Fig. 5: DALRD3 is required for efficient m3C formation in arginine tRNAs in vivo.

a Immunoblot verification for the loss of DALRD3 expression in human DALRD3-knockout (KO) cell lines compared to wild-type human HAP1 cells. Actin was used as a loading control. Asterisk denotes a non-specific band found in both wild-type and D3-KO cell lysates. b Schematic of the Positive Hybridization in the Absence of Modification (PHA) assay. c Northern blot analysis of PHA probes designed to detect m3C at position 32 and a control probe that hybridizes to a different area of the same tRNA. d Primer extension analysis of the tRNA-Arg-UCU and Arg-CCU harvested from the denoted human HAP1 cell lines. Length of primers; tRNA-Arg-UCU, 24 nt; tRNA-Arg-CCU, 23 nt. e Primer extension analysis of the tRNA-Ser-UGA and Thr-AGU harvested from the denoted human HAP1 cell lines. Length of primers; tRNA-Ser-UGA, 23 nt; tRNA-Thr-AGU, 20 nt. f Schematic of DALRD3 variants used for DALRD3 rescue experiments. g Immunoblot analysis confirming expression of DALRD3 variants in the indicated HAP1 cell lines. h Primer extension analysis of tRNA-Arg-CCU from WT or D3KO HAP1 cell lines stable integrated with the indicated DALRD3 expression constructs. (-RT) represents no reverse transcriptase was added; m3C- 3-Methylcytidine; D- dihydrouridine; t6A- threonylcarbamoyladenosine; > labelled probe. i Quantification of m3C formation in tRNA-Arg-CCU by primer extension. n = 3. Error bars represent standard deviation from the mean. Statistical analysis was performed using one-way ANOVA and significance calculated using Tukey’s multiple comparison test. ****P < 0.0001; ns, non-significant. P = 0.7430 for WT strain + vector versus D3-KO strain + WT-DALRD3. a, (c through e), g, h were repeated three times each with similar results. Source data are provided as a Source data file.