Using the tRNA-Arg-CCU variants, we detected no RT block when C32 was mutated to A, providing evidence that position 32 was the site of methylation (Fig. 4d, lanes 1–4). Notably, mutation of either U36 or A37 to a G residue in tRNA-Arg-CCU led to a major decrease in m3C formation (Fig. 4d, lanes 5–10, quantified in 4e). These studies provide evidence that DALRD3 serves as a discrimination factor to recognize distinct arginine tRNAs based upon sequence elements common between tRNA-Arg-CCU and tRNA-Arg-UCU.
