臺灣博碩士論文加值系統

Adenosine Deaminase Acting on RNA (ADAR)酵素能催化RNA腺苷至肌苷的轉化，此轉化過程是為RNA編輯。RNA 編輯能使轉錄本具多樣性並使生物體內的細胞調控機制多元。從腫瘤組織中發現RNA編輯的變化能影響腫瘤進程，然而此機制目前仍不清楚。藉由大範圍的RNA編輯標的分析，發現部分目標基因和細胞調節低氧壓力有關。其中，包含Hypoxia-inducible factor 1-alpha (HIF-1A)基因的兩個負調控角色-天然反股轉錄本HIF1A-AS2和泛素連接酶架橋的LIM domains containing 1 (LIMD1)蛋白。此研究發現HIF1A-AS2在細胞受到低氧壓力的初期會大量表現並透過對向轉錄的競爭壓力拮抗HIF-1A的轉錄本表達。ADAR1則是會透過影響其轉錄過程來抑制此反股轉錄本的表現。與之不同的，ADAR1對LIMD1的調控是透過干擾其mRNA易位至細胞質進而抑制其後續轉譯過程。最終，ADAR1能在氧氣缺乏時大量且迅速的幫助HIF-1α累積和其下游所影響的基因，並促進血管新生等。本篇文章提供一個全新的機制幫助了解細胞遭受低氧壓力的情況下，ADAR1對於HIF-1α的調控及其重要性。
Transmembrane protein 63B (Tmem63b)是一個11次穿膜之蛋白，會被Adenosine Deaminase Acting on RNA 2 (ADAR2)蛋白進行腦部特有的A-to-I之RNA編輯，導致轉譯出的氨基酸重編碼由麩醯胺酸轉變成精氨酸。然而此蛋白的功能以及被編輯的影響目前仍不清楚。為了探討Tmem63b本身之分子功能與機制，實驗室先前利用質譜儀分析了Tmem63b之交互作用蛋白並找到Adaptor-related protein complex 2, alpha 1 (AP2A1)。並進一步發現Tmem63b會幫助格形蛋白依賴性胞吞作用及胞吐作用的過程。為了進一步探討此穿膜蛋白的生理意義，本篇研究利用 CRISPR/ Cas9 技術將小鼠的Tmem63b基因突變或修改成編輯後的序列，並探討老鼠生理及行為上的差異。發現Tmem63b基因突變會導致其有過動或是學習不佳的狀況。其可能的調控機制，推測是因多巴胺受體可能透過囊泡運輸作用而影響在細胞膜的分布或表現，最後影響多巴胺訊號的傳遞。總結，此研究為Tmem63b的細胞和生理功能提供初步證據，也拓展ADAR2影響蛋白重編碼的功能意義。

Adenosine deaminase acting on RNA (ADAR)-catalyzed adenosine- to-inosine RNA editing is dysregulated in neoplastic progression. However, how this transcriptome recoding process is functionally correlated with tumorigenesis remains largely elusive. RNA editome datasets identify hypoxia-related genes as A-to-I editing targets. In particular, two negative regulators of HIF-1A – the natural antisense transcript HIF1A-AS2 and the ubiquitin ligase scaffold-LIMD1 – are directly but differentially modulated by ADAR1. We show that HIF1A-AS2 antagonizes the expression of HIF-1A in the immediate-early phase of hypoxic challenge, likely through a convergent transcription competition in cis. ADAR1 in turn suppresses transcriptional progression of the antisense gene. In contrast, ADAR1 affects LIMD1 expression post-transcriptionally, by interfering with the cytoplasmic translocation of LIMD1 mRNA and thus protein translation. This multi-tier regulation coordinated by ADAR1 promotes robust and timely accumulation of HIF-1α upon oxygen depletion and reinforces target gene induction and downstream angiogenesis. Our results pinpoint ADAR1-HIF-1α axis as a hitherto unrecognized key regulator in hypoxia.
Transmembrane protein 63B (Tmem63b) is an eleven-transmembrane protein, which is targeted by adenosine deaminase acting on RNA 2 (ADAR2) for brain-specific RNA recoding (Q to R). However, the cellular role of this protein and its RNA editing event remains largely unresolved. LC-MS/MS-based interactome analysis previously done by our laboratory has uncovered adaptor-related protein complex 2, alpha 1 (AP2A1) as one of associated proteins of Tmem63. In line with this interaction, Tmem63b was found to promote the processes of clathrin-dependent endocytosis and exocytosis. To further explore the biological significant of this transmembrane protein, we have created using the CRISPR/Cas9 technique Tmem63b mutant mice. Behavioral characterization then revealed that the heterozygous Tmem63bI/+ mice exhibits hyperactivity and altered learning ability. These neurological defects could be attributed to a dysregulated distribution and expression of the dopamine receptor in neurons. In summary, our findings in this part provided early evidence for the cellular and physiological function of Tmem63b and for the functional implications of ADAR2-mediated RNA recoding event.

Table of Contents
指導教授推薦書
口試委員會審定書
誌謝 iii
摘要 iv
Abstract vi
Table of Contents viii
List of Figures xi
List of Tables xiii
Chapter 1 Background and Significance - 1 -
1.1 RNA editing - 1 -
1.2 ADAR family - 1 -
1.3 The correlation between ADAR and hypoxia - 3 -
1.4 Hypoxia-inducible factor 1 alpha (HIF-1α) and the natural antisense transcript (HIF1A-AS2) - 3 -
1.5 Transmembrane protein 63B (Tmem63b) - 4 -
1.6 Significance - 6 -
Chapter 2 Materials and Methods - 8 -
2.1 Cell culture - 8 -
2.2 Plasmid constructs for transient gene overexpression - 9 -
2.3 Manipulation of endogenous ADAR1 and HIF1A-AS2 abundance by RNAi and RNase H-dependent degradation - 9 -
2.4 Reagents and antibodies - 10 -
2.5 RNA extraction, reverse transcription (RT)-PCR and quantitative PCR (qPCR) - 10 -
2.6 RNA immunoprecipitation (RIP) - 11 -
2.7 Poly(A)+ RNA fractionation and detection - 12 -
2.8 Nuclear and cytoplasmic fractionation - 12 -
2.9 Rapid amplification of cDNA ends (RACE) - 13 -
2.10 RNA fluorescence in situ hybridization (FISH) - 14 -
2.11 Apoptosis assay - 15 -
2.12 Detection of nascent RNA synthesis rate - 15 -
2.13 Chromatin immunoprecipitation (ChIP) - 16 -
2.14 Tube formation assay - 17 -
2.15 RNA stability assay - 17 -
2.16 Isolation of cytosolic ribosome nascent chain (RNC) - 18 -
2.17 RNA-sequencing - 18 -
2.18 Gene expression analysis - 19 -
2.19 Tmem63b knockout mice - 19 -
2.20 Open field test and locomotion - 20 -
2.21 T maze - 20 -
2.22 Morris water maze - 21 -
2.23 Fear-condition - 21 -
2.24 Statistical analysis - 22 -
Chapter 3 Results - 23 - 
3.1 ADAR1 promotes robust hypoxia signaling via distinct regulation of multiple HIF-1α-inhibiting factors - 23 -
3.1.1 To characterize the HIF1A-AS2 transcript - 23 -
3.1.2 To explore the regulation of HIF1A-AS2 on HIF-1Α - 26 -
3.1.3 To explore the role of ADAR1 on HIF1A-AS2/HIF-1Α regulation - 30 -
3.1.4 To evaluate the pathophysiologycal significance of the ADAR1- HIF1A-AS2 regulatory network - 34 -
3.2 Functional roles of Tmem63b and the RNA editing events - 37 -
3.2.1 To find the differences in animal behavior between wild type and heterozygous Tmem63bI/+ mice - 37 -
3.2.2 To dissect the molecular mechanism of Tmem63b regulates in mouse brain - 44 -
Chapter 4 Discussion - 46 -
References - 52 -
Figures - 64 -
Tables - 96 -








List of Figures
Figure 1. Characterization of the HIF1A-AS2 transcript and associated RNA editing events. - 65 -
Figure 2. Definition of HIF1A-AS2 transcript sub-cellular localization. - 67 -
Figure 3. RNA expression of HIF1A-AS2 shows inverse correlation to the HIF-1A mRNA under hypoxic conditions. - 68 -
Figure 4. Gene expression changes in cells under different doses of CoCl2 treatments. - 70 -
Figure 5. HIF1A-AS2 acts as an in-cis negative regulator of HIF-1A expression. - 71 -
Figure 6. HIF1A-AS2 has no effect on the HIF-1A mRNA stability or sub-cellular translocation. - 73 -
Figure 7. Hypoxia-induced transcription of HIF1A-AS2 triggers HIF-1A transcriptional silencing. - 74 -
Figure 8. ADAR1 p110 isoform decreases HIF1A-AS2 expression under hypoxia. - 76 -
Figure 9. Roles of ADAR1 in the regulation of HIF-1A/HIF1A-AS2 expression under hypoxia. - 78 -
Figure 10. ADAR1 has no effect on the HIF1A-AS2 mRNA stability or sub-cellular translocation. - 80 -
Figure 11. ADAR1 antagonizes HIF1A-AS2-dependent suppression of HIF-1A. - 82 -
Figure 12. Gene expression analysis of the double-knockdown experiments. - 84 -
Figure 13. Clinical relevance and a schematic model of ADAR1’s roles in maintaining HIF-1A expression and the associated signalling in hypoxia. - 85 -
Figure 14. Tmem63bI/+ mice display hyperactivity and anxiety-like behavior. - 88 -
Figure 15. Tmem63bI/+ mice show impaired spatial learning. - 90 -
Figure 16. Tmem63bI/+ mice exhibit normal in fear memory test. - 91 -
Figure 17. Behavioural characterization of Tmem63bA/A, A/G and Tmem63bG/G mice. - 92 -
Figure 18. Tmem63bG/G mice display delay spatial learning compare to Tmem63bA/A and Tmem63bA/G. - 93 -
Figure 19. Dopamine-related protein expression and distribution. - 94 -
List of Tables
Table 1. Oligonucleotide primers used in this study - 96 -
Table 2. Sequence of HIF1A-AS2(m) isoforms - 101 -
Table 3. Hypoxia up-regulated genes - 104 -
Table 4. Hypoxia down-regulated genes - 106 -
Table 5. Down-regulated genes in ADAR1-depletion cells - 108 -
Table 6. HIF1A-AS2 RNA editing degree - 110 -
Table 7. HIF1A-AS2 RNA editing degree under hypoxic condition - 111 -

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