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

Abstract

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’SS) but the mechanism of their selection is not clear. To understand how cryptic 3’SS 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 to 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.

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

“…20 When 23T.G minigene was cotransfected with any of the constructs splicing was observed within exon 10 ( Figure 2C, lanes 8-12). Whereas, when 224C.G minigene was used, only canonical splicing was observed in all cases (lanes [13][14][15][16][17], suggesting that the 23 position relative to cryptic AG is also important for its recognition during the second step of splicing. Further to test branch site utilization, we used ZDHHC16 minigenes with mutated branch sites (WT, 230A.G; mutant, 233 to 235 AAA.GGG).…”

“…[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.…”

DDX54 regulates transcriptome dynamics during DNA damage response

“…S9D). Similarly, introns with increased retention in the mutant compared to wild-type SF3B1 K562 cell line (Kesarwani et al 2016) or in SF3B1-depleted compared to mock-transfected HeLa cells (Kfir et al 2015) showed higher retention upon IR exposure and DDX54 knockdown (Supplemental Fig. S9E,F).…”

“…In this study, we made use of nucleolar proteome data sets (Andersen et al 2005;Moore et al 2011), RBP census (Gerstberger et al 2014), DDR genes (Wood et al 2001;Milanowska et al 2011;Mjelle et al 2015), TP53 target genes (Nikulenkov et al 2012;Menendez et al 2013), spliceosomal subcomplexes (Hegele et al 2012), exon-intron-exon regions (Braunschweig et al 2014), and RNA-seq data sets (Kfir et al 2015;Kesarwani et al 2016;Yoshimoto et al 2017). RNA-seq analysis of TCGA data sets (Sebestyén et al 2016) is described in Supplemental Methods.…”

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

“…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.…”