Using Northern blot hybridization, we detected low levels of tRNA-Arg-CCU and UCU copurifying with METTL2A or B alone but not tRNA-Arg-CCG, ACG or UCG (Fig. 3c, lanes 8 and 9). In addition, we found that tRNA-Thr-AGU also copurified with METTL2A or 2B when expressed alone (Fig. 3c, lanes 8 and 9, Thr-AGU). As a comparison, we detected only background levels of tRNA-Ser-UGA, tRNA-Gln-UUG or U6 snRNA in any of the purifications (Fig. 3c, lanes 7–12). The interaction of METTL2A and 2B with tRNA-Thr-AGU, Arg-CCU and Arg-UCU but not the other tRNA-Arg isoacceptors nor tRNA-Ser-UGA is consistent with the substrate specificity of METTL2A/B32. Remarkably, the amount of tRNA-Arg-CCU or UCU was greatly increased in the METTL2A or 2B purification when co-expressed with DALRD3 (Fig. 3c, tRNA-Arg-CCU or UCU, compare lanes 8 and 9 with lanes 11 and 12). In contrast, the interaction between METTL2A or 2B with tRNA-Thr-AGU was disrupted by co-expression with DALRD3 (Fig. 3c, compare lanes 8 and 9 with lanes 11 and 12).
