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

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Title:	Functional replacement of yeast nuclear and mitochondrial RNase P by a protein-only RNase P
Authors:	賽提威;Setiawibawa, Aditya Aryandi
Contributors:	生命科學系
Keywords:	核糖核酸?P;轉錄後修飾;蛋白質生物合成;轉譯;轉運核糖核酸;RNase P;posttranscriptional modification;protein synthesis;translation;tRNA
Date:	2021-07-20
Issue Date:	2021-12-07 11:22:44 (UTC+8)
Publisher:	國立中央大學
Abstract:	核糖核酸?P (RNase P)屬於核酸內切?/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?。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。;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.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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