To dissect the regions of DALRD3 that play a role in m3C formation, we tested the ability of DALRD3 variants to rescue m3C formation in the HAP1-D3KO cell line. We generated stable cell lines expressing either wildtype DALRD3 or DALRD3 variants lacking either the DALR domain or the N-terminal extension (Fig. 5f, WT, ΔDALR, and ΔNterm). The expression of each DALRD3 variant in the stable cell lines was confirmed by immunoblotting (Fig. 5g). As expected, HAP1-D3KO cells with vector alone exhibited no m3C stop at position 32 in tRNA-Arg-CCU when compared to HAP1-WT cells (Fig. 5h, compare lanes 1 and 2). Re-expression of wildtype DALRD3 in HAP1-D3KO cells was able to rescue m3C formation in tRNA-Arg-CCU as evidenced by the re-introduction of the m3C RT block and the absence of read-through products (Fig. 5h, lane 3). In contrast, expression of the DALRD3 mutant lacking the DALR domain or the N-terminal extension was unable to restore m3C formation in tRNA-Arg-CCU (Fig. 5h, lanes 4 and 5; quantified in 5i). Altogether, these results demonstrate that m3C formation at position 32 in specific arginine tRNAs requires the DALRD3 protein with both the DALR domain and N-terminal region contributing key roles in methyltransferase activity.
