Causal Role for p.Arg291His and p.Met386Val TRMT5 Variants in mt-tRNA Modification and Oxidative Metabolism Deficiency
(A) RT-PEx assay performed on RNA derived from individual 73901 and control (C2) skin fibroblasts (FB) with and without the expression of wild-type TRMT5. Quantification values are displayed as per Figure 4B. Error bars = 1 SEM; n = 4, ∗∗p < 0.01, unpaired two-tailed Student’s t test.
(B) Complementation assays in yeast. TRM5 was cloned under its natural promoter upon PCR amplification. PCR-based mutagenesis was performed to obtain the trm5R270H and trm5M368V mutant alleles. The trm5Δ1-33 allele was constructed as previously described.21 Disruption of genomic TRM5 gene was performed in the presence of TRM5 on the pFL38 plasmid, given that the deletion is lethal. The TRM5, trm5R270H, trm5M368V, and trm5Δ1-33 alleles cloned into the pFL39 vector were introduced into this strain, and then pFL38-TRM5 was lost through plasmid shuffling. Oxygen consumption rate was recorded on intact cells grown at 28°C in synthetic complete medium without tryptophan, supplemented with 0.5% glucose. Values were normalized to the rate of oxygen consumption of the TRM5 transformant and represented as the mean of at least three values. Error bars = 1 SD; ∗p < 0.05, paired Student’s t test.