Defects in Mitochondrial Protein Synthesis and Respiratory Chain Activity Segregate with the tRNA^Leu(UUR) Mutation Associated with Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Strokelike Episodes

TLDR

Cybrid cell lines were created by fusing patient-derived cytoplasts carrying the 3243A>G tRNA(Leu(UUR)) mutation with rho0 cells, and lines with <15 % or ≥95 % mutant mtDNA were examined for genetic, biochemical, and morphological differences. Cybrids with ≥95 % mutant mtDNA showed reduced mitochondrial protein synthesis, altered ND1 mobility, severe respiratory chain deficiency, and increased 16S rRNA + tRNA(Leu)+ND1 transcript levels, supporting a direct link between the mutation and MELAS pathogenesis.

Abstract

Cytoplasts from two unrelated patients with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) harboring an A----G transition at nucleotide position 3243 in the tRNA(Leu(UUR)) gene of the mitochondrial genome were fused with human cells lacking endogenous mitochondrial DNA (mtDNA) (rho 0 cells). Selected cybrid lines, containing less than 15 or greater than or equal to 95% mutated genomes, were examined for differences in genetic, biochemical, and morphological characteristics. Cybrids containing greater than or equal to 95% mutant mtDNA, but not those containing normal mtDNA, exhibited decreases in the rates of synthesis and in the steady-state levels of the mitochondrial translation products. In addition, NADH dehydrogenase subunit 1 (ND 1) exhibited a slightly altered mobility on polyacrylamide gel electrophoresis. The mutation also correlated with a severe respiratory chain deficiency. A small but consistent increase in the steady-state levels of an RNA transcript corresponding to 16S rRNA + tRNA(Leu(UUR)) + ND 1 genes was detected. However, there was no evidence of major errors in processing of the heavy-strand-encoded transcripts or of altered steady-state levels or ratios of mitochondrial rRNAs or mRNAs. These results provide evidence for a direct relationship between the tRNALeu(UUR) mutation and the pathogenesis of this mitochondrial disease.