温進德Han-Yu Chang張翰彧2025-11-142025-11-142025https://scholars.lib.ntu.edu.tw/handle/123456789/733694https://ntu.primo.exlibrisgroup.com/permalink/886NTU_INST/14poklj/alma991039401170204786獎項：校長獎；指導教授：温進德The human C9orf72 hexanucleotide (GGGGCC) repeat expansion (HRE) is a common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Past studies indicated RNA foci formed by C9orf72 HRE are strongly related to the diseases in different aspects. One of the known features of RNA foci was to sequester RNA-binding proteins (RBPs). Several RBPs were found to colocalize with the RNA foci found in neuron cells of c9ALS patients. Among them, hnRNP H1, a splicing factor with a preference for binding G-rich sequences, has been found to be predominant in ALS/FTD patients with C9orf72 HRE. Although the common cause of these diseases has been identified, the underlying pathogenic mechanisms are yet unclear. Not to mention the function of hnRNP H1 in ALS/FTD. To peek into the structural features of r(G4C2)n and the role of hnRNP H1 in the diseases, optical tweezers were applied in this research to monitor the real-time conformational change of RNA and its interaction with hnRNP H1. Through the unique single-molecule manipulations and measurements, we could determine the dynamic conformations of the RNA. Furthermore, hnRNP H1-N1, a truncation mutant of hnRNP H1, was added to determine its effect on the conformation of GGGGCC tandem repeats. Our results indicate that the short repeat of GGGGCC RNA was unable to form intramolecular G quadruplexes but still exhibited structural variations. In the presence of the protein, the GGGGCC RNA could be remodeled to new conformations, suggesting that hnRNP H1-N1’s binding has a structural specificity. Moreover, the protein-targeted structure tends to be destabilized by hnRNP H1-N1. The emergence of the protein-induced structure provides a great insight into the function of hnRNP H1. We will tackle these issues in future experiments.人類第九號染色體第72開讀框六核苷酸(GGGGCC)重複擴增（C9orf72 HRE）是導致肌萎縮性側索硬化症（漸凍症，ALS）和額顳葉癡呆症（FTD）的常見遺傳致病因素。過去的研究指出，由C9orf72 HRE形成的核醣核酸凝集體（RNA foci）在疾病的多個方面具有重要關聯性。核醣核酸凝集體的一個已知特徵是能夠捕獲核醣核酸結合蛋白（RBPs），，在C9orf72導致的漸凍症患者神經元細胞中發現有多種核醣核酸結合蛋白與核醣核酸凝集體共定位，是中，hnRNP H1是一種剪接因子，能夠結合富含鳥嘌呤的序列，被認為在C9orf72導致的ALS/FTD患者中占主導地位。儘管疾病的是中一種常見致病原因已被發現，但是實際致病機制仍不清楚，更不用說hnRNP H1在疾病中的角色。 為了探究 r(G4C2)n核醣核酸重複序列的結構特徵以及hnRNP H1扮演的角色，，我們應用單分子光鉗技術以監測核醣核酸的即時構形變化及是與hnRNP H1的相互作用。通過這種獨特的單分子操控與測量方法，我們可以使結構反覆解開與摺疊來確定核醣核酸重複序列的構型，。外，，我們將hnRNP H1的截短突變體hnRNP H1-N1添加到單分子實驗系統中，以研究是對GGGGCC串聯重複序列所形成之結構的影響。我們的結果表明，低重複次數的GGGGCC核醣核酸重複序列無法形成單分子的G四疊體，但仍然表現出結構變異性。 在hnRNP H1-N1存在的情況下，核醣核酸結構似乎被干擾，且形成新的結構。數據顯示，hnRNP H1-N1的結合對特定結構具有專一性，。外，，標結結構在hnRNP H1-N1的作用下傾向不穩定且易被解開。對蛋白誘導產生的新結構的分析提供了我們對於hnRNP H1的功能的新理解，在後續的研究中我們將做進一步的探討。C9orf72 HREamyotrophic lateral sclerosisfrontotemporal dementiaRNA foci, hnRNP H1optical tweezers人類第九號染色體第72開讀框六核苷酸重複擴增漸凍症額顳葉癡呆症核醣核酸凝集體hnRNP H1光鉗技術thesis