As additional validation, we monitored m3C formation in specific cellular tRNAs using the primer extension assay. As expected, an RT stop indicative of m3C at position 32 of tRNA-Arg-CCU and Arg-UCU was detected in wildtype human 293T and HAP1 cells (Fig. 5d). In contrast, the m3C block in both tRNA-Arg-CCU and Arg-UCU was greatly reduced in the D3-KO cell line with subsequent readthrough to the next RT-blocking modification (Fig. 5d, HAP1-D3KO lanes). Consistent with a role for DALRD3 only in arginine tRNA modification, the m3C modification in tRNA-Ser-UGA and Thr-AGU was unaffected in the D3-KO cell line (Fig. 5e). These results demonstrate that DALRD3 is required for efficient m3C formation in specific arginine tRNAs.
