RESUMO
Expanded non-coding RNA repeats of CUG and CCUG are the underlying genetic causes for myotonic dystrophy type 1 (DM1) and type 2 (DM2), respectively. A gain-of-function of these pathogenic repeat expansions is mediated at least in part by their abnormal interactions with RNA-binding proteins such as MBNL1 and resultant loss of activity of these proteins. To study pathogenic mechanisms of CCUG-repeat expansions in an animal model, we created a fly model of DM2 that expresses pure, uninterrupted CCUG-repeat expansions ranging from 16 to 720 repeats in length. We show that this fly model for DM2 recapitulates key features of human DM2 including RNA repeat-induced toxicity, ribonuclear foci formation and changes in alternative splicing. Interestingly, expression of two isoforms of MBNL1, MBNL135 and MBNL140, leads to cleavage and concurrent upregulation of the levels of the RNA-repeat transcripts, with MBNL140 having more significant effects than MBNL135. This property is shared with a fly CUG-repeat expansion model. Our results suggest a novel mechanism for interaction between the pathogenic RNA repeat expansions of myotonic dystrophy and MBNL1.
Assuntos
Expansão das Repetições de DNA , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Alelos , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Núcleo Celular/genética , Modelos Animais de Doenças , Drosophila , Expressão Gênica , Genes Letais , Estudos de Associação Genética , Fenótipo , Estabilidade de RNARESUMO
Spinocerebellar ataxia type 3 is one of the polyglutamine (polyQ) diseases, which are caused by a CAG-repeat expansion within the coding region of the associated genes. The CAG repeat specifies glutamine, and the expanded polyQ domain mutation confers dominant toxicity on the protein. Traditionally, studies have focused on protein toxicity in polyQ disease mechanisms. Recent findings, however, demonstrate that the CAG-repeat RNA, which encodes the toxic polyQ protein, also contributes to the disease in Drosophila. To provide insights into the nature of the RNA toxicity, we extracted brain-enriched RNA from flies expressing a toxic CAG-repeat mRNA (CAG100) and a non-toxic interrupted CAA/G mRNA repeat (CAA/G105) for microarray analysis. This approach identified 160 genes that are differentially expressed specifically in CAG100 flies. Functional annotation clustering analysis revealed several broad ontologies enriched in the CAG100 gene list, including iron ion binding and nucleotide binding. Intriguingly, transcripts for the Hsp70 genes, a powerful suppressor of polyQ and other human neurodegenerative diseases, were also upregulated. We therefore tested and showed that upregulation of heat shock protein 70 mitigates CAG-repeat RNA toxicity. We then assessed whether other modifiers of the pathogenic, expanded Ataxin-3 polyQ protein could also modify the CAG-repeat RNA toxicity. This approach identified the co-chaperone Tpr2, the transcriptional regulator Dpld, and the RNA-binding protein Orb2 as modifiers of both polyQ protein toxicity and CAG-repeat RNA-based toxicity. These findings suggest an overlap in the mechanisms of RNA and protein-based toxicity, providing insights into the pathogenicity of the RNA in polyQ disease.
Assuntos
Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Redes Reguladoras de Genes/genética , Genes de Insetos/genética , RNA/toxicidade , Expansão das Repetições de Trinucleotídeos/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Testes Genéticos , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Degeneração Neural/genética , Degeneração Neural/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/toxicidade , RNA não Traduzido/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Trinucleotide repeat instability underlies >20 human hereditary disorders. These diseases include many neurological and neurodegenerative situations, such as those caused by pathogenic polyglutamine (polyQ) domains encoded by expanded CAG repeats. Although mechanisms of instability have been intensely studied, our knowledge remains limited in part due to the lack of unbiased genome-wide screens in multicellular eukaryotes. Drosophila melanogaster displays triplet repeat instability with features that recapitulate repeat instability seen in patients with disease. Here we report an enhanced fly model with substantial instability based on a noncoding 270 CAG (UAS-CAG(270)) repeat construct under control of a germline-specific promoter. We find that expression of pathogenic polyQ protein modulates repeat instability of CAG(270) in trans, indicating that pathogenic-length polyQ proteins may globally modulate repeat instability in the genome in vivo. We further performed an unbiased genetic screen for novel modifiers of instability. These studies indicate that different aspects of repeat instability are under independent genetic control, and identify CG15262, a protein with a NOT2/3/5 conserved domain, as a modifier of CAG repeat instability in vivo.