{"title":"通过内切酶介导的 qPCR 对腺苷酸转肌苷酸 RNA 编辑的位点特异性定量","authors":"Wan-Bing Tao , Jun Xiong , Bi-Feng Yuan","doi":"10.1016/j.bmc.2024.117837","DOIUrl":null,"url":null,"abstract":"<div><p>RNA molecules contain diverse modified nucleobases that play pivotal roles in numerous biological processes. Adenosine-to-inosine (A-to-I) RNA editing, one of the most prevalent RNA modifications in mammalian cells, is linked to a multitude of human diseases. To unveil the functions of A-to-I RNA editing, accurate quantification of inosine at specific sites is essential. In this study, we developed an endonuclease-mediated cleavage and real-time fluorescence quantitative PCR method for A-to-I RNA editing (EM-qPCR) to quantitatively analyze A-to-I RNA editing at a single site. By employing this method, we successfully quantified the levels of A-to-I RNA editing on various transfer RNA (tRNA) molecules at position 34 (I<sub>34</sub>) in mammalian cells with precision. Subsequently, this method was applied to tissues from sleep-deprived mice, revealing a notable alteration in the levels of I<sub>34</sub> between sleep-deprived and control mice. The proposed method sets a precedent for the quantitative analysis of A-to-I RNA editing at specific sites, facilitating a deeper understanding of the biological implications of A-to-I RNA editing.</p></div>","PeriodicalId":255,"journal":{"name":"Bioorganic & Medicinal Chemistry","volume":"110 ","pages":"Article 117837"},"PeriodicalIF":3.3000,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Site-specific quantification of Adenosine-to-Inosine RNA editing by Endonuclease-Mediated qPCR\",\"authors\":\"Wan-Bing Tao , Jun Xiong , Bi-Feng Yuan\",\"doi\":\"10.1016/j.bmc.2024.117837\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>RNA molecules contain diverse modified nucleobases that play pivotal roles in numerous biological processes. Adenosine-to-inosine (A-to-I) RNA editing, one of the most prevalent RNA modifications in mammalian cells, is linked to a multitude of human diseases. To unveil the functions of A-to-I RNA editing, accurate quantification of inosine at specific sites is essential. In this study, we developed an endonuclease-mediated cleavage and real-time fluorescence quantitative PCR method for A-to-I RNA editing (EM-qPCR) to quantitatively analyze A-to-I RNA editing at a single site. By employing this method, we successfully quantified the levels of A-to-I RNA editing on various transfer RNA (tRNA) molecules at position 34 (I<sub>34</sub>) in mammalian cells with precision. Subsequently, this method was applied to tissues from sleep-deprived mice, revealing a notable alteration in the levels of I<sub>34</sub> between sleep-deprived and control mice. The proposed method sets a precedent for the quantitative analysis of A-to-I RNA editing at specific sites, facilitating a deeper understanding of the biological implications of A-to-I RNA editing.</p></div>\",\"PeriodicalId\":255,\"journal\":{\"name\":\"Bioorganic & Medicinal Chemistry\",\"volume\":\"110 \",\"pages\":\"Article 117837\"},\"PeriodicalIF\":3.3000,\"publicationDate\":\"2024-07-11\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bioorganic & Medicinal Chemistry\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0968089624002517\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bioorganic & Medicinal Chemistry","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0968089624002517","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
RNA 分子中含有多种修饰的核碱基,它们在许多生物过程中发挥着关键作用。腺苷-肌苷(A-to-I)RNA 编辑是哺乳动物细胞中最常见的 RNA 修饰之一,与多种人类疾病有关。要揭示 A 到 I RNA 编辑的功能,必须对特定位点的肌苷进行精确定量。在这项研究中,我们开发了一种内切酶介导的A-I RNA编辑裂解和实时荧光定量PCR方法(EM-qPCR)来定量分析单个位点的A-I RNA编辑。利用这种方法,我们成功地精确定量了哺乳动物细胞中各种转移 RNA(tRNA)分子第 34 位(I34)上的 A 对 I RNA 编辑水平。随后,我们将这种方法应用于睡眠不足小鼠的组织,结果发现睡眠不足小鼠和对照组小鼠的 I34 水平发生了显著变化。该方法开创了在特定位点定量分析A-to-I RNA编辑的先例,有助于深入了解A-to-I RNA编辑的生物学意义。
Site-specific quantification of Adenosine-to-Inosine RNA editing by Endonuclease-Mediated qPCR
RNA molecules contain diverse modified nucleobases that play pivotal roles in numerous biological processes. Adenosine-to-inosine (A-to-I) RNA editing, one of the most prevalent RNA modifications in mammalian cells, is linked to a multitude of human diseases. To unveil the functions of A-to-I RNA editing, accurate quantification of inosine at specific sites is essential. In this study, we developed an endonuclease-mediated cleavage and real-time fluorescence quantitative PCR method for A-to-I RNA editing (EM-qPCR) to quantitatively analyze A-to-I RNA editing at a single site. By employing this method, we successfully quantified the levels of A-to-I RNA editing on various transfer RNA (tRNA) molecules at position 34 (I34) in mammalian cells with precision. Subsequently, this method was applied to tissues from sleep-deprived mice, revealing a notable alteration in the levels of I34 between sleep-deprived and control mice. The proposed method sets a precedent for the quantitative analysis of A-to-I RNA editing at specific sites, facilitating a deeper understanding of the biological implications of A-to-I RNA editing.
期刊介绍:
Bioorganic & Medicinal Chemistry provides an international forum for the publication of full original research papers and critical reviews on molecular interactions in key biological targets such as receptors, channels, enzymes, nucleotides, lipids and saccharides.
The aim of the journal is to promote a better understanding at the molecular level of life processes, and living organisms, as well as the interaction of these with chemical agents. A special feature will be that colour illustrations will be reproduced at no charge to the author, provided that the Editor agrees that colour is essential to the information content of the illustration in question.