Abstract
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.
基金项目
国家重点研发计划(2022YFC3400700)
国家重点研发计划(2022YFA0806600)
国家自然科学基金(22277093)
国家自然科学基金(22074110)
国家自然科学基金(21721005)
国家自然科学基金(22207090)
Interdisciplinary Innovative Talents Foundation from Renmin Hospital of Wuhan University(JCRCGW-2022-008)
Translational Medicine and Interdisciplinary Research Joint Fund of Zhongnan Hospital of Wuhan University(ZNJC202208)
万方数据