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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 羅凱尹 | zh_TW |
| dc.contributor.advisor | Kai-Yin Lo | en |
| dc.contributor.author | 趙曼伶 | zh_TW |
| dc.contributor.author | Man-Ling Chao | en |
| dc.date.accessioned | 2025-08-19T16:16:08Z | - |
| dc.date.available | 2025-08-20 | - |
| dc.date.copyright | 2025-08-19 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-08-07 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/98804 | - |
| dc.description.abstract | 核糖體具有轉譯與蛋白質合成的關鍵功能，此複合體的生合成需依賴一系列有序的pre-rRNA裁切與大小次單元組裝。因此這個動態過程需眾多輔助因子協同參與，尤其DEAD-box RNA解旋酶能夠解開RNA二級結構，促進核糖核蛋白重塑，是核糖體生合成中不可或缺的角色。本研究探討ATP依賴性RNA解旋酶Fal1與兩個含MIF4G結構的調控因子Sgd1及eIF4G1，探討其於90S前驅核糖體組裝過程中的功能調控機制。我們發現抑制FAL1與SGD1表現，會導致細胞生長遲緩、pre-rRNA裁切異常及40S小次單元生成受阻。進一步透過Fal1 A67V (ATP 結合缺陷) 與D173N (解旋缺陷)突變，證實Fal1的ATPase與helicase活性，均為其功能必須。前人研究顯示Fal1、Sgd1與eIF4G1在體外可以形成穩定三聚體，三者亦具遺傳交互作用，缺失皆會影響pre-rRNA裁切與轉譯效率，且雙突變條件下更為嚴重。功能分析顯示，Sgd1能促進Fal1與RNA結合的能力，而eIF4G1則主要協助Fal1釋放RNA，促進完成解旋循環並從90S上離開。綜合研究結果，我們提出Fal1、Sgd1與eIF4G1可形成一個時序性的調控複合體，協助Fal1於90S核糖體中完成功能性循環，以維持40S小次單元生成與整體轉譯活性。 | zh_TW |
| dc.description.abstract | The ribosome has a key function in translation and protein synthesis in the cell. Its biogenesis relies on a series of orderly pre-rRNA cleavages and the assembly of both small and large subunits. This dynamic process requires the coordinated action of numerous accessory factors, among which DEAD-box RNA helicases play an essential role by unwinding RNA secondary structures and promoting the remodeling of ribonucleoproteins. In this study, we investigate the regulatory mechanism of the ATP-dependent RNA helicase Fal1 and two regulatory factors containing MIF4G domains, Sgd1 and eIF4G1, during the assembly of the 90S pre-ribosome. We found that repression of FAL1 or SGD1 expression led to growth retardation, abnormal pre-rRNA processing, and impaired formation of the 40S small subunit. Furthermore, mutations in Fal1, A67V (ATP-binding deficient) and D173N (helicase-deficient), also resulted in similar defects, confirming that both ATPase and helicase activities of Fal1 are essential for its function. Previous studies have shown that Fal1, Sgd1, and eIF4G1 can form a stable trimeric complex in vitro. These three proteins also exhibit genetic interactions, and their depletion affects pre-rRNA cleavage and translation efficiency. Moreover, the defects are more severe under double-mutant conditions. Functional analyses revealed that Sgd1 enhances Fal1’s RNA binding ability, whereas eIF4G1 primarily facilitates the release of RNA from Fal1, thereby promoting completion of the helicase cycle and release from the 90S pre-ribosome. Taken together, our results suggest that Fal1, Sgd1, and eIF4G1 form a temporally regulated complex that assists Fal1 in completing its functional cycle on the 90S ribosome, ensuring proper formation of the 40S small subunit downstream and maintenance of overall translational activity. | en |
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| dc.description.provenance | Made available in DSpace on 2025-08-19T16:16:08Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 致謝 i
摘要 ii Abstract iii 目次 iv 圖次 vii 表次 viii 第壹章、前言 1 一、核糖體 (Ribosome) 1 1. 核糖體的組成 1 2. 核糖體在轉譯作用中的角色 1 二、核糖體生合成 (Ribosome biosynthesis) 3 1. 核糖體生合成過程 3 2. 核糖體生合成的調控機制 6 3. 核糖體病變 (Ribosomopathies) 10 三、本研究相關的核糖體生合成因子 11 1. eIF4G (eukaryotic translation initiation factor 4G) 11 2. Fal1 12 3. Sgd1 14 4. eIF4G1、Sgd1與Fal1的關係 14 第貳章、研究動機 16 第參章、材料與方法 17 一、菌株與質體 17 二、生長測試 (Growth test) 17 三、轉型作用 (Transformation) 17 1. 快速酵母菌轉型 (Quick yeast transformation) 17 2. 高效酵母菌轉型 (High-efficiency yeast transformation) 17 3. 大腸桿菌轉型作用 (E. coli transformation) 18 四、螢光顯微鏡 (Fluorescence microscopy) 18 五、氫氧化鈉破菌法 (Post-alkaline extraction from yeast) 18 六、西方墨點法 (Western blotting) 19 七、北方墨點法 (Northern blotting) 19 1. 樣品製備及RNA電泳 19 2. RNA轉印 20 3. 探針置備及顯影 20 八、蛋白質免疫沉澱法 (Immunoprecipitation, IP) 20 九、多核糖體圖譜分析 (Polysome profile analysis) 21 十、蛋白質純化 22 1. 蛋白質大量表現 22 2. 蛋白質純化 22 3. 蛋白質交互作用測試 (protein-protein interaction) 23 十一、RNA assay 23 1. RNA結合活性實驗 (RNA Binding Assay) 23 2. RNA解旋活性實驗 (RNA Unwinding Assay) 23 十二、ATP 水解活性測試 (ATPase Assay) 24 第肆章、實驗結果 26 一、Fal1與Sgd1缺失阻礙90S前驅核糖體pre-rRNA的裁切 26 二、Fal1與Sgd1缺失會阻礙下游40S核糖體的生合成 27 三、Fal1功能突變破壞其ATP水解活性並影響90S組裝 28 四、Fal1與輔助因子Sgd1及eIF4G1組成功能關聯的複合物 30 五、Fal1、Sgd1及eIF4G1具基因遺傳交互作用 32 六、eIF4G1與Fal1及Sgd1雙突變體會影響90S前驅核糖體pre-rRNA裁切 33 七、tif4631∆會造成Fal1及Sgd1滯留在90S前驅核糖體上 34 八、Fal1、Sgd1及eIF4G1缺失阻礙晚期pre-40S核糖體成熟 34 九、eIF4G1抑制Fal1的RNA結合能力 35 十、不同MIF4G結構域對Fal1 ATPase具不同的調控模式 36 第伍章、討論 38 一、Fal1、Sgd1缺失影響pre-rRNA 的A0、A1及A2位點裁切 38 二、Sgd1與eIF4G共同調控Fal1與RNA結合及ATPase活性 39 三、Fal1、Sgd1與eIF4G1缺失導致90S前驅核糖體組裝異常 41 四、三者缺失造成pre-40S生成受阻，並改變Tsr1在細胞中的分布 42 第陸章、結論 44 參考文獻 45 附錄 83 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 核糖體生合成 | zh_TW |
| dc.subject | 90S核糖體 | zh_TW |
| dc.subject | 調控因子 | zh_TW |
| dc.subject | MIF4G結構 | zh_TW |
| dc.subject | RNA解旋酶Fal1 | zh_TW |
| dc.subject | RNA helicase Fal1 | en |
| dc.subject | MIF4G domain | en |
| dc.subject | regulatory factors | en |
| dc.subject | ribosome biogenesis | en |
| dc.subject | 90S pre-ribosome | en |
| dc.title | 探討RNA解旋酶Fal1在90S前驅核糖體生合成中的功能及調控 | zh_TW |
| dc.title | Study the function and regulation of the RNA helicase Fal1 in 90S pre-ribosome biogenesis | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 陳美瑜;何孟樵;溫進德;蕭貴陽 | zh_TW |
| dc.contributor.oralexamcommittee | Mei-Yu Chen ;Meng-Chiao Ho;Jin-Der Wen;Kuei-Yang Hsiao | en |
| dc.subject.keyword | 90S核糖體,核糖體生合成,RNA解旋酶Fal1,MIF4G結構,調控因子, | zh_TW |
| dc.subject.keyword | 90S pre-ribosome,ribosome biogenesis,RNA helicase Fal1,MIF4G domain,regulatory factors, | en |
| dc.relation.page | 86 | - |
| dc.identifier.doi | 10.6342/NTU202504044 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2025-08-12 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 農業化學系 | - |
| dc.date.embargo-lift | 2030-08-05 | - |
| 顯示於系所單位： | 農業化學系 | |
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|---|---|---|---|
| ntu-113-2.pdf 此日期後於網路公開 2030-08-05 | 4.6 MB | Adobe PDF |
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