Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes

Abstract

The splicing factor SF3B1 is the most frequently mutated gene in the myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA-sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3’ splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3’ splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide, showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

ASpediaFI: Functional interaction analysis of alternative splicing events

“…RI (37.9□53.7%) was most frequently detected in three cases, and the frequency of A3 events was next (Additional File 2: Table S2). The dominant occurrence of RI and A3 events in our result is consistent with a previous MDS analysis study using rMATS [4,6,13,23–26]. However, in the previous study, a more refined final DAS set from two comparisons using both WT and healthy control samples as controls, were selected [7,23].…”

“…In normal cells, splicing factors induce tissue-specific mRNA expression and embryonic stem cell differentiation [2,3]. In contrast, splice site mutations or splicing factor (SF) variants reprogram global splicing events and induce aberrant junctions in cancer cells and other diseased cells [4–6]. Aberrant AS events in cancer cells disrupt the function of tumor suppressor genes and activate the oncogenic pathways [6].…”

Splicing Factor Mutations in Myelodysplasias: Insights from Spliceosome Structures

“…A K700E substitution is the most frequent SF3B1 mutation among hematologic malignancies as well as pancreatic and breast cancers, whereas mutations of R625 (and in some cases K666) predominate in uveal melanomas. Several groups have demonstrated that SF3B1 mutations in cancers and hematologic malignancies are associated with altered selection of 3′ splice sites for a subset of transcripts [47, 49–52], including splicing-induced down-regulation of an iron transporter that may increase iron accumulation in RARS [53]. …”

RNA splicing during terminal erythropoiesis

“…Spliceosomal mutations could exert their effects by perturbing the expression of many genes, or only a few key genes essential for red cell physiology. One attractive candidate is the mitochondrial transporter encoded by ABCB7, which is itself mutated in some patients with X-linked sideroblastic anemia with ataxia [63,64]. Expression of ABCB7 is reduced in SF3B1-mutant MDS patients, apparently due to splicing at an abnormal 3′ splice site that alters translational reading frame, introduces a PTC, and activates NMD [64].…”