Demethylase-assisted site-specific detection of N1-methyladenosine in RNA

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外文摘要：The dynamic RNA modifications have been viewed as new posttranscriptional regulator in modulating gene expression as well as in a broad range of physiological processes.N1-methyladenosine(m1A)is one of the most prevalent modifications existing in multiple types of RNAs.In-depth investigation of the func-tions of m1A requires the site-specific assessment of m1A stoichiometry in RNA.Herein,we established a demethylase-assisted method(DA-m1A)for the site-specific detection and quantification of m1A in RNA.N1-methyl group in m1A could result in the stalling of reverse transcription at m1A site,thus produc-ing the truncated cDNA.E.coli AlkB is a demethylase that can demethylate m1A to produce adenine in RNA,thus generating full-length cDNA from AlkB-treated RNA.Evaluation of the produced amounts of full-length cDNA by quantitative real-time PCR can achieve the site-specific detection and quantification of m1A in RNA.With the DA-m1A method,we examined and successfully confirmed the previously well-characterized m1A sites in various types of RNAs with low false positive rate.In addition,we found that the level of m1A was significantly decreased at the bromodomain containing 2(BRD2)mRNA position 1674 and CST telomere replication complex component 1(CTC1)mRNA position 5643 in human hepa-tocellular carcinoma tissues.The results suggest that these two m1A sites in mRNA may be involved in liver tumorigenesis.Taken together,the DA-m1A method is simple and enables the rapid,cost-effective,and site-specific detection and quantification of m1A in RNA,which provides a valuable tool to decipher the functions of m1A in human diseases.

外文关键词：

N1-methyladenosineRNA modificationAlkB demethylaseRNA epigeneticsQuantitative real-time PCRHepatocellular carcinoma

作者：

Jun Xiong、Ke-Ke Chen、Neng-Bin Xie、Wei Chen、Wen-Xuan Shao、Tong-Tong Ji、Si-Yu Yu、Yu-Qi Feng、Bi-Feng Yuan

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作者单位：

College of Chemistry and Molecular Sciences,Research Center of Public Health,Renmin Hospital of Wuhan University,School of Public Health,Wuhan University,Wuhan 430071,China

Department of Laboratory Medicine,Zhongnan Hospital of Wuhan University,Wuhan University,Wuhan 430071,China

基金：

国家重点研发计划国家重点研发计划国家自然科学基金国家自然科学基金国家自然科学基金国家自然科学基金Interdisciplinary Innovative Talents Foundation from Renmin Hospital of Wuhan UniversityTranslational Medicine and Interdisciplinary Research Joint Fund of Zhongnan Hospital of Wuhan University

项目编号：

2022YFC34007002022YFA080660022277093220741102172100522207090JCRCGW-2022-008ZNJC202208

出版年：

2024

DOI：

10.1016/j.cclet.2023.108953

中国化学快报(英文版)

中国化学会

中国化学快报(英文版)

CSTPCD

影响因子：0.771

ISSN：1001-8417

年,卷(期)：2024.35(5)

参考文献量50