臺灣博碩士論文加值系統

　　Inosine 出現在哺乳類動物中是經 RNA 轉錄後修飾產生，此類編輯事件可調節基因的表現，並使該蛋白具有豐富的多樣性與功能性，在生物體中是必要過程，其中 ADAR 家族蛋白具有此編輯功能，可使雙股 RNA 序列上的 A 轉變成 I。透過次世代定序 (NGS) 資料庫分析，發現 TMEM63B 具有編輯的位點，會使第 619 個胺基酸由 glutamine (Q) 變成arginine (R)，但此影響目前仍未知。本篇主要目的是研究 TMEM63B 本身之功能以及 TMEM63B 受到 ADAR 編輯後的意義。在此證明 ADAR 主要藉由 double-strand binding domain 2 連接目標的 RNA 序列，此編輯事件專一性發生於腦部。為了探討 TMEM63B 在腦部的功能，選用小鼠神經瘤母細胞(Neuro-2A) 並抑制 TMEM63B ，分析後發現 TMEM63B 可能會抑制神經之分化。透過 Biological General Repository for Interaction Datasets (BioGRID) 的分析中找出 TMEM63B 與 PMCA2 兩者可能為交互作用蛋白，經由免疫沉澱法證實結合。進一步探討於活體的影響，以 CRISPR/Cas9 技術建立 Tmem63b 之基因轉殖鼠，去探討此編輯事件是否影響小鼠生長、神經以及認知相關疾病。在 Tmem63bI/+ 小鼠觀察到有過動症的傾向，而以 PTZ 施打在Tmem63bA/A 小鼠觀察到較為敏感且症狀加劇，推測 TMEM63B 會藉由調控鈣離子濃度，使神經分化而影響生理功能。

Inosine is an abundant RNA modification in mammal’s transcriptome and is necessary for sundry biological processes in modulating gene expression at the post-transcriptional level. Adenosine deaminase acting on RNA (ADAR) catalyzes the hydrolytic deamination of adenosines to inosines (A-to-I editing) in double-stranded RNA regions. Our previous Next Generation Sequencing (NGS) data have revealed that the transmembrane protein 63b (Tmem 63b) undergoes an editing event that is catalyzed by Adar2. This editing event resulted in its amino acid changed from glutamine (Q) into arginine (R), but its effect is still unknown. In this research, we performed a series of experiments to identify the Tmem63b functional and its editing consequence. We demonstrated that the RNA double-strand binding domain 2 of Adar2 is important, the Tmem63b RNA editing focus in brain. To explore, we would study Tmem63b function in brain, Tmem63b was silenced by siRNA in N2a cells, found it roles in neuronal differentiation. We analysis Tmem63b interaction proteins via Biological General Repository for Interaction Datasets (BioGRID) and confirm Tmem63b interaction proteins-PMCA2 by immunoprecipitation. To further confirm the biological role of Tmem63b and the editing. We establish Tmem63b knock-out and knock-in mice by CRISPR/Cas9 system. Identify Tmem63b editing to effect for neuronal differentiation and social behavior disorder. The result showed Tmem63bI/+ mice exhibited neuropsychiatric disorders compared to wild type mice. In addition, Tmem63bA/A mice showed strong seizures by PTZ induced. Our research will elucidate a biological meaning for RNA editing event.

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
圖目錄 xi
表目錄 xii
1.1 RNA 編輯 1
1.2 ADAR family 1
1.3 TMEM63 family 3
1.4 Plasma membrane calcium pump (PMCA) 4
1.5 PMCA2 之生理意義 5
第二章 實驗動機 8
第三章 實驗目標(Specific Aims)及設計 9
AIM 1 確認 TMEM63B 的編輯事件 10
1.1 確定 ADAR2 結合區域和催化區域對於 TMEM63B 編輯事件的重要性 10
1.2 確定 Tmem63b 在小鼠的各個組織編輯程度 10
AIM 2 鑑定 TMEM63B 的功能，且確認編輯對於TMEM63B 功能的改變 11
2.1 製備TMEM63B的抗體 11
2.2 建構人類 TMEM63B 表現載體 11
2.3 確定 TMEM63B 蛋白定位至細胞的位置 12
2.4 尋找 TMEM63B 的交互蛋白，以利分析上下游調控蛋白 12
2.5 探討 TMEM63B 對神經分化影響 13
AIM3 以基因轉殖鼠探討TMEM63B缺失以及編輯事件在生理的意義 13
3.1 探討 TMEM63B 缺失對於社交行為的影響 13
3.2 探討 TMEM63B 編輯事件對癲癇和聽覺的影響 14
第四章 實驗技術與方法 15
4.1 小鼠神經瘤母細胞 (Mouse neuroblastoma, Neuro-2A) 15
4.2 大鼠腎上腺髓質嗜鉻細胞瘤 (pheochromocytoma) 15
4.3 細胞轉染(Transfection) 16
4.4 RNA萃取 16
4.5 反轉錄聚合酶連鎖反應 ( Reverse transcription ) 17
4.6 即時定量聚合酶連鎖反應 (Quantitative real-time PCR) 17
4.7 蛋白質萃取及定量 18
4.8 SDS 聚丙烯醯胺膠體電泳 (SDS-PAGE) 18
4.9 西方墨點法(Western Blotting) 19
4.10 聚合酶連鎖反應 (Polymerase chain reaction , PCR) 20
4.11 建構人類 TMEM63B表現載體 20
4.12 免疫螢光染色(immunofluorescence staining) 21
4.13 免疫沉澱法(Immunoprecipitation) 22
4.14 抗體製備 22
4.15 動物社交行為測試 23
4.16 小鼠癲癇症狀測試 25
4.17 前脈衝抑制作用(Pre-pulse inhibition ) 25
第五章 實驗結果 26
5.1 確定ADAR2對TMEM63B會產生編輯 26
5.2 建構 TMEM63B 表現載體 26
5.3 製備TMEM63B專一性抗體 27
5.4 確定編輯事件對於 Tmem63b 蛋白在細胞中位置的定位 28
5.5 確定TMEM63B交互作用蛋白 29
5.6 確定Tmem63B是否影響神經分化 30
5.7 TMEM63B 之功能對於癲癇、聽覺障礙及社交行為障礙在小鼠動物模式實驗 30
5.8 TMEM63B 編輯事件對於癲癇、聽覺障礙及社交行為障礙在小鼠動物模式實驗 31
第六章 討論及未來方向 33
6.1 探討 TMEM63B 本身為何種離子通道且如何進行調控，並觀察在編輯前後的改變 33
6.2 確定 Tmem63b對於神經分化影響 34
6.3 利用實驗動物探討 TMEM63B 之生理功能影響 35
參考文獻 38
圖表 42


圖目錄

圖 一、ADAR2 各種突變組對於TMEM63B 的編輯程度 43
圖 二、測試專一性TMEM63B抗體用於西方墨點法實驗的可行性 46
圖 三、以 Immunofluorescence 鑑定TMEM63B 在細胞中位置 48
圖 四、以Immunoprecipitation鑑定TMEM63B 的交互蛋白 50
圖 五、抑制TMEM63B 對於神經分化的影響 52
圖 六、TMEM63B 之功能對於癲癇、聽覺障礙及社交行為障礙在小鼠動物模式實驗 55
圖 七、TMEM63B 編輯事件對於癲癇、聽覺障礙及社交行為障礙在小鼠動物模式實驗 59

 
表目錄

表 一、每次施打抗原及採集血清之時間表 61

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