As a positive control, we performed primer extension on RNA harvested from human 293T cells which results in an extension stop at position 32 of tRNA-Arg-CCU indicative of the m3C modification that was absent when no RT was added (Fig. 4c, lanes 1 and 2). No RT block at position 32 was detected for in vitro transcribed tRNA-Arg-CCU when pre-incubated with either water or negative control purification (Fig. 4c, lanes 3 and 4). In contrast, pre-incubation of tRNA-Arg-CCU with either purified METTL2A or 2B with or without DALRD3, followed by primer extension, revealed the appearance of a RT block at position 32 indicative of m3C formation (Fig. 4c, lanes 5–8). We note that METTL2A/B purified from human cells without overexpression of DALRD3 exhibited m3C modification activity, consistent with the co-purification of endogenous DALRD3 with the over-expressed METTL2A or 2B to form an active methyltransferase complex. Thus, the purified METTL2-DALRD3 complex is active for m3C formation on an in vitro transcribed tRNA substrate lacking any other modifications.
