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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 詹世鵬(Shih-Peng Chan) | |
dc.contributor.author | Mao-Feng Chen | en |
dc.contributor.author | 陳懋鋒 | zh_TW |
dc.date.accessioned | 2021-06-15T11:25:21Z | - |
dc.date.available | 2021-08-26 | |
dc.date.copyright | 2016-08-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-18 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49363 | - |
dc.description.abstract | DDX17 (p72) 是DEAD-box核糖核酸解旋酶 (RNA helicases) 的一員。DEAD-box核糖核酸解旋酶從細菌到人類間具有保留性,它們可以利用水解三磷酸腺苷 (ATP) 產生的能量解開雙股螺旋核糖核酸,或調節核糖核酸與蛋白質間的交互作用 (interaction) 。在真核生物中,幾乎所有的核糖核酸代謝都需要DEAD-box核糖核酸解旋酶參與。之前的研究指出,DDX17和它的同源蛋白DDX5 (p68) 形成的異二聚體 (heterodimer) 與負責微小核醣核酸生合成重要步驟的DROSHA共同作用,促進一群微小核糖核酸前驅物 (precursor microRNA) 的生成。DDX17也與癌症產生 (carcinogenesis) 和癌細胞的增生 (proliferation) 有關,不過機制尚未明瞭。此外,DDX17也參與抗病毒反應,做為zinc-finger antiviral protein (ZAP或ZC3HAV1) 的輔因子 (cofactor) ,徵召核糖核酸去頭蓋複合體 (decapping complex) 到目標病毒的信使核糖核酸 (messenger RNA) 上並將之降解。綜上所述,我們認為DDX17具有參與許多細胞反應的可能性,在細胞生理上也可能扮演著重要角色。所以我們決定用蛋白質體學的方法來尋找嶄新的DDX17交互作用蛋白,並探討可能的功能。我們在HeLa細胞中表現有HA標記 (tag) 的DDX17蛋白,經過免疫共沉澱及聚丙烯醯胺膠體電泳後進行蛋白質鑑定。在蛋白質鑑定的結果中除了前述的ZAP與DDX5外 (兩個已知的DDX17交互作用蛋白) ,我們發現十四個可能和DDX17交互作用的候選蛋白。候選蛋白之一的PABPC1是多聚腺苷酸結合蛋白 (poly-A tail binding protein) ,我們藉由免疫共沈澱法確認PABPC1和DDX17間的交互作用,並發現該作用依賴核糖核酸 (RNA-dependent) 的存在,推測DDX17可能與調控轉譯或信使核糖核酸的脫腺苷化 (deadenylation) 有關。本論文中,我們建構了帶有標記的質體在細胞中表現數個候選蛋白進行進一步分析,包括核糖核酸結合蛋白OASL,核糖核酸結合蛋白hnRNP K與E3泛素連接酶TRIM21。我們利用免疫共沉澱分析確認了OASL與hnRNP K各自和DDX17間存在著非依賴核糖核酸的 (RNA-independent) 交互作用;但我們沒有在免疫共沉澱分析觀察到TRIM21與DDX17的交互作用。我們對DDX17-OASL蛋白質複合體的功能進行進一步的研究,發現負責微小核醣核酸生合成重要步驟的DROSHA也可能存在於此蛋白質複合體內。我們透過核糖核酸免疫沉澱 (RIP) 與即時定量聚合酶連鎖反應 (qPCR) 發現,DDX17-OASL-DROSHA蛋白質複合體的功能與微小核醣核酸生合成可能無關。前人研究指出,此三個蛋白質各自被認為與先天免疫有關,而根據本論文的發現,我們推測DDX17-OASL-DROSHA可能以形成一個蛋白複合體的方式來運行其與先天免疫相關的功能。 | zh_TW |
dc.description.abstract | DDX17 (p72) belongs to the DExD/H-box family of RNA helicases that are conserved from bacteria to human and are required for almost all RNA metabolisms. The DExD/H-box RNA helicases promote unwinding of RNA duplexes or rearrangement of RNA-protein interaction using the energy from hydrolyzing ATP. Previously, DDX17 has been found to play a role in control of transcription and splicing. Recently, DDX17 and its homologous protein DDX5 (p68) were demonstrated to form a heterodimer that facilitates the DROSHA-mediated processing for a set of primary microRNAs (pri-miRNAs). On the other hand, DDX17 has been implicated in cell proliferation and carcinogenesis in a variety of cancers by studies from several laboratories while the mechanism remains largely unknown. Moreover, DDX17 has been demonstrated to participate in antiviral responses as a cofactor of the zinc-finger antiviral protein (ZAP or ZC3HAV1) that recruits RNA depredating enzymes to target viral mRNAs. Thus, DDX17 has a potential to function in multiple cellular processes and play important roles in many aspects of cell biology. In this work, we seek to identify novel pathway partners of DDX17 by proteomic approaches to discover its new biological implication. We expressed HA-tagged DDX17 in the cells and precipitated DDX17-containing complexes from the lysates following MALDI-TOF. So far, a few candidates were identified from the precipitates, including previously reported DDX5 and ZAP. Interestingly, we found that cytosolic poly(A)-tail binding protein PABPC1, one of these candidates, interacts with DDX17 in an RNA-dependent manner, suggesting a role of DDX17 in translational control and/or mRNA deadenylation. We also expressed tag-fused protein for several candidates, including RNA binding protein OASL, hnRNP K and E3 ubiquitin ligase TRIM21, to investigate their interaction with DDX17. By immunoprecipitation assays, we demonstrated that both OASL and hnRNP K interact with DDX17 in an RNA-independent manner whereas no interaction between TRIM21 and DDX17 could be proven. We further found the presence of DROSHA in DDX17-OASL complexes but exiled the possibility that the DDX17-OASL-DROSHA is involved in miRNA biogenesis. Previous studies from different groups have separately indicated that these three proteins could be all related to autoimmunity. Here, we provide evidence to support the notion that DDX17, OASL and DROSHA form a complex with RNAs in contributing to autoimmune responses. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T11:25:21Z (GMT). No. of bitstreams: 1 ntu-105-R03445127-1.pdf: 4232706 bytes, checksum: 73283d1c087647f94a52fc9c02e436b7 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | CONTENTS
口試委員會審定書 I 誌謝 II 中文摘要 III ABSTRACT V CONTENTS VII Chapter 1 Introduction 1 1.1 DEAD-box核糖核酸解旋酶 (RNA helicases) 1 1.2 DEAD-box核糖核酸解旋酶的功能 1 1.2.1 解旋活性 2 1.2.2 黏合 (annealing) 活性 2 1.2.3 自身構形改變 2 1.2.4 蛋白質交互作用 2 1.3 DDX17 3 1.3.1 轉錄 3 1.3.2 信使核醣核酸 (messenger RNA) 選擇性剪接 4 1.3.3 人類細胞內的先天免疫 (innate immunity) 4 1.3.4 癌細胞的存活和複製 4 1.3.5 微小核醣核酸 (microRNA) 的生合成 5 1.4 實驗動機 5 Chapter 2 Materials and Methods 7 2.1 細菌品系 7 2.2 線蟲品系 (Caenorhabditis elegans strains) 7 2.3 線蟲裂解液 (worm lysate) 8 2.3.1 均質機 (homogenizer) 8 2.3.2 磨缽 9 2.4 顯微注射 9 2.4.1 注射針製備 9 2.4.2 注射 9 2.5 細胞株 10 2.6 質體 11 2.6.1 非本人所構築之質體 11 2.6.2 本人所構築之質體 12 2.7 細胞轉染 (transfection) 14 2.8 細胞裂解液 (cell lysate) 14 2.8.1 抽取 15 2.8.2 蛋白質濃度測量 15 2.9 細胞核質分離 (cell fractionation) 16 2.10 核糖核酸干擾 (RNA interference) 16 2.10.1 慢病毒 (lenti virus) 包裹 16 2.10.2 細胞感染及抗生素篩選 17 2.11 免疫螢光法 (immunofluorescence assay) 17 2.12 免疫共沉澱法 (co-immunoprecipitation) 18 2.13 西方墨點法 19 2.13.1 聚丙烯醯胺膠體製備 19 2.13.2 樣品配製、膠體電泳及轉漬 19 2.13.3 抗體及冷光受質成像 20 2.14 聚丙烯醯胺膠體染色及蛋白質鑑定 (protein identification) 21 2.14.1 考馬斯藍染色 (coomassie blue staining) 21 2.14.2 基質輔助雷射脫附游離-飛行時間 (MALDI-TOF) 21 2.14.3 液態層析質譜儀 (LC-MS/MS) 21 2.15 蔗糖密度梯度 (sucrose gradient) 超高速離心 21 2.16 即時定量聚合酶連鎖反應 (quantitative real time polymerase chain reaction) 22 2.16.1 抽取核糖核酸 22 2.16.2 反轉錄 22 2.16.3 樣本配製與上機 23 Chapter 3 Results 24 3.1 人類細胞系統可以做為實現我們目的的材料 24 3.2 DDX17與OASL在細胞質進行不依賴核糖核酸的交互作用 26 3.3 OASL與DROSHA之間具有不依賴核糖核酸的交互作用 27 3.4 DDX17-OASL-DROSHA蛋白質複合體可能參與對抗核糖核酸病毒 28 3.5 我們無法確認extra band是否為DDX17的同功型 (isoform) 28 3.6 DDX17與hnRNP K之間具有不依賴核糖核酸的交互作用 29 3.7 TRIM21與rat IgG之間具有不依賴核糖核酸的交互作用 29 Chapter 4 Discussion 30 4.1 DDX17、OASL與DROSHA間的交互作用 30 4.2 DDX17抗體的extra band 30 4.3 hnRNP K與DDX17間的交互作用 31 4.4 無法偵測到TRIM21與DDX17間的交互作用 31 Chapter 5 Figures 32 Fig. 1 線蟲系統內的免疫沉澱實驗具有高背景值 32 Fig. 2 在人類細胞系統中可以免疫共沉澱下許多DDX17的交互作用候選蛋白 37 Fig. 3 DDX17以不依賴核糖核酸的方式與OASL交互作用 40 Fig. 4 DDX17與OASL在細胞質有共區域化 (co-localization) 現象 42 Fig. 5 DDX17和OASL各自以不依賴核糖核酸的方式與DROSHA交互作用 45 Fig. 6 OASL和pri-let-7a-1或pri-let-7-c無交互作用,DDX17則有 47 Fig. 7 DDX17和OASL的蛋白質表現量在Huh7.5與H7-HCVR細胞株中有上升的現象 49 Fig. 8 無法確認DDX17抗體的extra band是否為DDX17的同功型 51 Fig. 9 DDX17與hnRNP K有交互作用,DROSHA則無 54 Fig. 10 無法偵測到DDX17與TRIM21間的交互作用 56 Chapter 6 References 58 Chapter 7 Appendix 64 附圖一、 真核生物中有DEAD-box核糖核酸解旋酶參與的核醣核酸代謝 64 附圖二、 DEAD-box核糖核酸解旋酶結構 65 附圖三、 DDX17與DDX5胺基酸序列比較 66 附圖四、 降低DDX-17的蛋白表現量會使線蟲產生較虛弱的形態 67 附圖五、 小髮夾核糖核酸 (shRNA) 的目標位置 68 附表一、 質體構築之限制酶切位 70 | |
dc.language.iso | zh-TW | |
dc.title | DEAD-box核糖核酸解旋酶DDX17 (p72)新穎結合蛋白之鑑定 | zh_TW |
dc.title | Identification of novel pathway partners of the DEAD-box RNA helicase DDX17 (p72) | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 余明俊(Ming-Jiun Yu),蔡欣祐(Hsin-Yue Tsai) | |
dc.subject.keyword | DDX17,p72,OASL,DROSHA,hnRNP K,antiviral function, | zh_TW |
dc.relation.page | 70 | |
dc.identifier.doi | 10.6342/NTU201601827 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-18 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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