RESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Síndromes Mielodisplásicas/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Sequência de Bases , Cicloeximida/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Ferro/metabolismo , Mitocôndrias/metabolismo , Splicing de RNA , Células Tumorais CultivadasRESUMO
The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndrome (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). We investigated the functional effects of SF3B1 disruption in myeloid cell lines: SF3B1 knockdown resulted in growth inhibition, cell cycle arrest and impaired erythroid differentiation and deregulation of many genes and pathways, including cell cycle regulation and RNA processing. MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34(+) cells from MDS patients with SF3B1 mutations using RNA sequencing. Genes significantly differentially expressed at the transcript and/or exon level in SF3B1 mutant compared with wild-type cases include genes that are involved in MDS pathogenesis (ASXL1 and CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7 and SLC25A37) and RNA splicing/processing (PRPF8 and HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expression/splicing in SF3B1 mutant cases. This is the first study to determine the target genes of SF3B1 mutation in MDS CD34(+) cells. Our data indicate that SF3B1 has a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processes/pathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.
Assuntos
Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Síndromes Mielodisplásicas/genética , Fosfoproteínas/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Células-Tronco/citologia , Processamento Alternativo , Anemia Refratária com Excesso de Blastos/genética , Anemia Refratária com Excesso de Blastos/metabolismo , Antígenos CD34/metabolismo , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Éxons , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Heterozigoto , Homeostase , Humanos , Células K562 , Masculino , Mutação , Síndromes Mielodisplásicas/metabolismo , Fosfoproteínas/metabolismo , Mutação Puntual , RNA/genética , Splicing de RNA , Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Análise de Sequência de RNARESUMO
BACKGROUND: Genes underlying circadian rhythm generation are expressed in many tissues. We explore a role for circadian rhythms in the timing and efficacy of mouse reproduction and development using a genetic approach. METHODS: We compare fecundity in Clock(Delta19) mutant mice (a dominant-negative protein essential for circadian rhythm activity) and in Vipr2-/- null mutant mice (affecting the generation and output of the circadian rhythm of the hypothalamic suprachiasmatic nucleus) with wild type (WT) litter mates under both a 12 h:12 h light:dark cycle and continuous darkness. RESULTS: Uteri from Clock(Delta19) mice show no circadian rhythm and Vipr2-/- mice show a phase-advanced rhythm compared to WT uteri. In neither mutant line were homozygous or heterozygous fetuses lethal. Sexually mature adults of both mutant lines showed mildly reduced male in vivo (but not in vitro) fertility and irregular estrous cycles exacerbated by continuous darkness. However, pregnancy rates and neonatal litter sizes were not affected. The Clock(Delta19) mutant line was distinguishable from the Vipr2-/- null mutant line in showing more peri-natal delivery problems and very poor survival of offspring to weaning. CONCLUSIONS: A fully functional central and peripheral circadian clock is not essential for reproduction and development to term, but has critical roles peri-natally and post-partum.