Mammalian genomes express five different tRNA isoacceptors that decode arginine codons but only tRNA-Arg-CCU and Arg-UCU are modified to contain m3C at position 32 (Fig. 4a)17,20,41. Intriguingly, tRNA-Arg-UCG and Arg-ACG also contain C32 but are not modified by METTL2A/B in either human or mouse cells. Inspection of the isoacceptor stem loops reveals that tRNA-Arg-CCU and Arg-UCU contain U-A at positions 36 and 37 while tRNA-Arg-CCG, UCG, and ACG contain G-G at positions 36 and 37 (Fig. 4a). We also note that tRNA-Thr-AGU, which is a substrate of METTL2A/B, also contains a U-A dinucleotide at positions 36 and 37 (Fig. 4a, Thr-AGU). Moreover, A37 is modified to N6-threonylcarbamoyladenosine (t6A) in tRNA-Arg-CCU and Arg-UCU along with tRNA-Thr-AGU. Previous studies in S. cerevisiae and S. pombe have shown that the identity of residues in the anticodon loop along with the i6A/t6A modification at position 37 play key roles in the recognition and modification of seryl- and threonyl-tRNAs in m3C formation by the yeast Trm140 enzyme17,25. Combined with the tRNA interaction studies described above, these observations suggest the possibility that DALRD3 recognizes specific arginine tRNAs, in part, through sequence elements in the anticodon loop that include positions 36 and 37 to facilitate METTL2A-dependent methylation.
