Overexpressed mitochondrial leucyl-tRNA synthetase suppresses the A3243G mutation in the mitochondrial tRNA^Leu(UUR) gene

Abstract

The A3243G mutation in the human mitochondrial tRNA Leu(UUR) gene causes a number of human diseases. This mutation reduces the level and fraction of aminoacylated tRNA Leu(UUR) and eliminates nucleotide modification at the wobble position of the anticodon. These deficiencies are associated with mitochondrial translation defects that result in decreased levels of mitochondrial translation products and respiratory chain enzyme activities. We have suppressed the respiratory chain defects in A3243G mutant cells by overexpressing human mitochondrial leucyl-tRNA synthetase. The rates of oxygen consumption in suppressed cells were directly proportional to the levels of leucyl-tRNA synthetase. Fifteenfold higher levels of leucyl-tRNA synthetase resulted in wild-type respiratory chain function. The suppressed cells had increased steady-state levels of tRNA Leu(UUR) and up to threefold higher steady-state levels of mitochondrial translation products, but did not have rates of protein synthesis above those in parental mutant cells. These data suggest that suppression of the A3243G mutation occurred by increasing protein stability. This suppression of a tRNA gene mutation by increasing the steady-state levels of its cognate aminoacyl-tRNA synthetase is a model for potential therapies for human pathogenic tRNA mutations.

Human Mitochondrial Leucyl-tRNA Synthetase Corrects Mitochondrial Dysfunctions Due to the tRNALeu(UUR) A3243G Mutation, Associated with Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-Like Symptoms and Diabetes

“…Indeed, the degree of increase in the rate of oxygen consumption in 43B-LARS2 cells correlated well with the increase in the rate of mitochondrial protein synthesis, suggesting that the improvement of mitochondrial translation was responsible for the increasing oxidative phosphorylation. However, this result was not in good agreement with the observation that the full recovery of respiration chain function occurred by means of overexpression of LARS2 in a mutant cybrid cell line carrying the A3243G mutation (39). This discrepancy may be attributed to different nuclear backgrounds, as in the case of cell lines carrying other mtDNA mutations (7,15).…”

“…In addition, the modified human FARS2 displayed significantly improved aminoacylation efficiency for the mitochondrial tRNA Phe G611A mutation (32). Alternatively, an increasing LARS2 expression in 43B cells may facilitate the correct folding and stabilization of the hypomodified tRNA Leu(UUR) (39). Moreover, an improvement of aminoacylation of tRNA Leu(UUR) by high levels of LARS2 appeared to lead to more efficient processing of the longer RNA precursors (22,24), evidenced by the fact that a reduction in the RNA19 was observed in the 43B-LARS2 cell line compared with the parental 43B cell line.…”

Structural and functional role of bases 32 and 33 in the anticodon loop of yeast mitochondrial tRNAIle

“…Importantly, by transforming the mutants with multicopy plasmids overexpressing tRNA interactors (e.g., mt protein elongation factor EF-Tu or cognate aaRS) we were able to suppress the defective phenotypes determined by the mutations and partially restore the steady-state levels of mutated tRNAs (Feuermann et al 2003;De Luca et al 2006. More recently, analogous results have been obtained in patient cell lines and cybrids (Park et al 2008;Rorbach et al 2008;Sasarman et al 2008;Li and Guan 2010). Additionally, we have shown that ValRS and LeuRS have cross-suppressive activities on mt tRNA Leu and tRNA Val yeast mutants and that suppression of yeast defects can be obtained by overexpression of orthologous human factors (Montanari et al 2010).…”

Trends in Mitochondrial Therapeutics for Neurological Disease

“…Accordingly, modification of the mt-tRNA binding domain of human mitochondrial phenylalanyl-tRNA synthetase significantly improved the aminoacylation efficiency of mttRNA Phe carrying the m611G>A MERRF mutation [70]. Overexpression of human mitochondrial valyl-or leucyl-tRNA synthetases increased, respectively, the steady-state levels of mutant mt-tRNA Val [71] or mt-tRNA Leu(UUR) [72], consistent with increased stability of the charged mt-tRNA. Also, overexpression of mitochondrial leucyl-tRNA synthetase rescued mitochondrial translation and respiration in cells carrying the A3243G MELAS mutation in mt-tRNA Leu(UUR) [73].…”