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

Abstract

Hotspot mutations in the spliceosome gene SF3B1 are reported in B20% of uveal melanomas. SF3B1 is involved in 3 0 -splice site (3 0 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 0 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.

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

“…(D) Intron mutations that reduce pairing with U2 snRNA (U257C and A258C) are sensitive to hsh155 mutations analogous to those found in human disease, whereas the 5 ′ SS, 3 ′ SS, and branch nucleophile mutations are not. Darman et al 2015;DeBoever et al 2015;Alsafadi et al 2016), similar to prp5 mutants in yeast (Xu and Query 2007).…”

“…However, in the presence of mutant SF3B1-K666N (hsh155-K335N or hsh155-K700E), an upstream cryptic BS sequence (CUAAC) was used in which the UA is exactly our yeast wild-type reporter sequence (Darman et al 2015). Similarly, the weak SF3B1-K666T (hsh155-K335T) and SF3B1-R625G mutants prefer cryptic branchpoint sequences that have stronger pairing with U2 snRNA (Alsafadi et al 2016). Together, these examples demonstrate that weaker Prp5-SF3B1 interaction resulted in higher fidelity at the BS region in the human system.…”

“…Disease-related mutations cluster in proteins involved in spliceosome assembly, particularly ones important for intron recognition, such as SF3B1 (also known as SAP155, SF3b155, and Hsh155p), SRSF1, and U2AF (Yoshida et al 2011;Quesada et al 2012). In disease-related SF3B1 mutants, changes in branch site (BS) usage have been identified (Darman et al 2015;Alsafadi et al 2016;Kesarwani et al 2016); however, the mechanisms by which these mutations affect splicing are largely unknown.…”

Chronic lymphocytic leukemia: Time to go past genomics?

“…Most mutations are missense mutations localized within the HEAT domain repeats lying in the carboxyterminal moiety of the protein, indicating a gain of function. Indeed, SF3B1 mutations in various cellular contexts are associated with abnormal splice events [51], which result from promotion of alternative branchpoint usage [52][53][54] (Fig. 2).…”

A sequence-based, deep learning model accurately predicts RNA splicing branchpoints

“…Together, these factors have caused the characterization of branchpoints to lag far behind that such as splicing factor 3b associated cancers 7 and recent reports that expression levels of splicing factor 1 play a vital role in aging 8 .…”