以線蟲為模式系統探討同質多胺基酸的毒性及尋找重複序列表達修飾基因

Investigation of the toxicity of trinucleotide repeat-encoded homopolymers and screening for genetic modifiers required for repeat expression using the C. elegans model

陳伯軒(Po-Hsuan Chen)

指導教授 ： 蕭光明

國立中正大學/理學院/生命科學系分子生物研究所/博士(2016年)

摘要

染色體中三聯核酸重複序列發生擴增突變，已知會造成超過20種神經退化(neurodegenerative)和神經肌肉(neuromuscular)性疾病。這類疾病依重複序列發生擴增突變的位置可分為兩大類:密碼區(coding region)和非密碼區(non-coding region)。大部分發生在密碼區的擴增突變為CAG重複序列，其轉譯後會造成多麩胺醯氨基酸症(PolyQ disease)，這類疾病的致病機制被認為是因為蛋白質的聚集造成gain-of-function，使得蛋白功能改變或是細胞死亡。近年來的研究發現擴增的CAG和CTG重複序列，會透過非轉譯起始點進行不同讀序框架(reading frames)轉譯，而產生多絲胺酸(polyserine)、多丙胺酸(polyalanine)和多亮胺酸(polyleucine)，這些同質多胺基酸(homopolymeric amino acid)可能都參與了多麩胺醯氨基酸症致病機轉。為了解同質多胺基酸的相對毒性和致病潛力，我們利用綠螢光蛋白(GFP)作為報導基因，評估同質多胺基酸表達在線蟲體內所造成的毒性效應。我們的結果顯示，多絲胺酸、多丙胺酸和多亮胺酸都會在線蟲的觸摸神經元和體壁肌肉細胞形成聚集。在神經方面，表達同質多胺基酸會因為長度越長，造成更嚴重的神經功能缺失；然而擴增的CAG和CTG RNA不會對線蟲的觸摸神經元造成功能上的異常。將這些同質多胺基酸表達在線蟲體壁肌肉細胞可明顯的影響細胞的型態；此外，這些線蟲也出現了壽命縮短和後代隻數減少的表型。其中，表達多亮胺酸的線蟲出現最嚴重的症狀，因此我們推測在這些同質多胺基酸當中，多亮胺酸對線蟲所造成的毒性最強。綜合上述結果我們發現， CAG和CTG RNA重複序列會造成組織特異性的毒性效應，而由CAG和CTG重複序列不同讀序框架所轉譯出的同質多胺基酸可能參與了多麩胺醯氨基酸症的致病機轉。
另一方面，CTG重複序列在非密碼區的擴增突變會造成一種顯性遺傳疾病:第一型強直型肌肉萎縮症(myotonic dystrophy type 1)。先前實驗室已利用線蟲建立了一個強直型肌肉萎縮症動物模式，並證實減少CUG125 RNA的表現可回復轉殖線蟲的異常症狀。利用此模式我進一步篩選線蟲RNAi資料庫，尋找可幫助表達擴增CTG重複序列所需要的修飾基因。在篩選到的13個候選基因中，我挑選了F11C3.1和RFC-1做後續的研究。我們發現將F11C3.1和RFC-1弱化後，CUG125 RNA的表現會顯著下降，但不會影響轉殖線蟲短片段重複序列的表達。而由CUG125 RNA所造成的表型，如:壽命縮短、後代隻數減少、爬行速率減低和肌肉型態異常，都因為減少F11C3.1或RFC-1的表現而獲得部分改善。同樣的，在CAG重複序列轉殖線蟲減少F11C3.1或RFC-1的表現也可改善其表型，顯示CAG RNA和CUG RNA有類似的致病機轉。這些結果顯示，F11C3.1和RFC-1可專一的幫助擴增的重複序列表達，亦為擴增的重複序列造成毒性所需的基因。我們將帶有重復序列的哺乳類肌纖維母細胞 (C2C12-CUG200)利用shRNA弱化RFC1的表達，同樣可以減少擴增CUG RNA的表達，並改善由CUG 重複序列RNA所造成的分化停滯現象。我們的結果顯示，從線蟲模式所篩選到的重複序列修飾基因確實可調控CTG和CAG重復序列RNA的毒性，且此調控機制在高等哺乳類細胞中是被保留的。

關鍵字

多麩胺醯氨基酸症 ； 同質多胺基酸 ； 第一型強直型肌肉萎縮症 ； CTG重複序列 ； CAG重複序列 ； F11C3.1基因 ； RFC-1基因

並列摘要

More than 20 neurodegenerative and neuromuscular diseases were found to be caused by expanded trinucleotide repeats which have been identified within specific genes. These diseases can be grouped into two classifications according to the location of the repeats in the non-coding region or coding region of the disease genes. Most expanded trinucleotide repeats found in the coding region of disease genes are CAG repeat and the resultant diseases are called polyglutamine diseases (PolyQ disease). The aggregation of mutant protein causes gain-of-function effect and is thought to be the toxic agents that cause dysfunction or death of cells. Emerging evidences indicate that polyserine (polyS), polyalanine (polyA), and polyleucine (polyL) can be translated from the second and third reading frames of the CAG and CUG repeats by repeat-associated non-AUG translation and that these homopolymeric amino acid (HPAA) repeats also contribute to the pathogenesis of the polyQ diseases. To investigate the relative toxicity and pathogenic potential of the homopolymeric amino acid (HPAA) repeats, we expressed HPAA-tethered GFP in C. elegans and assessed the pathogenic effect of the ectopically expressed protein to the worms. Our result showed that polyS, polyA, and polyL all form aggregates in the touch neurons and the body wall muscle cells, indicating that tethering of either of these HPAA repeats leads to protein aggregation. Expression of the HPAA-GFP caused length-dependent toxicity on neuron function. However, expanded CAG and CUG RNA showed minimal impact to the function of the touch neurons. Expression of all expanded HPAA-GFP in the body wall muscle significantly disrupted the muscle morphology of the organisms. These worms also displayed shortened lifespan and decreased brood size. Furthermore, expression of polyL leads to the most severe functional effect, suggesting a more toxic nature of polyL than other HPAAs. These results indicated that the CAG and CUG RNA expansion exhibits tissue-specific toxicity and HPAAs translated from alternative reading frames of CAG and CUG expansion may also participate in the pathogenesis of the polyQ diseases.
On the other hand, expansion of non-coding CTG repeats is associated with myotonic dystrophy type 1 (DM1), a dominant neuromuscular disease. Our laboratory has established a C. elegans model of DM1 and further demonstrates that the DM1 phenotypes could be alleviated by decreasing the expression of CUG repeat RNA. In this study, we aimed to identify the DM1 genetic modifiers required for efficient expression of expanded CTG repeats using genome-wide RNAi approach. 13 genes were identified from screens of RNAi clones. Among theses, F11C3.1 (function-unknown protein) and RFC-1 (replication factor C (activator 1) 1) RNAi clones are picked up for further study. Our results showed that RNAi treatment of F11C3.1 or RFC-1 reduced the expression of CUG125 RNA, but did not affect the expression of reporter gene in control worms. Moreover, the phenotype of worms expressing toxic CUG repeats, including shortened life span, decreased broodsize, reduced motility rate and abnormal muscle morphology were partially reversed by F11C3.1 or RFC-1 knockdown. In addition, F11C3.1 or RFC-1 knockdown also rescued the phenotype of worms expressing CAG repeats, consistent with previous notion that CAG repeats can be toxic at the RNA level similar to CUG repeats. These results suggest that F11C3.1 and RFC-1 are specifically required for efficient expression of expanded CTG/CAG repeats and can function as a genetic modifier of CTG/CAG RNA toxicity. We also infected mammalian myoblast cells which express EGFP gene with expanded CTG repeats (C2C12-CUG200) with RFC1 shRNA. The result indicated that knockdown of RFC1 decreased EGFP RNA and protein expression in C2C12-CUG200 cells and promoted cell differentiation which was compromised by expanded CUG repeats. Although the underlying mechanism of how F11C3.1 and RFC-1 genes alleviating the toxicity of CUG/CAG repeats remains unclear, our results indicate that the CTG/CAG RNA toxicity can be suppressed by adjusting the expression level of certain genetic modifiers, and this mechanism is conserved in mammalian cells.

並列關鍵字

Polyglutamine disease ； PolyQ disease ； homopolymeric amino acid ； HPAA ； myotonic dystrophy type 1 ； DM1 ； CTG repeats ； CAG repeats ； F11C3.1 ； RFC-1

參考文獻

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