Structural basis for substrate binding and catalytic mechanism of a human RNA:m(5)C methyltransferase NSun6

5-methylcytosine (m(5)C) modifications of RNA are ubiquitous in nature and play important roles in many biological processes such as protein translational regulation, RNA processing and stress response. Aberrant expressions of RNA:m(5)C methyltransferases are closely associated with various human diseases including cancers. However, no structural information for RNA-bound RNA:m(5)C methyltransferase was available until now, hindering elucidation of the catalytic mechanism behind RNA:m(5)C methylation. Here, we have solved the structures of NSun6, a human tRNA:m(5)C methyltransferase, in the apo form and in complex with a full-length tRNA substrate. These structures show a non-canonical conformation of the bound tRNA, rendering the base moiety of the target cytosine accessible to the enzyme for methylation. Further biochemical assays reveal the critical, but distinct, roles of two conserved cysteine residues for the RNA:m(5)C methylation. Collectively, for the first time, we have solved the complex structure of a RNA:m5C methyltransferase and addressed the catalytic mechanism of the RNA:m(5)C methyltransferase family, which may allow for structure-based drug design toward RNA:m(5)C methyltransferase–related diseases.