線蟲 RTEL -1是擴增的 CTG 與 CAG 重複序列表達的必需基因

RTEL-1 Is Required for the Expression of Expanded CTG and CAG Repeats in C. elegans

莊凱盛(Kai-Sheng Jhuang)

指導教授 ： 蕭光明 ； 李沁

國立中正大學/理學院/生命科學系生物醫學研究所/碩士(2015年)

摘要

第一型強直型肌肉萎縮症是一個由CUG擴增重複序列所造成的顯性遺傳疾病，目前並沒有根本的治療方式。先前實驗室已利用線蟲作為模式生物，建築了一個強直型肌肉萎縮症模式，並證實減少CUG125的表現可回復其異常的表現型。因此，我們利用線蟲的RNAi資料庫去尋找表達擴增的CTG重複序列所需要的基因修飾者。在本實驗中，候選RNAi clone－F25H2.13，其對應到的線蟲基因為rtel-1 (regulator of telomere elongation length 1)，進行弱化處理後，發現會顯著下降CUG125轉殖質體的表現，但不會影響表達短片段重複序列的線蟲。此外，更能有效改善CUG125轉殖線蟲的異常表現型包含：壽命長度縮短、異常肌肉型態與異常肌肉功能；而在CAG重複序列轉殖線蟲的實驗中也有相似的結果。進一步利用rtel-1剔除的線蟲(tm1866)進行確認，結果顯示完全無法在後代觀察到轉殖基因的表現，這結果更加證實了RTEL-1在CTG擴增重複序列表現中的重要性。另一方面， rtel-1基因在哺乳類動物上具有同源基因，因此我們利用CTG200 C2C12細胞弱化其同源基因，結果發現與控制組相比減少了40 %的轉殖基因表現，但卻無法回復其延緩分化的異常表現型。同時也利用DM1病人的初級纖維母細胞弱化rtel-1同源基因表現，也同樣的發現無法減少 RNA foci的表現型。上述結果表明，線蟲模式中RTEL-1是一個表達擴增的CTG與CAG重複序列所需要的基因修飾者，不過由於高等生物較為複雜，其對於三聯核酸重複序列表現的必要性仍有待進一步研究。

關鍵字

CTG重複序列 ； 第一型強直型肌肉萎縮症 ； RTEL-1 ； CAG重複序列

並列摘要

Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by expanded CUG repeats. A C. elegans model of DM1 has been established in our laboratory and used to demonstrat that the DM1 phenotypes could be alleviated by decreasing the expression level of CUG125 transgene. To search for the genetic modifiers of DM1, we have conducted a genome-wide screen using C. elegans RNAi library to identify those gene required for efficient expression of expanded CTG repeats. A candidate RNAi clone, F25H2.13, was selected for further analysis after screening. Sequencing data revealed that the target of clone F25H2.13 is rtel-1 (a regulator of telomere elongation length) gene. Knockdown of rtel-1 significantly reduced the expression level of CUG125, but showed no effect to the expression of CUG0 and CUG2. Phenotype analysis showed that suppression of rtel-1 alleviated the toxicity of CUG125 in C. elegans, including shortened life span, abnormal muscle morphology and reduced motility rate, without inducing appreciable toxicity in control worms. Moreover, rtel-1 knockdown reduced the expression and toxicity of expanded CAG repeats as well. When CUG125 transgene was introduceed into rtel-1 knockout worms (CUG125::rtel-1(-/-)), no transgene expression was detected, further confirming that RTEL-1 is required for the expression of expanded CTG repeats. To test whether RTEL-1 plays a similar function in mammals, shRNA targeting to Rtel1 was introduced into mouse C2C12 cells expressing EGFP gene with expanded CTG repeats (CUG200). Although the EGFP expression level decreased more than 40% compared to control cells, but the adverse effect of CUG200 RNA on C2C12 differentiation has not significantly reversed. In addition to the model C2C12 cell line, we also tested the function of RTEL1 in the primary fibroblast cells from DM1 patient. Preliminary result showed that RTEL1 knockdown didn’t clearly rescue RNA foci of DM1 primary cells. Based on our findings, we propose that RTEL-1 plays an important role in facilitating the expression of genes containing large CTG and CAG repeats in C. elegans. However, the effect of RTEL-1 on CUG repeats expression is not as prominent in mammalian cells, other factors may have overlapping function and supplement the deficiency of RTEL1 function when we knockdown RTEL1. Therefore, the role of RTEL1 of mammalian cell in trinucleotide repeats remains unclear and requires further investigation.

並列關鍵字

CTG repeats ； Myotonic Dystrophy type 1 ； RTEL-1 ； CAG repeats

參考文獻

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