Formation and removal of 1,N(6)-dimethyladenosine in mammalian transfer RNA

RNA molecules harbor diverse modifications that play important regulatory roles in a variety of biological processes. Over 150 modifications have been identified in RNA molecules. N(6)-methyladenosine (m(6)A) and 1-methyladenosine (m(1)A) are prevalent modifications occurring in various RNA species of mammals. Apart from the single methylation of adenosine (m(6)A and m(1)A), dual methylation modification occurring in the nucleobase of adenosine, such as N(6),N(6)-dimethyladenosine (m(6,6)A), also has been reported to be present in RNA of mammals. Whether there are other forms of dual methylation modification occurring in the nucleobase of adenosine other than m(6,6)A remains elusive. Here, we reported the existence of a novel adenosine dual methylation modification, i.e. 1,N(6)-dimethyladenosine (m(1,6)A), in tRNAs of living organisms. We confirmed that m(1,6)A is located at position 58 of tRNAs and is prevalent in mammalian cells and tissues. The measured level of m(1,6)A ranged from 0.0049% to 0.047% in tRNAs. Furthermore, we demonstrated that TRMT6/61A could catalyze the formation of m(1,6)A in tRNAs and m(1,6)A could be demethylated by ALKBH3. Collectively, the discovery of m(1,6)A expands the diversity of RNA modifications and may elicit a new tRNA modification-mediated gene regulation pathway.