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

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 (m1 A) is one of the most prevalent modifications existing in multiple types of RNAs. In-depth investigation of the functions of m1 A requires the site-specific assessment of m1 A stoichiometry in RNA. Herein, we established a demethylase-assisted method (DA-m1 A) for the site-specific detection and quantification of m1 A in RNA. N 1-methyl group in m1 A could result in the stalling of reverse transcription at m1 A site, thus producing the truncated cDNA. E. coli AlkB is a demethylase that can demethylate m1 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 m1 A in RNA. With the DA-m1 A method, we examined and successfully confirmed the previously wellcharacterized m1 A sites in various types of RNAs with low false positive rate. In addition, we found that the level of m1 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 m1 A sites in mRNA may be involved in liver tumorigenesis. Taken together, the DA-m1 A method is simple and enables the rapid, cost-effective, and site-specific detection and quantification of m1 A in RNA, which provides a valuable tool to decipher the functions of m1 A in human diseases. (c) 2024 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.