Search for differences in post-transcriptional modification patterns of mitochondrial DNA-encoded wild-type and mutant human tRNALys and tRNALeu(UUR)

Abstract

Post-transcriptional modifications are characteristic features of tRNAs and have been shown in a number of cases to influence both their structural and functional properties, including structure stabilization, amino-acylation and codon recognition. We have developed an approach which allows the investigation of the post-transcriptional modification patterns of human mitochondrial wild-type and mutant tRNAs at both the qualitative and the quantitative levels. Specific tRNA species are long-term labeled in vivo with [³²P]orthophosphate, isolated in a highly selective way, enzymatically digested to mononucleotides and then subjected to two-dimensional thin layer chromatographic analysis. The wild-type tRNA^Lys and the corresponding tRNA^Lys carrying the A8344G mutation associated with the MERRF (Myoclonic Epilepsy with Ragged Red Fibers) syndrome exhibit the same modified nucleotides at the same molar concentrations. By contrast, a quantitatively different modification pattern was observed between the wild-type tRNA^Leu(UUR) and its counterpart carrying the A3243G mutation associated with the MELAS (Mitochondrial Myopathy, Encephalopathy with Lactic Acidosis and Stroke-like episodes) syndrome, the latter exhibiting a 50% decrease in m²G content. Complementary sequencing of tRNA^Leu(UUR) has allowed the localization of this modification at position 10 within the D-stem of the tRNA. The decreased level of this modification may have important implications for understanding the molecular mechanism underlying the MELAS-associated mitochondrial dysfunction.