Degenerate minigene library analysis enables identification of altered branch point utilization by mutant splicing factor 3B1 (SF3B1)

Cancer-associated mutations of the core splicing factor 3 B1 (SF3B1) result in selection of novel 3splice sites (3SS), but precise molecular mechanisms of oncogenesis remain unclear. SF3B1 stabilizes the interaction between U2 snRNP and branch point (BP) on the pre-mRNA. It has hence been speculated that a change in BP selection is the basis for novel 3SS selection. Direct quantitative determination of BP utilization is however technically challenging. To define BP utilization by SF3B1-mutant spliceosomes, we used an overexpression approach in human cells as well as a complementary strategy using isogenic murine embryonic stem cells with monoallelic K700E mutations constructed via CRISPR/Cas9-based genome editing and a dual vector homology-directed repair methodology. A synthetic minigene library with degenerate regions in 3 intronic regions (3.4 million individual minigenes) was used to compare BP usage of SF3B1(K700E) and SF3B1(WT). Using this model, we show that SF3B1(K700E) spliceosomes utilize non-canonical sequence variants (at position -1 relative to BP adenosine) more frequently than wild-type spliceosomes. These predictions were confirmed using minigene splicing assays. Our results suggest a model of BP utilization by mutant SF3B1 wherein it is able to utilize non-consensus alternative BP sequences by stabilizing weaker U2-BP interactions.