To determine the tRNA-binding specificity of DALRD3, we probed the DALRD3 purifications for distinct tRNAs via Northern blotting. Notably, we found that DALRD3 purifications were greatly enriched for arginine tRNAs known to contain m3C generated by METTL2A/B (Fig. 2c, lanes 10–12, tRNA-Arg-CCU and UCU). In contrast, arginine tRNA isoacceptors lacking m3C exhibited considerably less co-purification with DALRD3 (Fig. 2c, lanes 10–12, tRNA-Arg-UCG, CCG, and ACG). Consistent with the predicted specificity of the DALR domain for arginine tRNAs, only background levels of tRNA-Thr-AGU or Ser-UGA was present in any of the DALRD3 purifications whether co-expressed with or without METTL2A/B (Fig. 2c, Thr-AGU and Ser-UGA, lanes 10–12). Collectively, these results uncover a distinct tRNA-binding specificity for DALRD3 that contrasts with arginyl-tRNA synthetases, which recognizes all arginine tRNA isoacceptors regardless of the anticodon.
