A disease-associated G5703A mutation in human mitochondrial DNA causes a conformational change and a marked decrease in steady-state levels of mitochondrial tRNA(Asn).

We introduced mitochondrial DNA (mtDNA) from a patient with a mitochondrial myopathy into established mtDNA-less human osteosarcoma cells. The resulting transmitochondrial cybrid lines, containing either exclusively wild-type or mutated (G5703A transition in the tRNA[Asn] gene) mtDNA, were characterized and analyzed for oxidative phosphorylation function and steady-state levels of different RNA species. Functional studies showed that the G5703A mutation severely impairs oxidative phosphorylation function and mitochondrial protein synthesis. We detected a marked reduction in tRNA(Asn) steady-state levels which was not associated with an accumulation of intermediate transcripts containing tRNA(Asn) sequences or decreased transcription. Native polyacrylamide gel electrophoresis showed that the residual tRNA(Asn) fraction in mutant cybrids had an altered conformation, suggesting that the mutation destabilized the tRNA(Asn) secondary or tertiary structure. Our results suggest that the G5703 mutation causes a conformational change in the tRNA(Asn) which may impair aminoacylation. This alteration leads to a severe reduction in the functional tRNA(Asn) pool by increasing its in vivo degradation by mitochondrial RNases.