Nucleic acids undergo naturally occurring chemical modifications. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified1. Despite recent progress, the mechanism for the biosynthesis of most modifications is not fully understood, owing, in part, to the difficulty associated with reconstituting enzyme activityin vitro. Whereas some modifications can be efficiently formed with purified components, others may require more intricate pathways2. A model for modification interdependence, in which one modification is a prerequisite for another, potentially explains a major hindrance in reconstituting enzymatic activityin vitro3. This model was prompted by the earlier discovery of tRNA cytosine-to-uridine editing in eukaryotes, a reaction that has not been recapitulatedin vitroand the mechanism of which remains unknown. Here we show that cytosine 32 in the anticodon loop ofTrypanosoma bruceitRNAThr is methylated to 3-methylcytosine (m3C) as a pre-requisite for C-to-U deamination. Formation of m3Cin vitrorequires the presence of both theT. bruceim3C methyltransferase TRM140 and the deaminase ADAT2/3. Once formed, m3C is deaminated to 3-methyluridine (m3U) by the same set of enzymes. ADAT2/3 is a highly mutagenic enzyme4, but we also show that when co-expressed with the methyltransferase its mutagenicity is kept in check. This helps to explain howT. bruceiescapes ‘wholesale deamination’5of its genome while harbouring both enzymes in the nucleus. This observation has implications for the control of another mutagenic deaminase, human AID, and provides a rationale for its regulation.