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Published on: October 9, 2014
Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic
H Dolatshad1,2, A Pellagatti1,2, F G Liberante3
1Bloodwise Molecular Haematology Unit, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
Abstract:
The splicing factor SF3B1 is the most frequently mutated gene in 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.
Insights
Mutations in the splicing factor SF3B1 cause aberrant RNA splicing in myelodysplastic syndromes (MDS). This leads to iron accumulation in red blood cells by downregulating the ABCB7 iron exporter via nonsense-mediated decay.
Area of Science:
- Hematology
- Molecular Biology
- Genetics
Background:
- SF3B1 mutations are common in myelodysplastic syndromes (MDS) and linked to ring sideroblasts (RS).
- Iron accumulation in mitochondria is a hallmark of MDS with RS.
Purpose of the Study:
- To investigate cryptic splicing abnormalities in SF3B1-mutant MDS with RS.
- To elucidate the mechanism of iron dysregulation in these patients.
Main Methods:
- RNA sequencing of hematopoietic stem cells (HSCs) from SF3B1-mutant MDS patients.
- Analysis of alternative splice site usage and downstream gene expression.
- Nonsense-mediated decay (NMD) inhibition experiments using cycloheximide.
Main Results:
- Identified widespread cryptic splicing events in SF3B1-mutant MDS HSCs.
- Demonstrated aberrant splicing of the iron transporter ABCB7, leading to premature termination codons.
- Showed that aberrant ABCB7 mRNA is degraded by NMD, reducing iron exporter levels.
Conclusions:
- Aberrant splicing of ABCB7 due to SF3B1 mutations causes its downregulation via NMD.
- This NMD-induced downregulation of ABCB7 contributes to mitochondrial iron accumulation in MDS with RS.
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