RESUMEN
Myotonic dystrophy type 1 (DM1) is caused by CUG triplet expansions in the 3' UTR of dystrophia myotonica protein kinase (DMPK) messenger ribonucleic acid (mRNA). The etiology of this multi-systemic disease involves pre-mRNA splicing defects elicited by the ability of the CUG-expanded mRNA to 'sponge' splicing factors of the muscleblind family. Although nuclear aggregation of CUG-containing mRNPs in distinct foci is a hallmark of DM1, the mechanisms of their homeostasis have not been completely elucidated. Here we show that a DEAD-box helicase, DDX6, interacts with CUG triplet-repeat mRNA in primary fibroblasts from DM1 patients and with CUG-RNA in vitro. DDX6 overexpression relieves DM1 mis-splicing, and causes a significant reduction in nuclear DMPK-mRNA foci. Conversely, knockdown of endogenous DDX6 leads to a significant increase in DMPK-mRNA foci count and to increased sequestration of MBNL1 in the nucleus. While the level of CUG-expanded mRNA is unaffected by increased DDX6 expression, the mRNA re-localizes to the cytoplasm and its interaction partner MBNL1 becomes dispersed and also partially re-localized to the cytoplasm. Finally, we show that DDX6 unwinds CUG-repeat duplexes in vitro in an adenosinetriphosphate-dependent manner, suggesting that DDX6 can remodel and release nuclear DMPK messenger ribonucleoprotein foci, leading to normalization of pathogenic alternative splicing events.
Asunto(s)
Regiones no Traducidas 3' , ARN Helicasas DEAD-box/metabolismo , Distrofia Miotónica/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/metabolismo , Núcleo Celular/química , Células Cultivadas , Citoplasma/química , ARN Helicasas DEAD-box/antagonistas & inhibidores , Fibroblastos/química , Fibroblastos/metabolismo , Humanos , Distrofia Miotónica/enzimología , Distrofia Miotónica/metabolismo , Proteína Quinasa de Distrofia Miotónica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Empalme del ARN , ARN Mensajero/análisis , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/análisis , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Expansión de Repetición de TrinucleótidoRESUMEN
BACKGROUND: Myotonic dystrophy type 1 (DM1) is the most common muscle dystrophy in adults. The disease is caused by a triplet expansion in the 3'end of the myotonic dystrophy protein kinase (DMPK) gene. In order to develop a human cell model for investigation of possible effects of antisense and RNAi effector molecules we have used lentiviral mediated myoD-forced myogenesis of DM1 patient fibroblasts. FINDINGS: Transduced fibroblasts show a multinuclear phenotype and express the differentiation marker myogenin. Furthermore, fluorescence in situ hybridization (FISH) analysis revealed a statistical significant increase in the amount of nuclear foci in DM1 patient fibroblasts after myogenesis. Finally, no nuclear foci were found after treatment with oligonucleotides targeting the repeat expansions. CONCLUSIONS: The abundance of nuclear foci in DM1 patient fibroblasts increase following myogenesis, as visualized by FISH analysis. Foci were eradicated after treatment with antisense oligonucleotides. Thus, we propose that the current cell model is suitable for testing of novel treatment modalities.
