New substrates and determinants for tRNA recognition of RNA methyltransferase DNMT2/TRDMT1

Methylation is a common post-transcriptional modification of tRNAs, particularly in the anticodon loop region. The cytosine 38 (C38) in tRNAs, such as tRNA^Asp-GUC, tRNA^Gly-GCC, tRNA^Val-AAC, and tRNA^Glu-CUC, can be methylated by human DNMT2/TRDMT1 and some homologs found in bacteria, plants, and animals. However, the substrate properties and recognition mechanism of DNMT2/TRDMT1 remain to be explored. Here, taking into consideration common features of the four known substrate tRNAs, we investigated methylation activities of DNMT2/TRDMT1 on the tRNA^Gly-GCC truncation and point mutants, and conformational changes of mutants. The results demonstrated that human DNMT2/TRDMT1 preferred substrate tRNA^Gly-GCC in vitro. L-shaped conformation of classical tRNA could be favourable for DNMT2/TRDMT1 activity. The complete sequence and structure of tRNA were dispensable for DNMT2/TRDMT1 activity, whereas T-arm was indispensable to this activity. G19, U20, and A21 in D-loop were identified as the important bases for DNMT2/TRDMT1 activity, while G53, C56, A58, and C61 in T-loop were found as the critical bases. The conserved CUXXCAC sequence in the anticodon loop was confirmed to be the most critical determinant, and it could stabilize C38-flipping to promote C38 methylation. Based on these tRNA properties, new substrates, tRNA^Val-CAC and tRNA^Gln-CUG, were discovered in vitro. Moreover, a single nucleotide substitute, U32C, could convert non-substrate tRNA^Ala-AGC into a substrate for DNMT2/TRDMT1. Altogether, our findings imply that DNMT2/TRDMT1 relies on a delicate network involving both the primary sequence and tertiary structure of tRNA for substrate recognition.