3’UTR CAG重複序列在RNA層次上的作用機制之探討

Abstract

Expanded CAG and CTG repeats are the widely accepted pathogenic agents in a variety of neuromuscular degenerative disorders. CAG expansions are almost found in translated regions whereas CTG expansions are located in untranslated regions. Previously, we showed that expanded CAG repeats in the 3’-untranslated region (UTR) of EGFP had pathogenic effects in muscle of transgenic C. elegans and mice.Moreover, we found that RNA foci were formed in Hela cells expressing expanded CAG repeats and the muscleblind-like (MBNL) proteins were colocolized to these foci. These results suggested that expanded CAG repeats may exert toxic gain-of-function mechanisms not only at protein levels but at RNA levels. All known CAG repeat disorders affect the central nervous system. Therefore, we established transgenic mice expressing neuron-specific transcripts with CAG repeats in the coding region or 3’UTR of the EGFP gene in the Purkinje cell of cerebellum. One transgenic line expressing EGFP-polyglutamine fusion protein displayed ataxia phenotype with neurodegeneration and cataract. However, no phenotypic changes were observed in the other lines. Two possibilities for the failure of phenotype presentation are low transgene expression and lack of tissue specific factors. In order to overcome the problem of insufficient transgene expression, and to investigate whether the CAG repeats bring about different effects in different tissues, we established cell lines expressing expanded CAG repeats in mouse myoblast cells (C2C12) and mouse neuroblast cells (Neuro-2a). We found the expanded CAG not only formed RNA foci which colocalized with the MBNL protein, they also inhibited the differentiation of cells and resulted in the abnormal splicing of RyR AS1､RyR AS1 and vinculin genes in C2C12 cells and of APP gene in Neuro-2a cells. These results suggest that expanded CAG repeats can have gain-of-function mechanism at the RNA level. Moreover, using CAG repeats RNA as a probe we detected the existence of CAG RNA binding protein, we also discovered that CAG and CUG binding proteins have the same molecular weight. This result suggests CAG and CUG RNA binding proteins are possibly the identical protein. In the future, we hope to further resolve the mechanism of RNA gain-of-function of CAG repeats, and identify the CAG RNA binding proten.