臺灣博碩士論文加值系統

真核細胞中的轉錄體修飾作用賦予遺傳多樣性，並做為基因調控機制；已被完善解析的作用範例包含剪切作用與A-to-I RNA編輯。mRNA修飾作用是動態性，並且與轉錄反應同時進行；因此其受時間與空間限制的調控。許多證據顯示剪接作用和RNA編輯之間的交互作用，包含對前軀體RNA的競爭性結合、對RNA結合蛋白進行差異性調節，而這些交互作用控制轉錄本的命運。我們的實驗結果顯示：RNA編輯與剪接機制會共同調控HNRPLL變異體表達。我們證實一個HNRPLL轉錄變異體的表現，其具備一個額外的外顯子（E12A）；並且此變異體為ADAR1與ADAR2的受質。ADAR依賴其脫氨酶活性調整E12A轉錄本的表達，且ADAR介導調控機制影響轉錄本的剪切作用，而不影響轉錄本的穩定性與細胞核質分佈。ADAR對於外顯子12A的修飾作用促進選擇性剪接頻率，並且會產生受剪接因子SRSF1辨識的增強子。抑制細胞的E12A表達會降低細胞克隆能力，並且增強其對於艾黴素的敏感性；顯現E12A對細胞的重要性。綜合上述，我們證明RNA編輯與剪接作用的相互作用能作為調控基因表現的機制，而這些結果拓展對轉錄動力學的理解。

Processing of the eukaryotic transcriptome is a regulatory mechanism that confers genetic diversity, and splicing and A-to-I RNA editing are well-characterized examples of such processing. mRNA processing is co-transcriptional and dynamic, and hence permits reciprocal regulation within time and space constraints. Growing evidence reveals the crosstalk between the splicing and RNA editing that controls a transcript’s fate, involving competitive binding to precursor mRNA and distinct regulation of RNA-binding proteins. Here, we demonstrate that RNA editing, in conjunction with splicing machinery, regulates the expression of HNRPLL variants. We discovered an HNRPLL transcript variant containing an additional exon 12A (E12A), which is a substrate of ADAR1 and ADAR2. ADARs direct deaminase-dependent expression of the E12A transcript, and ADAR-mediated regulation of E12A is wholly splicing-based, and does not affect the stability or nucleocytoplasmic distribution of the transcript. Further, ADAR-mediated modification of exon 12A promotes the frequency of alternative splicing and generates the recognized enhancer for the splicing factor SRSF1. Silencing E12A expression leads to impaired clonogenic ability and enhanced sensitivity to doxorubicin, thus highlighting the significance of E12A. In summary, we present the interplay of RNA editing and splicing as a regulatory mechanism of gene expression. These findings extend our understanding of transcriptional dynamics.

目錄
指導教授推薦書
口試委員審定書
誌謝 iii
Abstract iv
摘要 v
目錄 vi
List of figures viii
List of tables ix
Chapter 1 Background and Significance 1
1.1 ADAR modifies transcriptome by converting adenosines to inosines 1
1.2 A-to-I RNA editing regulates cancer development 2
1.3 Aberrant splicing patterns in cancers 3
1.4 Reciprocal regulation of RNA editing and splicing 4
1.5 HNRPLL functions as a splicing factor 5
1.6 RNA editing may regulate HNRPLL in cancers 6
Chapter 2 Material and methods 7
2.1 Cell culture 7
2.2 Gene knockdown and overexpression plasmids construction 7
2.3 RNA extraction, reverse transcription (RT)-PCR, and quantitative PCR (qPCR) 8
2.4 Chemicals and antibodies 8
2.5 RNA-immunoprecipitation (RNA-IP) experiment 9
2.6 Nuclear and cytoplasmic fractionation 9
2.7 Splicing luciferase reporter assay 10
2.8 Isolation of ribosome nascent chain-mRNA (RNC-mRNA) complex 10
2.9 Detection of nascent RNA synthesis rate 11
Chapter 3 Experimental Results 13
3.1 HNRPLL-E12A transcript is an ADAR substrate 13
3.2 The editing status of the E12A transcript is associated with its expression 14
3.3 ADARs do not affect stability or distribution of the E12A transcript 16
3.4 RNA editing regulates the splicing pattern of the E12A transcript 17
3.5 The E12A polypeptide may be undetected because it is targeted for proteolysis 18
3.6 Exon 12A inclusion is regulated by RNA editing and SRSF1 20
3.7 E12A expression is associated with clonogenicity and survival 21
Chapter 4 Discussion 24
4.1 Regulatory role of RNA editing in HNRPLL splicing 24
4.2 Oncogenic SRSF1 regulates HNRPLL alternative splicing 24
4.3 The functional form of E12A is a noncoding RNA 25
4.4 HNRPLL is not involved in E12A-mediated regulation 26
4.5 Dominant selection of editing and splicing 27
References 29
Figures 34
Tables 54


List of figures
Figure 1. The HNRPLL-E12A transcript variant is expressed in cells. 34
Figure 2. The HNRPLL-E12A transcript variant an ADAR1 substrate. 35
Figure 3. The HNRPLL-E12A transcript variant is edited by ADAR2. 36
Figure 4. ADAR1 p110 isoform is responsible for HNRPLL editing. 37
Figure 5. Editing degree of E12A transcript is associated with its expression. 38
Figure 6. ADAR2 overexpression enhances E12A RNA level. 39
Figure 7. ADAR2 regulates expression and editing of E12A transcript in a dose-dependent manner. 40
Figure 9. The sequences of E12A transcript are highly identical to reference transcript. 42
Figure 10. RNA editing regulates HNRPLL alternative splicing. 43
Figure 11. Endogenous E12A-encoded protein is not detected. 45
Figure 12. The E12A polypeptide may undergo proteolysis. 46
Figure 13. The editing degree in precursor mRNA and mature RNA of HNRPLL. 47
Figure 14. SRSF1 promotes HNRPLL alternative splicing. 48
Figure 15. Identification of E12A target genes by RNA sequencing analysis. 49
Figure 16. E12A mediate the transcriptional regulation of target genes. 50
Figure 17. E12A expression is associated with cell growth and death. 51
Figure 18. E12A editing is observed in most cell lines. 53



List of tables
Table 1. Primers used in this study 54
Table 2. MIQE checklist for quantitative real-time PCR 56

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