RESUMEN
Myotonic dystrophy (DM; also known as dystrophia myotonica) is an autosomal dominant disorder that affects the heart, eyes, brain and endocrine system, but the predominant symptoms are neuromuscular, with progressive muscle weakness and wasting. DM presents in two forms, DM1 and DM2, both of which are caused by nucleotide repeat expansions: CTG in the DMPK gene for DM1 and CCTG in ZNF9 (CNBP) for DM2. Previous studies have shown that the mutant mRNAs containing the transcribed CUG or CCUG repeats are retained within the nuclei of cells from individuals with DM, where they bind and sequester the muscleblind-like proteins MBNL1, MBNL2 and MBNL3. It has been proposed that the sequestration of these proteins plays a key role in determining the classic features of DM. However, the functions of each of the three MBNL genes are not completely understood. We have generated a zebrafish knockdown model in which we demonstrate that a lack of mbnl2 function causes morphological abnormalities at the eye, heart, brain and muscle levels, supporting an essential role for mbnl2 during embryonic development. Major features of DM are replicated in our model, including muscle defects and splicing abnormalities. We found that the absence of mbnl2 causes disruption to the organization of myofibrils in skeletal and heart muscle of zebrafish embryos, and a reduction in the amount of both slow and fast muscle fibres. Notably, our findings included altered splicing patterns of two transcripts whose expression is also altered in DM patients: clcn1 and tnnt2. The studies described herein provide broader insight into the functions of MBNL2. They also lend support to the hypothesis that the sequestration of this protein is an important determinant in DM pathophysiology, and imply a direct role of MBNL2 in splicing regulation of specific transcripts, which, when altered, contributes to the DM phenotype.
Asunto(s)
Distrofia Miotónica/patología , Proteínas de Pez Cebra/deficiencia , Pez Cebra/metabolismo , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Secuencia de Bases , Huesos/anomalías , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/patología , Embrión no Mamífero/anomalías , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Embrión no Mamífero/ultraestructura , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Datos de Secuencia Molecular , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/ultraestructura , Miocardio/metabolismo , Miocardio/patología , Distrofia Miotónica/metabolismo , Oligonucleótidos Antisentido/farmacología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disorder characterised by muscle weakness and wasting. There are two forms of DM; both of which are caused by the expansion of repeated DNA sequences. DM1 is associated with a CTG repeat located in the 3' untranslated region of a gene, DMPK and DM2 with a tetranucleotide repeat expansion, CCTG, located in the first intron of a different gene, ZNF9. Recent data suggest a dominant RNA gain-of-function mechanism underlying DM, as transcripts containing either CUG or CCUG repeat expansions accumulate as foci in the nuclei of DM1 and DM2 cells respectively, where they exert a toxic effect, sequestering specific RNA binding proteins such as Muscleblind, which leads to splicing defects and the disruption of normal cellular functions. Z-band disruption is a well-known histological feature of DM1 muscle, which has also been reported in Muscleblind deficient flies. In order to determine whether there is a common molecular basis for this abnormality we have examined the alternative splicing pattern of transcripts that encode proteins associated with the Z-band in both organisms. Our results demonstrate that the missplicing of ZASP/LDB3 leads to the expression of an isoform in DM1 patient muscle, which is not present in normal controls, nor in other myopathies. Furthermore the Drosophila homologue, CG30084, is also misspliced, in Muscleblind deficient flies. Another Z-band transcript, alpha actinin, is misspliced in mbl mutant flies, but not in DM1 patient samples. These results point to similarities but subtle differences in the molecular breakdown of Z-band structures in flies and DM patients and emphasise the relevance of Muscleblind proteins in DM pathophysiology.
Asunto(s)
Empalme Alternativo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Musculares/genética , Distrofia Miotónica/genética , Proteínas Nucleares/genética , Actinina/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Proteínas de Drosophila/deficiencia , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mutación , Distrofia Miotónica/embriología , Distrofia Miotónica/patología , Proteínas Nucleares/deficiencia , Isoformas de Proteínas/genética , ARN/genética , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
Type 1 myotonic dystrophy or DM1 (Steinert's disease), which is the commonest muscular dystrophy in adults, has intrigued physicians for over a century. Unusual features, compared with other dystrophies, include myotonia, anticipation, and involvement of other organs, notably the brain, eyes, smooth muscle, cardiac conduction apparatus, and endocrine system. Morbidity is high, with a substantial mortality relating to cardiorespiratory dysfunction. More recently a second form of multisystem myotonic disorder has been recognized and variously designated as proximal myotonic myopathy (PROMM), proximal myotonic dystrophy (PDM), or DM2. For both DM1 and DM2 the molecular basis is expansion of an unstable repeat sequence in a noncoding part of a gene (DMPK in DM1 and ZNF9 in DM2). There is accumulating evidence that the basic molecular mechanism is disruption of mRNA metabolism, which has far-reaching effects on many other genes, in part through the induction of aberrant splicing, explaining the multisystemic nature of the disease. The unstable nature of the expansion provides a molecular explanation for anticipation. This review emphasizes the clinical similarities and differences between DM1 and DM2. It examines current views about the molecular basis of these disorders, and contrasts them with other repeat expansion disorders that have increasingly been recognized as a cause of neurological disease.