The above results suggest that DALRD3 functions in the recognition and interaction of specific arginine tRNAs with METTL2A/B. To investigate the requirement for DALRD3 in m3C formation in vivo, we engineered human cell lines lacking DALRD3 using CRISPR/Cas9 gene editing. Using the HAP1 human haploid cell line42, we generated a cell clone containing a 14 base-pair deletion in exon 1 of the DALRD3 gene (Supplementary Fig. 4a). The deletion is predicted to produce a truncated DALRD3 missing the majority of the polypeptide or a translation frameshift that results in nonsense mediated decay (NMD). Indeed, immunoblotting revealed the absence of full-length DALRD3 protein in the DALRD3-knockout (D3-KO) cell line compared to the isogenic control wildtype (WT) cell line (Fig. 5a). While expression of DALRD3 was abolished in the D3-KO cell line, no change in METTL2A levels was detected between the WT or D3-KO cell lines (Supplementary Fig. 4b).
