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研究生：	董沛綺 Tung, Pei-Chi
論文名稱：	NSUN2甲基化轉運核糖核酸之分子結構研究 Structural study of NSUN2 in tRNA methylation
指導教授：	吳權娟 Wu, Chyuan-Chuan
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2025
畢業學年度：	113
語文別：	英文
論文頁數：	89
中文關鍵詞：	轉錄後表觀修飾、RNA m5C 甲基化、RNA 甲基轉移酶、NSUN2 、體外轉錄、X 光晶體學、冷凍電子顯微鏡（Cryo-EM）
外文關鍵詞：	epitranscriptomics, RNA m5C methylation, RNA methyltransferases, NSUN2, X-ray crystallography, cryo-EM
相關次數：	點閱：20 下載：0
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RNA 修飾在調控 RNA 功能中扮演關鍵角色，作為轉錄體與蛋白質體之間的緩衝機制，使細胞得以迅速應對環境變化。在人類中，NOL1/NOP2/Sun（NSUN）結構域家族與 DNA 甲基轉移酶 2（DNMT2）為催化 RNA 第五位碳甲基胞嘧啶（5-methylcytosine, m5C）修飾的酵素，分別作為 RNA m5C 的「書寫者」（writers），各自具有特定的受質選擇性。其中，NSUN2 可修飾多種類型的 RNA，包括 tRNA、rRNA、mRNA 與長鏈非編碼 RNA。NSUN2 活性上調會增加特定 mRNA 上的 m5C 修飾，進而促進癌症進程；相對地，NSUN2 功能缺失及 tRNA m5C 修飾下降則與神經功能異常相關，顯示其在維持 tRNA 穩定性中的重要角色。本研究旨在闡明 NSUN2 識別 RNA 受質的結構基礎。我們在體外證實 NSUN2 可專一性地修飾其已知 mRNA 與 tRNA 受質上的目標胞嘧啶，並發現其主要透過 RNA 的三級結構而非線性序列來辨識受質。為獲得 NSUN2 與受質複合體的結構資訊，我們純化了重組人類 NSUN2 蛋白，並製備體外轉錄的 RNA 作為受質進行複合體重組，後續利用 X 光晶體繞射與單顆粒冷凍電子顯微鏡（Cryo-EM）進行結構解析。此外，為探討 NSUN2 結合蛋白是否參與其受質特異性的調控，我們進一步以免疫沉澱結合質譜技術鑑定其互作蛋白群（interactome）。透過本研究，我們期望深入理解 NSUN2 的受質辨識機制，並為其於轉錄後表觀修飾中的功能奠定結構與理論基礎。

RNA modifications play a crucial role in modulating RNA function, acting as a buffering system between the transcriptome and proteome to enable cells to rapidly adapt to environmental changes. In humans, members of the NOL1/NOP2/Sun (NSUN) domain-containing family and DNA methyltransferase 2 (DNMT2) catalyze 5-methylcytosine (m5C) modifications on RNA, functioning as "writers" with distinct substrate specificities. Among them, NSUN2 is capable of methylating a wide range of RNA substrates, including tRNAs, rRNAs, mRNAs, and long non-coding RNAs. Aberrant upregulation of NSUN2 enhances m5C deposition on specific mRNAs, which has been implicated in cancer progression. Conversely, loss of NSUN2 function and the resultant reduction in tRNA m5C levels are associated with neurological abnormalities, underscoring NSUN2’s essential role in maintaining tRNA homeostasis. In this study, we aim to elucidate the structural basis of RNA substrate recognition by NSUN2. We have demonstrated that NSUN2 specifically methylates target cytosines within known mRNA and tRNA substrates in vitro and found that it primarily recognizes its RNA targets through tertiary structure rather than linear sequence context. To gain structural insights into the NSUN2-substrate complex, we purified recombinant human NSUN2 protein and prepared in vitro transcribed RNA substrates for complex assembly. Both X-ray crystallography and single-particle cryo-electron microscopy (cryo-EM) were employed for structural determination. Additionally, to investigate the contribution of NSUN2-associated proteins to substrate specificity, we performed immunoprecipitation coupled with mass spectrometry to characterize the NSUN2 interactome. Through this work, we hope to advance our understanding of NSUN2’s substrate recognition mechanism and provide a structural framework for its role in epitranscriptomic regulation.

Abstract	I
中文摘要	II
致謝	III
Table of Contents	IV
List of Tables	VII
List of Figures	VIII
Abbreviation	IX
1	Introduction	1
1.1	The Role of RNA Modifications in Epitranscriptomics	1
1.2	The Role of m5C Modification in tRNA Biology	3
1.3	RNA m5C Writers: the NSUN Family of RNA m5C Methyltransferases	5
1.4	The NSUN2-mediated tRNA m5C modification	8
1.5	Structural Basis of NSUN2-Mediated RNA Recognition	10
2	Specific Aims	14
2.1	Purify NSUN2-ΔN recombinant protein	14
2.2	Characterize NSUN2’s substrate specificity toward tRNAs	14
2.3	Determine the structure of NSUN2-tRNA complex	15
2.4	Investigate NSUN2’s interactome	15
3	Materials and Methods	16
3.1	Materials	16
3.1.1	Plasmids and Constructs	16
3.1.2	Host cell lines	16
3.1.3	Antibodies	17
3.1.4	Synthetic oligonucleotides	17
3.1.5	List of RNA used in this study.	18
3.1.6	Buffers and Solutions	18
3.2	Methods	19
3.2.1	Protein Expression and Purification of NSUN2-ΔN	19
3.2.2	Preparation of tRNAGly Construct	20
3.2.3	Preparation of C48-50U and C48-50A tRNAGly Variants	21
3.2.4	Purification of human tRNALeu and Its C34A, C48A, and C34/48A Variants	23
3.2.5	Size-exclusion chromatographic analysis of NSUN2-ΔN-tRNAGly complex	24
3.2.6	MTase-Glo methyltransferase assay	25
3.2.7	Crystallization of NSUN2-tRNA Complex	26
3.2.8	Negative-stain electron microscopy (EM) analysis	27
3.2.9	Cryo-electron microscopy (cryo-EM) analysis	28
3.2.10Cell Culture	29
3.2.11Transient Expression of GFP-fused NSUN2 in Mammalian Cells	29
3.2.12Immunoprecipitation (IP)	30
4	Results	32
4.1	Structural Prediction and Domain Organization of HsNSUN2-ΔN	32
4.2	Expression and Purification of Recombinant HsNSUN2-ΔN	33
4.3	Preparation of tRNAGly	34
4.4	Preparation of C48-50U tRNAGly variant	35
4.5	Preparation of C48-50A tRNAGly variant	36
4.6	Preparation tRNALeu(CAA)4-1 and its variants	36
4.7	Complex formation between NSUN2-ΔN and tRNAGly revealed by size-exclusion chromatography	37
4.8	Substrate specificity of NSUN2-ΔN toward tRNAGly and tRNALeu analyzed by MTase-Glo assay	38
4.9	Protein crystallization screening results	40
4.10	Negative-stain EM reveals the morphology of the NSUN2-ΔN-tRNAGly complex	41
4.11	Identification of NSUN2-interacting proteins by IP-MS	42
5	Discussions	46
5.1	Functional Characterization of NSUN2-ΔN	46
5.2	Substrate Specificity and Methylation Site Preference	47
5.3	Structural investigation of the NSUN2-tRNA complex	49
5.4	NSUN2 protein-protein interactions	50
6	Conclusions	51
Tables	52
Table 1. The NSUN RNA:m5C MTase family.	52
Table 2. Protein precipitation ratio in crystallization screen.	53
Table 3. Protein interactors identified exclusively in NSUN2-FL immunoprecipitation.	54
Table 4. Protein interactors identified exclusively in NSUN2-FL-CA immunoprecipitation.	56
Table 5. Protein interactors identified in both NSUN2-FL and FL-CA immunoprecipitation.	57
Figures	58
Figure 1. Structure-based domain annotation of HsNSUN2-ΔN with the AlphaFold-predicted model.	58
Figure 2. The expression and purification of NSUN2-ΔN.	59
Figure 3. The design and production of WT tRNAGly(GCC)1-1.	61
Figure 4. The design and production of C48-50U tRNAGly(GCC)1-1.	62
Figure 5. The design and production of C48-50A tRNAGly(GCC)1-1.	63
Figure 6. The design and production of tRNALeu WT and mutants.	65
Figure 7. NSUN2-ΔN forms a complex with tRNAGly as analyzed by size-exclusion chromatography.	66
Figure 8. MTase-Glo assay of NSUN2-ΔN with various tRNA substrates.	68
Figure 9. Negative-stain EM visualization of the NSUN2-ΔN-tRNAGly complex.	69
Figure 10. Identification of NSUN2-interacting proteins by Immunoprecipitation-Mass Spectrometry (IP-MS).	71
References	72
Appendix Figures	77
Appendix Figure 1. Optimization of in vitro transcription and refolding conditions for tRNAGly synthesis.	77
Appendix Figure 2. Purification of in vitro transcribed tRNAGly using different clean-up methods.	78
                                    

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