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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 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.

SUBMITTER: Dolatshad H

PROVIDER: S-EPMC5029572 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Coordinated missplicing of TMEM14C and ABCB7 causes ring sideroblast formation in SF3B1-mutant myelodysplastic syndrome.

Project description:SF3B1 splicing factor mutations are near-universally found in myelodysplastic syndromes (MDS) with ring sideroblasts (RS), a clonal hematopoietic disorder characterized by abnormal erythroid cells with iron-loaded mitochondria. Despite this remarkably strong genotype-to-phenotype correlation, the mechanism by which mutant SF3B1 dysregulates iron metabolism to cause RS remains unclear due to an absence of physiological models of RS formation. Here, we report an induced pluripotent stem cell model of SF3B1-mutant MDS that for the first time recapitulates robust RS formation during in vitro erythroid differentiation. Mutant SF3B1 induces missplicing of ∼100 genes throughout erythroid differentiation, including proposed RS driver genes TMEM14C, PPOX, and ABCB7. All 3 missplicing events reduce protein expression, notably occurring via 5' UTR alteration, and reduced translation efficiency for TMEM14C. Functional rescue of TMEM14C and ABCB7, but not the non-rate-limiting enzyme PPOX, markedly decreased RS, and their combined rescue nearly abolished RS formation. Our study demonstrates that coordinated missplicing of mitochondrial transporters TMEM14C and ABCB7 by mutant SF3B1 sequesters iron in mitochondria, causing RS formation.

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Altered splicing and cytoplasmic levels of tRNA synthetases in SF3B1-mutant myelodysplastic syndromes as a therapeutic vulnerability.

Project description:Myelodysplastic syndromes (MDS) are haematopoietic malignancies that are characterised by a heterogeneous clinical course. In recent years, sequencing efforts have uncovered recurrent somatic mutations within RNA splicing factors, including SF3B1, SRSF2, U2AF1 and ZRSR2. The most frequently mutated gene is SF3B1, mutated in 17% of MDS patients. While SF3B1 mutations and their effects on splicing have been well characterised, much remains to be explored about their more far-reaching effects on cellular homeostasis. Given that mRNA splicing and nuclear export are coordinated processes, we hypothesised that SF3B1 mutation might also affect export of certain mRNAs and that this may represent a targetable pathway for the treatment of SF3B1-mutant MDS. We used CRISPR/Cas9-genome editing to create isogenic cellular models. Comprehensive transcriptome and proteome profiling of these cells identified alterations in the splicing and export of components of the translational machinery, primarily tRNA synthetases, in response to the SF3B1 K700E mutation. While steady-state protein synthesis was unaffected, SF3B1 mutant cells were more sensitive to the clinically-relevant purine analogue, 8-azaguanine. In this study, we also demonstrated that 8-azaguanine affects splicing. Our results suggest that the simultaneous targeting of RNA metabolism and splicing by 8-azaguanine represents a therapeutic opportunity for SF3B1-mutant myelodysplastic syndromes.

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SF3B1 mutant-induced missplicing of MAP3K7 causes anemia in myelodysplastic syndromes.

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Coordinated mis-splicing of TMEM14C and ABCB7 causes ring sideroblast formation in SF3B1-mutant myelodysplastic syndrome

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2021-11-29 | GSE189546 | GEO

Splicing factor SF3B1 mutations and ring sideroblasts in myelodysplastic syndromes: a Brazilian cohort screening study.

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SF3B1 mutant-induced missplicing of MAP3K7 causes anemia in myelodysplastic syndromes

Project description:SF3B1 is the most frequently mutated RNA splicing factor in cancer, including in ~25% of myelodysplastic syndromes (MDS) patients. SF3B1-mutated MDS, which is strongly associated with ringed sideroblast morphology, is characterized by ineffective erythropoiesis, leading to severe, often fatal, anemia. However, functional evidence linking SF3B1 mutations to the anemia described in MDS patients harboring this genetic aberration is weak, and the underlying mechanism is completely unknown. Using isogenic SF3B1 wild-type and mutant cell lines, normal human CD34 cells and MDS patient cells, we define a previously unrecognized role of the kinase MAP3K7, encoded by a known mutant SF3B1-targeted transcript, in controlling proper terminal erythroid differentiation, and show how MAP3K7 missplicing leads to the anemia characteristic of SF3B1-mutated MDS, although not to ringed sideroblast formation. We found that p38 MAPK is deactivated in SF3B1 mutant isogenic and patient cells and that MAP3K7 is an upstream positive effector of p38 MAPK. We demonstrate that disruption of this MAP3K7-p38 MAPK pathway leads to premature downregulation of GATA1, a master regulator of erythroid differentiation, and that this is sufficient to trigger accelerated differentiation, erythroid hyperplasia and ultimately apoptosis. Our findings thus define the mechanism leading to the severe anemia found in MDS patients harboring SF3B1 mutations.

2021-11-06 | GSE187356 | GEO

SF3B1 haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic syndromes.

Project description:Whole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

| S-EPMC3476536 | biostudies-literature

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