Dipartimento di Biologia e
Biotecnologie Charles Darwin

Human mitochondrial (mt)DNA diseases are a group of clinically heterogeneous disorders that cause mainly muscle and neurodegenerative dysfunction and the ageing process itself. A large proportion of pathogenic mtDNA mutations are found in genes encoding mt tRNAs, resulting in the inhibition of mt protein synthesis and consequent oxidative phosphorylation machinery (OXPHOS) defects. Presently, no effective treatment exists for the large majority of patients with mtDNA diseases. This absence is partly due to the scarcity of suitable models and to the fact that it is not possible to manipulate mtDNA in human. This difficulty has been overcome in yeast, in which biolistic mt transformation is possible (Fox et al, 1991; Bonnefoy, Fox, 2007).

We have established the yeast S. cerevisiae model to analyse the molecular and cellular aspects of the mt defects due to mutations equivalent to human pathogenetic substitutions in mt tRNA genes. This was made possible by biolistically introducing into yeast mtDNA the equivalent human pathogenic mt tRNA mutations. We observed that the yeast mutants respiratory defects were comparable with the severity of human equivalent mt mutations. We perform phenotypic analyses on yeast mutants measuring their glycerol growth capability and O₂ consumption and we observed phenotypic variability dependent on the nuclear context of mutant cells. By Northern blot on high resolution gels we can study whether the defect might be related to the amount or to aminoacylation defects of mutated tRNA.

We then exploit the power of yeast genetics by identifying nuclearly encoded protein synthesis factors such as the mt protein synthesis elongation factor (EF-Tu) and cognate mt aminoacyl-tRNA synthetases (aaRS) that rescued, when overexpressed, the respiratory defects caused by mt tRNA mutations (Feuermann et al, 2003; De Luca et al, 2006, 2009; Montanari et al, 2010).Recently, the nuclear suppressors identified in our laboratory have also been used to alleviate the defective mt phenotypes in human cells (Rorbach et al, 2008; Sasarman et al, 2008; Park et al, 2008; Perli et al, 2012), indicating that results obtained in yeast can be transposed to human. Moreover, we found that the respiratory defects were relieved not only by overexpression of cognate mt aaRS but, unexpectedly, of non cognate mt aaRS. We also found that the orthologous human aaRS were active as suppressors in yeast mt tRNA mutants of our collection (Montanari et al, 2010). We hypothesised that the suppressor molecule could help to correct and protect the altered structure of mutated tRNA in a chaperone-like manner, and that this RNA stabilizing capability could be restricted to a conserved domain of the aaRS (De Luca et al, 2009). This indeed proved to be the case and we observed that the carboxy-terminal (C-term) region of human and yeast mt LeuRS is sufficient to maintain the full suppressing activity towards yeast mutants in mt tRNA^Leu, tRNA^Val and tRNA^Ile genes (Francisci et al, 2011).