Demethylase-assisted site-specific detection of N^(1)-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.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.