Aberrant splicing and defective mRNA production induced by somatic spliceosome mutations in myelodysplasia

Abstract

Spliceosome mutations are frequently found in myelodysplasia. Splicing alterations induced by these mutations, their precise targets, and the effect at the transcript level have not been fully elucidated. Here we report transcriptomic analyses of 265 bone marrow samples from myelodysplasia patients, followed by a validation using CRISPR/Cas9-mediated gene editing and an assessment of nonsense-mediated decay susceptibility. Small but widespread reduction of intron-retaining isoforms is the most frequent splicing alteration in SF3B1-mutated samples. SF3B1 mutation is also associated with 3′ splice site alterations, leading to the most pronounced reduction of canonical transcripts. Target genes include tumor suppressors and genes of mitochondrial iron metabolism or heme biosynthesis. Alternative exon usage is predominant in SRSF2- and U2AF1-mutated samples. Usage of an EZH2 cryptic exon harboring a premature termination codon is increased in both SRSF2- and U2AF1-mutated samples. Our study reveals a landscape of splicing alterations and precise targets of various spliceosome mutations.

Distinct and convergent consequences of splice factor mutations in myelodysplastic syndromes

“…A similar segregation was observed in our analysis of an independent cohort of MDS samples with U2AF1 mutations (accession number EGAS00001002346) 28 (Figure S4B), indicating distinct splicing consequences of these two hotspots, similar to the findings in previous reports. 10,11 Both S34 and Q157 mutations of U2AF1 generally affected low number of largely non-overlapping splicing events within each group (Figure 1A-D).…”

“…These genes may represent possible common mediators downstream of spliceosome mutations.3.4 | Splicing targets of U2AF1 S34 and Q157 mutations are distinctThe PCA analysis of splicing changes in U2AF1 mutant samples showed two distinct clusters with samples separated according to mutation sites -S34 and R156/Q157 (referred to as U2AF1 Q157 mut) (Figure S4A).A similar segregation was observed in our analysis of an independent cohort of MDS samples with U2AF1 mutations (accession number EGAS00001002346)28 (Figure S4B), indicating distinct splicing consequences of these two hotspots, similar to the findings in previous reports 10,11. Other than EZH2, 10 other genes were mis-spliced in more than two mutant group(Figure 2A).…”

“…30 More recently, splicing changes and transcriptional consequences of abnormal RNA splicing were also investigated in a large cohort of MDS samples. 28 Spliceosome mutations in MDS co-occur frequently with other somatic mutations typically affecting the epigenetic regulators. 6,7 In this study, we selected samples lacking mutations in 30 commonly mutated genes with an aim to uncover bona fide splicing alterations caused by each splice factor mutation.…”

Mutations in spliceosome genes and therapeutic opportunities in myeloid malignancies

“…In addition, the mechanistic basis of how splicing factor mutations confer selective advantage during the evolution of MDS pathogenesis remains a mystery. Although recent large‐scale transcriptomic analysis of mutations affecting SF3B1 , SRSF2 , and U2AF1 revealed global splicing dysregulation consistent with their predicted effects, there was minimal overlap between mis‐spliced transcripts altered by each of the mutant proteins . To identify key splicing abnormalities induced by mutant splicing factors, several studies provided functional evidence that expression of the abnormal splice isoforms in normal cells, or reintroduction of the canonical isoforms in spliceosomal mutant cells, can only partially phenocopy or rescue the effects of mutant spliceosome proteins .…”

“…73 Although recent large-scale transcriptomic analysis of mutations affecting SF3B1, SRSF2, and U2AF1 revealed global splicing dysregulation consistent with their predicted effects, there was minimal overlap between mis-spliced transcripts altered by each of the mutant proteins. 93,94 To identify key splicing abnormalities induced by mutant splicing factors, several studies provided functional evidence that expression of the abnormal splice isoforms in normal cells, or reintroduction of the canonical isoforms in spliceosomal mutant cells, can only partially phenocopy or rescue the effects of mutant spliceosome proteins. 72,80,95,96 These findings suggest that the pathological effects of spliceosome mutations are imparted through multiple mis-spliced products simultaneously.…”