The three human 3-methyl-cytosine (m³C) tRNA methyltransferases have been implicated in various cancers and diseases. Each methyltransferase modifies a distinct subset of tRNAs, yet the molecular mechanisms underlying their substrate selection remain poorly understood, posing a challenge for the targeted design of therapeutic strategies. Prior to this, we presented a high-resolution cryo-EM structure of METTL6 and revealed how it achieves substrate-specificity for serine tRNAs. We are now proceeding to analyze the substrate recognition mechanisms in the related m3C tRNA methyltransferases METTL2 and METTL8.