臺灣博碩士論文加值系統

RNA編輯為一個可改變特異性核苷酸的轉錄後修飾。其中Adenosine-to-Inosine編輯主要由ADAR酵素所主導，使Adenosine(A)脫氨作用變為Inosine ( I )，而I會與Cytosine (C)配對，所以A-to-I可以視為A-to-G的編輯。而A-to-I RNA編輯可能會造成核苷酸改變而影響胺基酸。已知ADAR會與雙股序列結合，發生編輯現象。在這些ADAR標的基因當中，得知老鼠組織之中，RNA編輯程度具有明顯差異、且與ADAR表現量無正向相關性，因此推測ADAR可能與未知作用蛋白結合，達到RNA編輯程度不同的能力。並以RNA pull-down assay分析過表達ADAR2的細胞，實驗結果顯示無找到相關蛋白。參考其他利用質譜儀鑑定ADAR相關蛋白實驗結果，未來使用初代培養老鼠組織並標定相關蛋白，分析ADAR之下游基因編輯程度。ADAR形成二聚體影響編輯程度，我們將探討蛋白質相互作用的調節之編輯程度。環形RNA生成需要Alu序列，ADAR可能會結合Alu序列產生編輯，調控環形RNA表現。現結果分析顯示，在口腔鱗狀癌臨床檢體中環形RNA與ADAR表現量不同。為了探討ADAR調控環形RNA，未來在口腔鱗狀癌分析環形RNA其扮演功能，也許能成為OSCC潛在癌症治療。

RNA editing is a post-transcriptional modification that alters specific nucleotides. Adenosine-to-Inosine (A-to-I) RNA editing is carried out by ADAR enzymes which converting adenosines converted to inosines through deamination. Inosine pairs with cytosine, and A-to-I can be considered A-to-G editing. A-to-I RNA editing may cause nucleotide change that affect amino acid coding. It is known that ADAR will bind double stranded sequence and have editing events. We observed differential editing degree in mouse tissues, and the difference is not associated with ADAR expression level. Therefore, we speculate that ADAR may interact with associated proteins and deliver different editing activity. RNA pull-down assay was analyzed with ADAR2 overexpression cell lysate, and no interaction protein was found. Other groups have identify several ADAR-associated proteins by mass spectrometry assay; we will target the associated protein expression in primary culture of mouse tissues, and analyze the editing level of ADAR downstream target genes. Dimerization of ADAR proteins affects the degree of editing, and we will explore the regulation of protein-protein interaction in editing activity. Alu sequence is required for the biogenesis of circular RNA, ADAR may bind Alu repeats and regulate circular RNA expression. Our current sequencing analysis suggested the differential expression of circular RNA and ADAR in oral squamous cell carcinoma clinical samples. To explore ADAR regulation in circular RNA, we will analyze the functions of circular RNA in OSCC cells, and which may serve the potential cancer therapy.

指導教授推薦書…………………………………………………………
論文口試委員審定書……………………………………………………
致謝 iii
中文摘要 iv
Abstract vi
目錄 viii
圖目錄 xii
附圖表目錄 xiii
第一章、緒論 - 1 -
1.1 ADAR蛋白簡介 - 1 -
1.2 Circular RNA - 4 -
1.3研究動機 - 5 -
實驗流程圖 - 7 -
1.4實驗設計 - 7 -
第二章、實驗方法與材料 - 10 -
2.1 細胞培養 - 10 -
2.2 細胞轉染 - 10 -
2.3 RNA萃取 - 11 -
2.4 反轉錄聚合酶鏈式反應 - 12 -
2.5即時定量聚合酶鏈式反應 - 12 -
2.6引子設計 - 12 -
2.7 聚合酶鏈式反應 - 13 -
2.8 膠體純化 - 13 -
2.9 載體連接 - 14 -
2.10轉型作用 - 14 -
2.11質體萃取 - 15 -
2.12 表現載體連接 - 15 -
2.13蛋白質萃取 - 16 -
2.14西方墨點法 - 16 -
2.15免疫沉澱 - 18 -
2.16核質分離 - 19 -
2.17 In vitro transcription - 19 -
2.18 RNA clean up - 20 -
2.19 Biotinylation RNA - 20 -
2.20 RNA-pull down assay - 21 -
2.21 Dot bloting - 22 -
2.22 Ribosome-nascent chain complex (RNC) - 23 -
第三章、實驗結果 - 24 -
3.1 確認 ADAR在老鼠部位中的編輯能力 - 24 -
3.2 探討與ADAR互相作用的蛋白 - 24 -
3.3 分析ADAR二聚體之編輯程度 - 28 -
3.4 分析ADAR 與circular RNA形成之調控關係 - 29 -
第四章、討論 - 32 -
4.1 利用蛋白質體方式以鑑定與ADAR互相作用的蛋白 - 32 -
4.2 分析ADAR二聚體之編輯程度 - 33 -
4.3分析ADAR 與circular RNA形成之調控關係 - 34 -
第五章、參考文獻 - 37 -
第六章、圖 - 40 -
附圖 - 57 -
附表 - 58 -

 
圖目錄
圖一、ADAR2標的基因在不同組織的編輯程度…………….………………………40
圖二、ADAR與其下游基因在不同組織的表現量…………………….……………41
圖三、利用西方墨點法分析抗體免疫沉澱效果…………………….……………….42
圖四、組織樣本分析抗體免疫沉澱效果………………………….…………………….….43
圖五、利用裂解液測試老鼠組織裂解效果……………………….…………….…………44
圖六、預測Tmem63b及Copa之RNA二級結構…….………….……….…….……45
圖七、Tmem63b載體編輯程度分析………………….…………….…………….……….……46
圖八、利用西方墨點法分析 Tmem63b 300片段之RNA pull-down assay結果………………….…………….…………….……….………….…………….…………….……………….…...47
圖九、分析相關蛋白表現量……….…………….………….…………………….….………………49
圖十、分析ADAR標的基因 之編輯程度……….….………………………………………51
圖十一、偵測環形RNA之MMP12與LIFR表現量…………………………………52
圖十二、分析circular RNA的形成和架構…………………………………………………53
圖十三、在核質分離中分析環形RNA表現量…………………………………………54
圖十四、建構環形RNA載體……….….……………………………………………………………55
圖十五、siRNA抑制效率………………………………………………56

附圖目錄
附圖…….….………………………………….….………………………………….….………………………………57
附圖一、環形RNA資料庫分析及Cir載體…………………………………..………..57

附表目錄
附表….………………………………….….………………………………….….…………………………………….58
Table 1. Oligonucleotides………………….….………………………………….….…………...…58

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