Functional replacement of yeast nuclear and mitochondrial RNase P by a protein-only RNase P

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賽提威(Aditya Aryandi Setiawibawa) 查詢紙本館藏

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生命科學系

論文名稱

(Functional replacement of yeast nuclear and mitochondrial RNase P by a protein-only RNase P)

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摘要(中)

核糖核酸酶P (RNase P)屬於核酸內切酶/核糖核蛋白家族，參與轉運核糖核酸(tRNA)的成熟作用。tRNA前驅物的成熟涉及 5′ 前導序列的切割、3′ 尾端序列的切割、內含子剪接、3′ 末端添加CCA、和轉錄後修飾。 RNase P 是負責在tRNA 前驅物的 +1 位置進行特定切割來去除 5′ 前導序列的關鍵酶。RNase P通常是由一個 RNA和一到多個蛋白質共同組成。然而，最近的研究發現了一種獨特形式的 RNase P，稱為純蛋白質 RNase P (protein-only RNase P, PRORP)，它在真核生物中非常普遍。這引起了我們對不含催化性 RNA 成分的 RNase P 如何有效切割tRNA 前驅物的興趣。研究 PRORP 切割tRNA前驅物，特別是 tRNAHis 前驅物的 5′ 前導序列的機制，我們建構了酵母菌細胞核和酵母菌線粒體 RNase P 剔除菌株，用於細胞內功能測定，並純化異源 PRORP 用於體外切割測定，尤其是它們在 tRNAHis 中的切割位點（+1 或 -1 位置）。我們的研究結果顯示: Trypanosoma brucei PRORP (TbPRORP)、Aquifex aeolicus PRORP (AqPRORP) 和Chlamydomonas reinhardtii PRORP (CrPRORP) 可以互補酵母菌細胞核 RNase P剔除菌株，但不能互補酵母菌線粒體 RNase P 剔除菌株。此外，這些 PRORP 都不能切割 tRNAsHis在 -1 位置。更多深入研究持續進行中以全面了解PRORP的作用機制。

摘要(英)

RNase P belongs to a universal endonuclease/ribonucleoprotein family which is involved in precursor tRNA maturation. Maturation of a precursor tRNA involves cleavage of the 5’ leader sequence, cleavage of the 3’ trailer sequence, intron splicing, CCA addition to the 3’ end, and post-transcriptional modifications. RNase P is the key enzyme responsible for removing the 5’ leader sequence by making a specific cut at the +1 position in the precursor tRNA. Normally, RNase P consists of an RNA subunit and one to several protein subunits. However, recent studies identified a unique form of RNase P, called protein-only RNase P (PRORP), that is highly ubiquitous in eukaryotic organisms. This attracts our attention as to how an RNase P without the catalytic RNA component can efficiently cleave and process precursor tRNA. To investigate the mechanism by which PRORP cleaves the 5’ leader sequence of a precursor tRNA, in particular tRNAHis, we constructed a yeast nuclear and a yeast mitochondrial RNase P knockout (KO) strain for in vivo functional assay, and purified the heterologous PRORPs for in vitro cleavage assay, with a particular emphasis on their cleavage site in tRNAHis (+1 or -1 position). We showed herein that Trypanosoma brucei PRORP (TbPRORP), Aquifex aeolicus PRORP (AqPRORP), and Chlamydomonas reinhardtii PRORP (CrPRORP) can rescue the yeast nuclear RNase P KO strain, but none of these PRORPs can rescue the yeast mitochondrial RNase P KO strain. Moreover, none of these PRORPs can cut tRNAsHis at -1 position. A more detailed analysis is underway to characterize these PRORPs.

關鍵字(中)

★ 核糖核酸酶P
★ 轉錄後修飾
★ 蛋白質生物合成
★ 轉譯
★ 轉運核糖核酸

關鍵字(英)

★ RNase P
★ posttranscriptional modification
★ protein synthesis
★ translation
★ tRNA

論文目次

ABSTRACT………………………………………………………………………ii
ABSTRACT (in Chinese) ………………………………………………….…...iii
ACKNOWLEDGEMENTS…………………………………………………….iv
TABLE OF CONTENT……………………………………………………. ….vi
LIST OF FIGURES……………………………………………………….........vii
ABBREVIATIONS…………………………………………………………….viii
CHAPTER I INTRODUCTION………………………………………….. ……1
1.1 Protein translation, transfer RNA, and aminoacyl tRNA synthetase……….1
1.2 Transfer RNA (tRNA) maturation process…………………………………1
1.3 Ribonuclease P (RNase P) ………………………………………………....2
1.4 Yeast RNase P………………………………………………………………3
1.5 Protein-only RNase P (PRORP) …………………………………………...4
1.6 Interesting PRORPs………………………………………………………...5
1.7 RNase P processing is part of tRNAHis……………………………………..6
1.8 Nanoarchaeum equitans…………………………………………………………..7
1.9 Specific Aims………………………………………………………………8
1.10 Hypothesis…………………………………………………………………8
CHAPTER II MATERIAL AND METHODS………………………………….9
2.1 Cloning of Protein-only RNase P (PRORP)……………………………….9
2.2 Cloning of E. coli RNase P………………………………………………..10
2.3 Cloning of N. equitans Histidyl-tRNA synthetase and its tRNA………….11
2.4 Cloning of tRNA for in vivo complementation and in vitro assay….........11
2.5 Construction of yeast RNase P knock-out strains…………………………12
2.6 Complementation assay for testing the replacement of yeast nuc………..15
2.7 Complementation assay for yeast THG1 KO…………………………….15
2.8 Complementation assay for NeHisRS using yeast HisRS KO strain…….16
2.9 Protein purification of PRORP…………………………………………...16
2.10 Western blotting………………………………………………………....17
2.11 In vitro transcription of tRNA……………………………………………17
2.12 In vitro RNase P cleavage assay…………………………………………19
2.13 In vitro aminoacylation assay…………………………………………....19
CHAPTER III RESULTS……………………………………………………....20
3 PRORP alone able to complement yeast nuclear, but not……………….20
3.2 Cicada RNA-only RNase P cannot complement………………………....21
3.3 None of heterologous tRNAHis able to complement………………………21
3.4 Using THG1 knockout strain, we tend to determine…………….……….23
3.5 HisRS alone able to complement yeast HisRS……………………………23
3.6 NeHisRS might be a dual function enzyme which prefers……………….24
3.7 PRORP can process pre-tRNAHis in vitro………………………………..25
CHAPTER IV DISCUSSIONS…………………………………………….…..27
4.1 PRORP able to replace yeast nuclear, but not mitochondrial……………27
4.2 Our study suggests that yeast mitochondrial RPM2…………...………...29
4.3 Cicada RNA-only might not the only subunit required for……………...31
4.4 None of tRNAHis plus PRORP complementation are able to……………32
4.5 NeHisRS was not able to rescue the loss of HisRS KO strain, but……...33
REFERENCES……………………………………………………………........35
APPENDIX……………………………………………………………………..40

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指導教授

王健家(Chien-Chia wang)

審核日期

2021-7-20

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