Investigation of the toxicity of trinucleotide repeat-encoded homopolymers and screening for genetic modifiers required for repeat expression using the C. elegans model
| Autor: | Chen, Po-Hsuan, 陳伯軒 |
|---|---|
| Rok vydání: | 2016 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 104 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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