Demethylase-assisted site-specific detection of N^(1)-methyladenosine in RNA  

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 

机构地区：[1]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 [2]Department of Laboratory Medicine,Zhongnan Hospital of Wuhan University,Wuhan University,Wuhan 430071,China

出　　处：《Chinese Chemical Letters》2024年第5期397-401,共5页中国化学快报（英文版）

基　　金：supported by the National Key R&D Program of China(Nos.2022YFC3400700 and 2022YFA0806600);the National Natural Science Foundation of China(Nos.22277093,22074110,21721005 and 22207090);the Interdisciplinary Innovative Talents Foundation from Renmin Hospital of Wuhan University(No.JCRCGW-2022-008);the Translational Medicine and Interdisciplinary Research Joint Fund of Zhongnan Hospital of Wuhan University(No.ZNJC202208)。

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.N^(1)-methyladenosine(m^(1)A)is one of the most prevalent modifications existing in multiple types of RNAs.In-depth investigation of the functions of m^(1)A requires the site-specific assessment of m^(1)A stoichiometry in RNA.Herein,we established a demethylase-assisted method(DA-m^(1)A)for the site-specific detection and quantification of m^(1)A in RNA.N^(1)-methyl group in m^(1)A could result in the stalling of reverse transcription at m^(1)A site,thus producing the truncated cDNA.E.coli AlkB is a demethylase that can demethylate m^(1)A 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 m^(1)A in RNA.With the DA-m^(1)A method,we examined and successfully confirmed the previously well-characterized m^(1)A sites in various types of RNAs with low false positive rate.In addition,we found that the level of m^(1)A 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 hepatocellular carcinoma tissues.The results suggest that these two m^(1)A sites in mRNA may be involved in liver tumorigenesis.Taken together,the DA-m^(1)A method is simple and enables the rapid,cost-effective,and site-specific detection and quantification of m^(1)A in RNA,which provides a valuable tool to decipher the functions of m^(1)A in human diseases.

关 键 词：N^(1)-methyladenosine RNA modification AlkB demethylase RNA epigenetics Quantitative real-time PCR Hepatocellular carcinoma 

分 类 号：Q503[生物学—生物化学]