RNA Methyltransferases Utilize Two Cysteine Residues in the Formation of 5-Methylcytosine

Abstract

Proteins that have sequence homology with known RNA m⁵C methyltransferases contain two conserved cysteines, each of which lies within a sequence that bears similarity to a methyltransferase active site. Other enzymes that transfer a methyl group to carbon 5 of a pyrimidine nucleotide, such as the bacterial DNA m⁵C methyltransferases, utilize their single conserved cysteine residue to form a covalent Michael adduct with carbon 6 of the pyrimidine ring during catalysis. We present a model for the utilization of two cysteines in catalysis by RNA m⁵C methyltransferases. It is proposed that one thiol acts in a classical fashion by forming a covalent link to carbon 6 of the pyrimidine base, while the other cysteine assists breakdown of the covalent adduct. Therefore, alteration of the assisting cysteine is anticipated to stabilize the covalent enzyme−RNA intermediate. The model was conceived as a possible explanation for the effects of mutations that change the conserved cysteines in Nop2p, an apparent RNA m⁵C methyltransferase that is essential for ribosome assembly and yeast viability. Evidence for the predicted accumulation of protein−RNA complexes following mutation of the assisting cysteine has been obtained with Nop2p and a known tRNA m⁵C methyltransferase called Ncl1p (Trm4).