3’UTR CAG重複序列在RNA層次上的作用機制之探討

Investigation on the mechnisms of effect at the RNA level of 3’UTR CAG repeats

林炯志(Chiung-Chih Lin)

指導教授 ： 潘惠錦

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2009年)

https://doi.org/10.6834/CSMU.2009.00123

摘要

三聯核酸重複序列擴增突變所引起的遺傳性神經及肌肉退化性疾病中，大部分是由於CAG以及CTG擴增突變所造成的。CAG擴增突變通常發生在轉譯區內，而CTG擴增突變通常發生在非轉譯區。先前我們實驗室在線蟲以及老鼠的動物模式上發現，在GFP基因的3’-端非轉譯區接上CAG重複序列擴增，表現在肌肉組織上會造成異常的表型，而在Hela細胞上也發現CAG重複序列擴增會形成RNA foci，且與muscleblind-like (MBNL)蛋白質有colocalize的情形。基於這些發現，我們推測CAG重複序列具有RNA gain-of-function的致病機轉。目前已知的CAG重複序列所導致的疾病大多與神經系統的病變有關，為此，我們實驗室建立在小鼠小腦的Purkinje cell表現EGFP基因分別在轉譯區以及3’-端非轉譯區帶有CAG重複序列的轉殖基因小鼠動物模式來探討這個推論。但除了重複序列表現在轉譯區的一個品系有產生運動失調、神經退化以及白內障的病徵，重複序列表現在非轉譯區的基因轉殖小鼠並沒有發現有任何生理上的異常。推論原因可能有兩個：一是由於送入的轉殖基因的表現量太少；二是組織特異性因子可能扮演重要的角色。為了解決轉殖基因表現量不足的問題，同時繼續探討在RNA層次上，CAG重複序列是否會因為組織特異性而有不同的影響，我們利用CMV promoter分別在小鼠肌肉纖維母細胞(C2C12)以及小鼠神經纖維母細胞(Neuro-2a)建立會大量表現CAG重複序列的細胞株。我們發現除了會形成與MBNL蛋白質colocalization的RNA foci，CAG重複序列的擴增突變還會影響細胞的分化，進而導致C2C12細胞中RyR AS1和vinculin以及Neuro-2a細胞中MAPT和APP等基因的剪接異常。這個結果暗示CAG重複序列擴增突變存在RNA gain-of-function的致病機轉的可能性。另外，利用CAG 重複序列的RNA probe探討CAG RNA binding protein存在與否的實驗中，我們發現CAG與CUG binding protein有相同的分子量，這暗示CAG 和CUG RNA binding protein 可能是同一個蛋白質。未來，我們希望能進一步去了解CAG重複序列RNA gain-of-function詳細的機轉，並進一步將CAG RNA binding protein純化出來，進一步去確定它的身分。

關鍵字

三聯核酸重複序列

並列摘要

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.

並列關鍵字

3’UTR CAG repeats

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