To determine the molecular impact of the DALRD3 mutation on m3C formation, we generated lymphoblastoid cell lines (LCLs) from the two sibling patients (referred to as D3-LCLs generated from patients 1 and 2; P1 and P2). LCLs generated from the affected patients were compared to control lymphoblasts from two ethnically matched, healthy, unrelated individuals (LCL-WT1 and WT2). Consistent with the nonsense mutation leading to a truncation of the DALRD3 protein and/or NMD, the levels of full-length DALRD3 protein were decreased in cell lysates prepared from either patient LCLs compared to WT-LCLs (Fig. 7a). Using the aforementioned PHA assay, we detected a substantial increase in PHA signal for tRNA-Arg-CCU and UCU in D3-LCLs compared to WT control cells, indicating the loss of m3C modification in these particular tRNAs (Fig. 7b). Follow up investigation using the primer extension assay confirmed the severe reduction of m3C modification in tRNA-Arg-CCU and UCU of both D3-LCLs from affected patients (Fig. 7c, d). In contrast, the levels of m3C modification detected by PHA probe hybridization or primer extension for tRNA-Ser-UGA and tRNA-Thr-AGU was similar between D3-
