臺灣博碩士論文加值系統

RNA的編輯是一種由ADAR蛋白家族催化進行脫胺作用，而使原本的adenosine 變成 inosine (A到I)的核苷酸改變。我們先前的次世代定序結果顯示穿膜蛋白63B (TMEM63B)會經由ADAR2的催化造成編輯修飾，並導致TMEM63B密碼子從麩醯胺酸(Q) 變成精胺酸 (R)。在本文中我們想要知道TMEM63B的生理意義並當編輯事件發生時會造成甚麼影響。透過質譜儀和免疫沉澱的實驗分析我們發現了TMEM63B會與AP2a1有交互作用，也由此推論TMEM63B可能參與了clathrin-dependent endocytosis的機制。為了探討TMEM63B的功能和ADAR媒介調控的內吞作用，我們建構了沉默與過度表現TMEM63B系統。當降低TMEM63B表現後會降低AP2a1於細胞質的分布，並且減少早期內吞體與晚期內吞體的範圍，而溶酶體作用亦受到影響。而後我們在HeLa細胞過度表現未編輯和編輯型的TMEM63B，發現過度表現編輯型的TMEM63B細胞中，其內吞作用的標的蛋白範圍會減少；因此推論當TMEM63B編輯時會對內吞作用產生負調控。由這些結果顯示TMEM63B可能參與clathrin-dependent endocytosis 的路徑，並且RNA編輯可能為一種平衡的調控機制。

RNA editing, a site-selective conversion of adenosine to inosine (A-to-I), which is catalyzed by adenosine deaminase acting on RNA (ADAR) family. Our previous NGS data have identified that the transmembrane protein 63b (TMEM63B) is catalyzed by ADAR2, which alters the codon glutamine (Q) to arginine (R) of TMEM63B protein. In this study, we want to know the physiological significance of TMEM63B and the impact of the editing event. Spectrometry-based analysis and co-immunoprecipitation assay showed the interaction between TMEM63B and AP2a1 suggesting the regulatory role of TMEM63B in clathrin-dependent endocytosis pathway. To investigate the functional role of TMEM63B and ADAR-mediated regulation in endocytosis, we analyzed the endocytosis process with the established silencing and overexpression systems of TMEM63B. Knockdown of TMEM63B reduce the distribution of AP2a1 and reduces early endosome and late endosomes formation, supporting the functional regulation of TMEM63B in endocytosis process. Furthermore, silencing TMEM63B expression will affect lysosomes function. We overexpressed unedited TMEM63B and edited TMEM63B in HeLa cells, and subsequently found the reduced formation of endocytosis marker in edited TMEM63B-expressing cells. Therefore, it suggests the negative regulatory role of ADAR editing in endocytosis. Taken together, these findings the functional role of TMEM63B in clathrin-dependent endocytosis pathway, and RNA editing on TMEM63B may serve the regulatory mechanism in homeostasis.

目錄
指導教授推薦書
口試委員會審定書
致謝 iii
摘要 iv
ABSTRACT v
目錄 vi
圖表目錄 viii
第一章 緒論 1
1-1 Adenosine deaminase acting on RNA (ADAR)介紹 1
1-2 TMEM63B(Transmembrane protein 63B)簡介 3
1-3 內吞作用(Endocytosis)簡介 4
1-4 多巴胺(dopamine)簡介 5
1-5研究動機 5
1-6實驗設計 6
第二章 實驗方法 7
2-1引子設計(Primer design) 7
2-2即時定量聚合酶連鎖反應(Quantitative real-time PCR) 7
2-3 DNA膠體純化(Gel extraction) 7
2-4質體製備 8
2-4-1接合反應(Ligation) 8
2-4-2質體轉型(Transform) 8
2-4-3質體萃取 9
2-4-4質體再轉型(retransform) 10
2-5 聚合酶連鎖反應(PCR) 10
2-6限制酶剪切反應 10
2-7 Sanger定序 10
2-8 細胞培養(Cell culture) 10
2-8-1 小鼠神經瘤母細胞(Mouse neuroblastoma cell line, Neuro-2A) 10
2-8-2 人類子宮頸癌細胞(HeLa cell) 11
2-8-3 細胞轉染(Cell transfection) 11
2-9 transferrin測試 11
2-10 Lysotracker 測試 12
2-11 免疫螢光染色法 (Immunofluorescence) 12
第三章 實驗結果 13
3-1-1 TMEM63B降低表現後對AP2a1蛋白所造成的影響 13
3-1-2 探討TMEM63B在內吞作用不同時期的角色 13
3-1-3 TMEM63B降低表現對細胞型態的影響 14
3-1-4利用transferrin探討TMEM63B對clathrin-depentant endocytosis的影響 15
3-1-5 利用Lysotracker測試TMEM63B受否會運送至溶酶體 15
3-1-6 探討TMEM63B之功能性區域 16
3-1-7 過度表現TMEM63B表現型對於細胞的型態之影響 16
3-1-8 測試TMEM63B編輯後在內吞作用不同時期之生理意義 17
3-1-9 利用Lysotracker測試TMEM63B編輯後運送至溶酶體 18
3-2 探討TMEM63B是否會影響到多巴胺的運送 18
第四章 實驗討論 20
4-1-1 TMEM63B使否影響到鈣離子通道活性並影響溶酶體功能 20
4-1-2 TMEM63B不同的functional domain所造成的生理意義 20
4-2 TMEM63B 是否參與了神經的運輸 21
參考文獻 22
實驗圖表 25

圖表目錄
圖一 TMEM63B降低表現後對AP2a1蛋白所造成的影響 26
圖二 探討TMEM63B在內吞作用不同時期的角色 30
圖三 TMEM63B降低表現對細胞型態的影響 31
圖四 利用transferrin探討TMEM63B對clathrin-depentant endocytosis的影響 32
圖五 利用Lysotracker測試TMEM63B受否會運送至溶酶體 35
圖六、探討TMEM63B之功能性區域 38
圖七 過度表現後測試TMEM63B編輯後在細胞中的型態與對AP2a1蛋白所造成的影響 41
圖八 測試TMEM63B編輯後在內吞作用不同時期有何生理意義 45
圖九 利用Lysotracker測試TMEM63B編輯後運送至溶酶體 47
圖十 探討TMEM63B是否會影響到多巴胺的運送 49

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