Figure 2.

Identification of a missense variant in the DALRD3 gene that impacts m3C modification status in arginine tRNAs

(A) DALRD3 exon and intron structure with encoded protein shown below. An asterisk represents the location of the variant in the mRNA (NM_001009996.3). WT and c.1549C>T alleles are shown, with the variant shown in red. The schematic depicts the domain structure of the DALRD3 protein and alignment of the region encompassing the R517C variant.

(B) Pedigree of the family harboring the missense variant in the DALRD3 gene.

(C) Immunoblot of lysates prepared from WT or R517 fibroblast cell lines. %DALRD3 represents the amount of DALRD3 protein relative to the WT fibroblast cell line and was quantified from three independent samples. ±, standard deviation from the mean.

(D) Primer extension analysis of tRNA-Arg-UCU and Arg-CCU extracted from WT or R517 fibroblast cell lines. (−) indicates that no RNA was added to the RT reaction. m3C₃₂, 3-methylcytosine. m1G₉, 1-methylguanine; >, labeled probe. Bar graphs represent quantification of m3C formation in tRNA-Arg-UCU or CCU by primer extension. n = 3. Error bars represent standard deviation from the mean. Significance was determined using an unpaired t test with two-tailed p value. ∗∗∗∗p ≤ 0.0001.