Elucidating the molecular mechanisms underlying the biological activity of rescuing peptides in MELAS mitochondrial disease

Mitochondrial Encephalopathy with Lactic Acidosis and Stroke like Episodes (MELAS) is a mitochondrial disease caused by point mutations in the tRNALeu(UUR) with a prevalence in position 3243 (A>G). These mutations cause severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA such as reduced aminoacylation and lack of post-transcriptional modification. It has been shown that overexpression of leucyl-tRNA-synthetase (mt-LeuRS) is able to rescue defects of pathogenic mutation of tRNALeu(UUR) in MELAS cybrids [1]. The rescuing activity of human mt-LeuRS resides in a small (<70 amino acids) part of the carboxy-terminal domain (Cterm) of the enzyme and in short (15-16 aa) C-term-derived peptides. However, the molecular mechanisms underlying the rescuing process are still unknown. On these bases, we started a project aimed at elucidating the ability of mt-LeuRS C-term and its derived peptides to correct the pathological phenotype in MELAS. To address this issue we intend: i) to investigate in vivo the interactions of rescuing molecules with mutated mt-tRNA; ii) to study the effect of overexpression of rescuing molecules on the rate of synthesis and stability of de novo synthesized mt-polypeptides; iii) to investigate the effect of rescuing molecules on the steady-state level, the aminoacylation level and the post-transcriptional modifications of the mutated tRNA. To date we have characterized the profile of the de novo synthesized mt-polypeptides from MELAS cybrids, finding an overall decreased rate of synthesis and the appearance of aberrant mitochondrial translation product. We have also observed a decreased steady-state level of NDUFB8 (Complex I) and COXI (Complex IV). Finally, we have demonstrated in MELAS cybrids constitutively expressing C-term, the existence of a preferential interaction between Cterm polypeptide and tRNALeu(UUR) by means of RNA immunoprecipitation experiments