AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N-6,N-6-Dimethyladenosine (m(6,6)A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m(6,6)A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m(6,6)A) method for the site-specific detection and quantification of m(6,6)A in RNA. The N-6,N-6-dimethyl groups in m(6,6)A can cause reverse transcription to stall at the m(6,6)A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m(6,6)A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m(6,6)A in RNA. Using the AD-m(6,6)A method, we successfully detected and quantified m(6,6)A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m(6,6)A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m(6,6)A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m(6,6)A in RNA, which can aid in uncovering the functions of m(6,6)A in human diseases.