The tRNAs stably associated with METTL2 and/or DALRD3 could represent substrates that have not yet been modified with m3C and/or have been modified but not dissociated. To ascertain the relative m3C modification state of copurifying tRNAs, we used an oligonucleotide probe that displays differential hybridization based upon m3C modification status (described in further detail below). Using this probe, we found that DALRD3 co-purifies with both modified and unmodified tRNA-Arg-UCU and Arg-CCU (Supplementary Fig. 3a). In contrast, we found that the majority of tRNA-Arg-UCU and Arg-CCU that copurified with METTL2 was modified with m3C (Supplementary Fig. 3b). These results are consistent with DALRD3 protein binding unmodified tRNA-Arg-UCU or CCU for subsequent methylation upon interaction with METTL2.
