RESUMO
The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families--muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins--are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5' end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.
Assuntos
Processamento Alternativo , Canais de Cloreto/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas CELF , Canais de Cloreto/metabolismo , Expansão das Repetições de DNA , Éxons , Humanos , Camundongos , Músculo Esquelético/metabolismo , Sítios de Splice de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidoresRESUMO
The muscleblind-like (MBNL) protein family is thought to be involved in the molecular mechanism of myotonic dystrophy (DM). Although it has been shown to have splicing activity, a broader function in cellular RNA metabolism has been implicated. In this study, we attempted to find the binding proteins of MBNL1 in order to elucidate its physiological function. First, we performed a GST pull-down assay using GST-MBNL1-6xHis as bait. Several proteins were identified, including YB-1, a multifunctional DNA/RNA-binding protein, and DDX1, a DEAD box RNA helicase. MBNL1 formed an RNP complex with YB-1 and DDX1 in binding assays. YB-1 also showed a weak but significant effect on alpha-actinin splice site selection. Interestingly, in response to stress, MBNL1 moved to cytoplasmic stress granules, where it colocalized with YB-1, which was previously reported to be a component of stress granules. We found that DDX1 also colocalized with MBNL1 at stress granules. These results provide new insight into the dynamics of MBNL1 in response to stress, and they suggest a role for MBNL1 in mRNA metabolism in the cytoplasm.
Assuntos
Grânulos Citoplasmáticos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Músculo Esquelético/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , RNA Helicases DEAD-box/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Amplificação de Genes , Humanos , Proteínas Nucleares/genética , Proteínas de Ligação a Poli(A) , Polirribossomos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Antígeno-1 Intracelular de Células T , Proteína 1 de Ligação a Y-BoxRESUMO
A neuropathological hallmark of Alzheimer's disease is the presence of amyloid plaques in the brain. Amyloid-beta peptide (Abeta) is the major constituent of the plaques and is generated by proteolytic cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. Growing evidence shows that lipid rafts are critically involved in regulating the Abeta generation. In support of this, APP, Abeta, and presenilins have been found in lipid rafts. Although cholesterol plays a crucial role in maintaining lipid rafts, functions of other components in the generation of Abeta are unknown. Caveolins (CAVs) and flotillins (FLOTs) are principal proteins related to lipid rafts and have been suggested to be involved in APP processing. Here, we report that FLOT-1 binds to BACE1 (beta-site APP cleaving enzyme 1) and that overexpression of CAV-1 or FLOT-1 results in recruiting BACE1 into lipid rafts and influence on beta-secretase activity in cultured cells. Our results show that both CAV-1 and FLOT-1 may modulate beta-secretase activity by interacting with BACE1.