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

Cybrids were created by fusing patient cytoplasts carrying the 3243A>G tRNA(Leu(UUR)) mutation with rho0 cells, and lines with low (<15%) or high (≥95%) mutant load were compared for genetic, biochemical, and morphological traits. Cybrids with ≥95% mutant mtDNA exhibited reduced mitochondrial protein synthesis, lower steady‑state translation products, altered ND1 mobility, and severe respiratory chain deficiency, while transcript levels of 16S rRNA + tRNA(Leu(UUR)) + ND1 were modestly increased, supporting a direct link between the 3243A>G 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.