Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage

Hotspot mutations in the spliceosome gene SF3B1 are reported in ~20% of uveal melanomas. SF3B1 is involved in 3'-splice site (3'ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear. Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1^R625/K666 mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3'ss. Modelling the differential junctions in SF3B1^WT and SF3B1^R625/K666 cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3'ss-sequence context. SF3B1^WT knockdown or overexpression do not reproduce the SF3B1^R625/K666 splice pattern, qualifying SF3B1^R625/K666 as change-of-function mutants. Mutagenesis of predicted branchpoints reveals that the SF3B1^R625/K666-promoted splice pattern is a direct result of alternative branchpoint usage. Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease.