNA-seq, we show that Prp8Q1594A enables use of non-consensus 3SS by relaxing sequence requirements at the -3 and -2 positions. Consequently, this can change how adjacent 3SS compete with one another during mRNA formation. The ability for 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 -finger acts as a sensor of 3SS accommodation within the spliceosome active site. We propose that conformational change of the -finger either allows or inhibits binding of the Prp22 c-terminal domain. This may provide a mechanism for regulating Prp22 activity in response to 3SS binding.

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Liu, Y., Paulson, J. C., Hoskins, A. A.. 2025-06-20. Control of 3' Splice Site Selection in S. cerevisiae by a Highly Conserved Amino Acid within the Prp8 α-finger Domain. https://doi.org/10.1101/2025.06.16.659908

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