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Prp8 impacts cryptic but not alternative splicing frequency

Significance The spliceosome selects, and in the case of alternative splicing, contextually distinguishes between, splice sites with extremely high fidelity. However, when a splice site becomes mutated, as in many human diseases, the spliceosome can aberrantly select nearby “cryptic” splice sites, which produce nonfunctional protein. It is unclear how the spliceosome routinely selects against cryptic sequences. Here, we implicate core components of the spliceosome itself in its ability to select against cryptic splice sites and show that how the spliceosome selects against cryptic splice sites is different than how it selects between bona fide splice sites during alternative splicing. In addition to providing insight into an essential cellular process, our work is relevant to the development of therapeutics that act by modulating splice-site choice. Pre-mRNA splicing must occur with extremely high fidelity. Spliceosomes assemble onto pre-mRNA guided by specific sequences (5′ splice site, 3′ splice site, and branchpoint). When splice sites are mutated, as in many hereditary diseases, the spliceosome can aberrantly select nearby pseudo- or “cryptic” splice sites, often resulting in nonfunctional protein. How the spliceosome distinguishes authentic splice sites from cryptic splice sites is poorly understood. We performed a Caenorhabditis elegans genetic screen to find cellular factors that affect the frequency with which the spliceosome uses cryptic splice sites and identified two alleles in core spliceosome component Prp8 that alter cryptic splicing frequency. Subsequent complementary genetic and structural analyses in yeast implicate these alleles in the stability of the spliceosome’s catalytic core. However, despite a clear effect on cryptic splicing, high-throughput mRNA sequencing of these prp-8 mutant C. elegans reveals that overall alternative splicing patterns are relatively unchanged. Our data suggest the spliceosome evolved intrinsic mechanisms to reduce the occurrence of cryptic splicing and that these mechanisms are distinct from those that impact alternative splicing.