To test this hypothesis, we probed the methyltransferase activity of METTL2-DALRD3 complexes purified from human cells on in vitro transcribed tRNA-Arg-CCU substrates. For monitoring m3C formation, we used a primer extension assay in which the presence of m3C leads to a reverse transcriptase (RT) block at position 32 while the lack of m3C allows for read-through and generation of an extended product. As a control to ensure that methylation was occurring at the correct position, we tested a tRNA-Arg-CCU substrate in which C32 was mutated to A (C32A). To probe the requirement for positions 36 and 37 in METTL2-DARLD3 methylation, we generated the tRNA-Arg-CCU substrates U36G and A37G (Fig. 4b).
