SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing

Abstract

Mutations in the U2 snRNP component SF3B1 are prominent in myelodysplastic syndromes (MDSs) and other cancers and have been shown recently to alter branch site (BS) or 3 ′ splice site selection in splicing. However, the molecular mechanism of altered splicing is not known. We show here that hsh155 mutant alleles in Saccharomyces cerevisiae, counterparts of SF3B1 mutations frequently found in cancers, specifically change splicing of suboptimal BS pre-mRNA substrates. We found that Hsh155p interacts directly with Prp5p, the first ATPase that acts during spliceosome assembly, and localized the interacting regions to HEAT (Huntingtin, EF3, PP2A, and TOR1) motifs in SF3B1 associated with disease mutations. Furthermore, we show that mutations in these motifs from both human disease and yeast genetic screens alter the physical interaction with Prp5p, alter branch region specification, and phenocopy mutations in Prp5p. These and other data demonstrate that mutations in Hsh155p and Prp5p alter splicing because they change the direct physical interaction between Hsh155p and Prp5p. This altered physical interaction results in altered loading (i.e., "fidelity") of the BS-U2 duplex into the SF3B complex during prespliceosome formation. These results provide a mechanistic framework to explain the consequences of intron recognition and splicing of SF3B1 mutations found in disease.

How do messenger RNA splicing alterations drive myelodysplasia?

“…As noted earlier, RNA splicing alterations imparted by mutations in SF3B1, 12,15,17,18 SRSF2, 6,14 U2AF1, 10,11,20 and ZRSR2 19 are each distinct, and thus, it is not clear what precise splicing changes would be expected in MDS Figure 1 (continued) function required in every cell, overt phenotypic effects of these mutations are only apparent within the retina. In contrast to the U4/U6.U5 tri-snRNP mutations in autosomal dominant retinitis pigmentosa, mutations in the RNA splicing factors SF3B1, U2AF1, SRSF2, and ZRSR2 are enriched in leukemias and subsets of epithelial malignancies.…”

Novel SF3B1 in-frame deletions result in aberrant RNA splicing in CLL patients

“…[6][7][8] Although the majority of SF3B1 hotspot mutations are located in the Py tract interacting region, the reason for inducing aberrant 39 ss selection through reduced branch site fidelity remains unclear. [9][10][11][12][13] The unique signature of aberrant 39 ss junction usage by SF3B1 mutations suggests that these events can be used as biomarkers to discover additional genomic alterations that lead to similar splicing defects. To this end, we analyzed RNA sequencing (RNA-seq) from 215 CLL patients.…”

Splicing factor mutations in MDS RARS and MDS/MPN-RS-T

“…The regions of SF3B1 affected by mutations in cancer are remarkably conserved amongst species and this has allowed two groups to generate mutations in the yeast orthologue of SF3B1, termed Hsh155 [51,52]. In both of these studies, MDS disease-associated mutations in SF3B1 altered interaction between SF3B1 and the protein Prp5, which is a helicase responsible for the first ATP-dependent step in splicing.…”