Pathogenic mechanism of a human mitochondrial tRNA ^Phe mutation associated with myoclonic epilepsy with ragged red fibers syndrome

Abstract

Human mitochondrial tRNA (hmt-tRNA) mutations are associated with a variety of diseases including mitochondrial myopathies, diabetes, encephalopathies, and deafness. Because the current understanding of the precise molecular mechanisms of these mutations is limited, there is no efficient method to treat their associated mitochondrial diseases. Here, we use a variety of known mutations in hmt-tRNA Phe to investigate the mechanisms that lead to malfunctions. We tested the impact of hmt-tRNA Phe mutations on aminoacylation, structure, and translation elongation-factor binding. The majority of the mutants were pleiotropic, exhibiting defects in aminoacylation, global structure, and elongation-factor binding. One notable exception was the G34A anticodon mutation of hmt-tRNA Phe (mitochondrial DNA mutation G611A), which is associated with MERRF (myoclonic epilepsy with ragged red fibers). In vitro, the G34A mutation decreases aminoacylation activity by 100-fold, but does not affect global folding or recognition by elongation factor. Furthermore, G34A hmt-tRNA Phe does not undergo adenosine-to-inosine (A-to-I) editing, ruling out miscoding as a possible mechanism for mitochondrial malfunction. To improve the aminoacylation state of the mutant tRNA, we modified the tRNA binding domain of the nucleus-encoded human mitochondrial phenylalanyl-tRNA synthetase, which aminoacylates hmt-tRNA Phe with cognate phenylalanine. This variant enzyme displayed significantly improved aminoacylation efficiency for the G34A mutant, suggesting a general strategy to treat certain classes of mitochondrial diseases by modification of the corresponding nuclear gene.aminoacyl-tRNA synthetase ͉ translation ͉ mitochondria

Mechanistic insights into cognate substrate discrimination during proofreading in translation

“…The study also implies that these mechanisms are designed in such a tipping point that a mutation in the editing modules even far away from the editing site could lead to deleterious consequences especially in the light of a recent study that showed that proofreading modules of aaRS recheck the fidelity of the aminoacylation process by capturing the elongation factor bound aminoacylated-tRNA before it reaches the ribosomes (19). Because cognate aa-tRNAs are normally enriched in the cellular pool, in conjunction with our study, it would imply that a small mutational perturbation in the editing domain may result in hydrolysis of the cognate amino acid from tRNA thus depleting the correctly charged tRNA pool which results in disease conditions (28). Generally, editing defects are defined on the basis of efficiency of deacylation of mischarged tRNA.…”

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

“…(40). 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).…”

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.…”