U2AF1 mutations alter splice site recognition in hematological malignancies

Abstract

Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 39 splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 39 splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allelespecific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1's zinc finger domains.

The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations

“…We observed, by hierarchical clustering analysis, that all mutations in SRSF2 and U2AF35 induced large-scale changes in alternative splicing upon Dox induction (Figure 1E). Remarkably, even two separate mutant alleles in U2AF35 invoked completely different sets of splicing changes, as reported earlier (Ilagan et al, 2015). We further confirmed these data on a subset of genes by RT-PCR (Figures S2A–S2C).…”

“…Taking advantage of all mutations in the same cellular background, we first revisited an important problem with respect to diverse splicing responses induced by different splicing factor mutations (Ilagan et al, 2015; Qiu et al, 2016). We extracted total RNA from individual cell lines and used highly quantitative RNA annealing selection ligation followed by deep sequencing (RASL-seq), which we developed in the lab, to measure a large number (>5,000) of annotated alternative splicing events in humans (Li et al, 2012).…”

“…In particular, disease development has been functionally linked to the inclusion of a “toxic” exon in the Ezh2 gene in Srsf2 (P95H) knockin mice (Kim et al, 2015) and a polyadenylation switch event in the autophagy gene Atg7 caused by U2af1 (S34F) overexpression on a murine pro-B cell model (Park et al, 2016). Puzzling, however, is the observation that each splicing factor mutation appears to cause a rather unique set of splicing changes in both cellular models and patients (Ilagan et al, 2015; Kim et al, 2015; Qiu et al, 2016; Shirai et al, 2015). Even the same mutation, such as that in SF3B1 , induced largely non-overlapping splicing changes in mouse versus human cells (Mupo et al, 2017).…”

Somatic Mutational Landscape of Splicing Factor Genes and Their Functional Consequences across 33 Cancer Types

“…U2AF1 , like SF3B1 , is associated with the U2 complex, and it is known to recognize the 3′ dinucleotide AG; and, along with its partner U2AF2 that binds to the 3′ poly-Y tract, it promotes assembly of the pre-catalytic spliceosome (Wu et al, 1999). Hotspot mutations at amino acid positions p.S34 and p.Q157 are common in hematological malignancies (Papaemmanuil et al, 2013; Lindsley et al, 2015) and confer distinct splicing phenotypes (Ilagan et al, 2015), affecting exon inclusion rates based on the nucleotide in the −1 and +1 position relative to the 3′ AG dinucleotide, respectively. In TCGA, p.S34F/Y is the dominant hotspot mutation and is observed in multiple tumor types, most notably LAML, LUAD, and UCEC (Figure 3A; Table S2).…”

RNA splicing factors as oncoproteins and tumour suppressors

“…3b). Interestingly, the molecular consequences of U2AF1 mutations are allele-specific 88 . S34 and Q157 mutations respectively affect recognition of the −3 (pyrimidine normally preferred) and +1 (purine normally preferred) positions, where the coordinates are defined with respect to the intron-exon boundary to induce different changes in 3′ splice site recognition.…”