Trm4 and Nsun2 RNA:m5C methyltransferases form metabolite-dependent, covalent adducts with previously methylated RNA.

Trm4p from Saccharomyces cerevisiae and its mammalian orthologue Nsun2 fabricate 5-methylcytosine (m(5)C) in RNA molecules utilizing a dual-cysteine catalytic mechanism. These enzymes are now shown to form covalent complexes with previously methylated RNA. Enzyme linkage to methylated RNA requires S-adenosylhomocysteine (AdoHcy), and the removal of this metabolite results in the disassembly of preexisting complexes. The fraction of Trm4p linked to modified RNA is influenced by the AdoHcy concentration and by the pH of the solution, with maximal formation of Trm4p-RNA complexes observed in the pH range of 5.5-6.5. Four active-site residues critical for Trm4p-mediated tRNA methylation are also required for the formation of the denaturant-resistant complexes with m(5)C-containing RNA. On the basis of these findings, it is proposed that formation of a covalent complex between dual-cysteine RNA:m(5)C methyltransferases and methylated RNA provides a unique means by which metabolic factors can influence RNA. By controlling the degree of formation of the enzyme-RNA covalent complex, AdoHcy and pH are likely to influence the extent of m(5)C formation and the rate of release of methylated RNA from RNA:m(5)C methyltransferases. Metabolite-induced covalent complexes could plausibly affect the processing and function of m(5)C-containing RNAs.