METTL6 is an S-adenosyl-L-methionine-dependent RNA methyltransferase that specifically catalyzes N3-methylcytidine (m3C) modification at position 32 in the anticodon loop of serine tRNAs, including tRNA(Ser)(UGA) and tRNA(Ser)(GCU) 123. The enzyme operates through a substrate-specific mechanism requiring interaction with seryl-tRNA synthetase (SerRS), which acts as a tRNA substrate selection factor and enhances METTL6's methylation activity 14. Structural studies reveal that METTL6 uses a flipped-base mechanism for methylation, with the cytosine base binding into a positive cavity 2. This modification is crucial for translation fidelity, particularly affecting serine codon-biased mRNAs involved in cell cycle and DNA repair pathways 5. Loss of METTL6 function leads to altered translation, defective cell proliferation, increased cisplatin sensitivity, and compromised cellular proteostasis 52. In cancer contexts, METTL6 is upregulated in hepatocellular carcinoma where it promotes cell progression, migration, and invasion through regulation of cell adhesion molecules 6. The enzyme represents part of a mutually exclusive substrate selection system among mammalian m3C methyltransferases, ensuring precise tRNA modification specificity 1.