Pathophysiology of the MELAS 3243 Transition Mutation

Abstract

Single base substitutions of the mitochondrial genome are associated with a variety of metabolic disorders. The myopathy, encephalopathy, lactic acidosis, stroke-like episodes syndrome, most frequently associated with an A to G transition mutation at position 3243 of the mitochondrial tRNA Leu(UUR) gene, is characterized by biochemical and structural alterations of mitochondria. To investigate the pathophysiology of the mutation, we established distinct Epstein-Barr virus-transformed B-cell lines for analyses that harbored 30 -70% of the mutated genome. Interestingly, neither an alteration of the processing of primary transcripts nor a general impairment of individual mitochondrial protein subunit synthesis rates could be observed. Nevertheless a marked decrease of cytochrome-c oxidase activity and reduced content of mitochondrial encoded subunits in the assembled respiratory complex IV was recorded on the cell line harboring 70% mutated mtDNA. Quantitative analysis of incorporation rates of the amino acid leucine into newly synthesized mitochondrial proteins, representing the functionality of the tRNA Leu(UUR) in protein biosynthesis, revealed a specific decrease of this amino acid in distinct mitochondrial translation products. This observation was supported by a variation in the proteolytic fingerprint pattern. Our results suggest that the malfunctioning mitochondrial tRNA Leu(UUR) leads to an alteration of amino acid incorporation into the mitochondrially synthesized subunits of the oxidative phosphorylation system, thus altering it's structure and function.

Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease

“…A number of analyses have suggested that the extent of the deficiency in protein synthesis did not seem to parallel the decline in the enzymatic activity of the respiratory complexes in the case of MELAS (12)(13)(14)35). Thus, a quantitative decrease in functional mt tRNA Leu(UUR) alone seems insufficient to be the direct cause of the mitochondrial dysfunction, although the mitochondrial dysfunction in MELAS arises from multiple causes.…”

“…These dysfunctions include impaired termination (34), impaired pre-tRNA processing (35), decreased stability and aminoacylation (36), and abnormal conformation (37), and commonly lead to a decreased steady-state level of the normal aminoacylated tRNA, which in turn leads to reduced protein synthesis. Some of these biochemical tRNA analyses were performed by using an unmodified tRNA transcribed in vitro, because it is difficult to obtain a sufficient number of native mt tRNAs with the MELAS mutation for a series of analyses.…”

Structural probing of a pathogenic tRNA dimer

“…Thus, the A14G mutant may not function correctly with tRNA processing and maturation enzymes, and accordingly, defects have been observed in a variety of biochemical pathways (Jacobs and Holt 2000;Florentz 2002;. Cellular studies investigating the A3243G mutant have revealed deficiencies in RNA processing (Masucci and Schon 1996;Rossmanith and Karwan 1998;Koga et al 2003), aminoacylation (El Meziane et al 1998;Janssen et al 1999;Borner et al 2000;Chomyn et al 2000;Wittenhagen and Kelley 2002;Park et al 2003), post-transcriptional tRNA modifi-cation (Helm et al 1999;Yasukawa et al 2000), and translation (Chomyn et al 1992;King et al 1992;Schon et al 1992;Flierl et al 1997).…”

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

“…Diseases associated with hmt-tRNA point mutations include MERRF (myoclonic epilepsy with ragged red fibers), MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), and deafness (2)(3)(4). Pathogenic mutations of hmt-tRNAs have been shown to affect global tRNA structure (5)(6)(7)(8), tRNA processing (9-13), modification (14)(15)(16)(17)(18)(19)(20), aminoacylation (7,(21)(22)(23)(24)(25)(26), and translation efficiency (27)(28)(29). However, little is known about the precise pathogenic mechanisms of the vast majority of hmt-tRNA mutations, hindering the development of appropriate treatments.…”