C 2 -methyladenosine in tRNA promotes protein translation by facilitating the decoding of tandem m 2 A-tRNA-dependent codons.

RNA modification C 2 -methyladenosine (m 2 A) exists in both rRNA and tRNA of Escherichia coli (E. coli), installed by the methyltransferase RlmN using a radical-S-adenosylmethionine (SAM) mechanism. However, the precise function of m 2 A in tRNA and its ubiquity in plants have remained unclear. Here we discover the presence of m 2 A in chloroplast rRNA and tRNA, as well as cytosolic tRNA, in multiple plant species. We identify six m 2 A-modified chloroplast tRNAs and two m 2 A-modified cytosolic tRNAs across different plants. Furthermore, we characterize three Arabidopsis m 2 A methyltransferases-RLMNL1, RLMNL2, and RLMNL3-which methylate chloroplast rRNA, chloroplast tRNA, and cytosolic tRNA, respectively. Our findings demonstrate that m 2 A37 promotes a relaxed conformation of tRNA, enhancing translation efficiency in chloroplast and cytosol by facilitating decoding of tandem m 2 A-tRNA-dependent codons. This study provides insights into the molecular function and biological significance of m 2 A, uncovering a layer of translation regulation in plants.