RESUMO
Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Processamento Alternativo/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Debilidade Muscular/genética , Distrofia Miotônica/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Linhagem Celular , Éxons/genética , Humanos , Camundongos , Debilidade Muscular/fisiopatologia , Distrofia Miotônica/fisiopatologia , Proteínas Nucleares/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Whereas the role of thyroid hormone is clearly established in the regulation of cholesterol homeostasis, its involvement in the control of serum triglyceride (TG) levels remains largely debated. Angiopoietin-like proteins 3 and 4 have recently been characterized as potent lipoprotein lipase inhibitors and therefore as important components of plasma triglyceride homeostasis. In the present study, the role of thyroid hormone in the regulation of both ANGPTL4 and ANGPTL3 gene expression was investigated. In vivo studies revealed that thyroid hormone down-regulates ANGPTL3 but not ANGPTL4 gene expression in hypothyroid rats. Using thyroid hormone receptor (TR)-deficient mice, we show that thyroid hormone regulates ANGPTL3 gene expression in a TRbeta-dependent manner. Transfection studies revealed that this inhibition occurs at the transcriptional level in a DNA binding-independent fashion and requires the proximal (-171 to +66) region of the ANGPTL3 gene promoter. Moreover, site-directed mutagenesis experiments indicate that the HNF1 site within this proximal region mediates this TRbeta-dependent repression. Finally, co-transfection studies and electrophoretic mobility shift assays suggest that TRbeta antagonizes the HNF1alpha signaling pathway by inhibiting its transcriptional activity without interfering with its DNA-binding capacity. Taken together, our results lead to the identification of ANGPTL3 as a novel TRbeta target gene and provide a new potential mechanism to explain the hypotriglyceridemic properties of TRbeta agonists in vivo.