Cancer-associated SF3B1 mutants recognize otherwise inaccessible cryptic 3′ splice sites within RNA secondary structures

Recurrent mutations in core splicing factors have been reported in several clonal disorders, including cancers. Mutations in SF3B1, a component of the U2 splicing complex, are the most common. SF3B1 mutations are associated with aberrant pre-mRNA splicing using cryptic 3' splice sites (3' SSs), but the mechanism of their selection is not clear. To understand how cryptic 3' SSs are selected, we performed comprehensive analysis of transcriptome-wide changes to splicing and gene expression associated with SF3B1 mutations in patient samples as well as an experimental model of inducible expression. Hundreds of cryptic 3' SS were detectable across the genome in cells expressing mutant SF3B1. These 3' SS are typically sequestered within RNA secondary structures and poorly accessible compared with their corresponding canonical 3' SS. We hypothesized that these cryptic 3' SS are inaccessible during normal splicing catalysis and that this constraint is overcome in spliceosomes containing mutant SF3B1. This model of secondary structure-dependent selection of cryptic 3' SS was found across multiple clonal processes associated with SF3B1 mutations (myelodysplastic syndrome and chronic lymphocytic leukemia). We validated our model predictions in mini-gene splicing assays. Additionally, we found deregulated expression of proteins with relevant functions in splicing factor-related diseases both in association with aberrant splicing and without corresponding splicing changes. Our results show that SF3B1 mutations are associated with a distinct splicing program shared across multiple clonal processes and define a biochemical mechanism for altered 3' SS choice.