Exploring the ability of LARS2 carboxy-terminal domain in rescuing the MELAS phenotype

The m.3243A>G mutation within mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to Mitochondrial Encephalopathy with Lactic Acidosis and Stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A>G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme; however, the precise molecular mechanisms underlying this process have not been fully elucidated. To address this issue, we investigated the effect of Cterm overexpression on a wide range of cellular processes, using MELAS cybrids with a high mutation load (95%). First, we demonstrated that Cterm domain is able to contact in vivo the cognate mutated mt-tRNALeu(UUR) tRNA and its precursor RNA19, thus corroborating the "chaperonic" hypothesis previously formulated mainly on the basis of in vitro findings (Perli, E., Giordano, C., Pisano, A. et al. EMBO Mol Med 2014, 6,169). Moreover, we observed a complete recovery of cell viability to the level of the healthy controls. Next, we evaluated the effect of Cterm overexpression on a range of mitochondrial processes such as protein synthesis, steady-state level and aminoacylation efficiency of mttRNALeu(UUR), bioenergetic competence and mitophagy. We found that Cterm was able to ameliorate de novo mitochondrial protein synthesis, whilst it had no effect on mttRNALeu(UUR) level and aminoacylation. However, despite the complete recovery of cell viability and the increase in mitochondrial translation, we could not detect any improvement in mitochondrial oxygen consumption rate, as well as in the level and activity of respiratory chain complexes. Furthermore, Cterm expression did not affect the higher glycolytic flux peculiar of MELAS cells, as shown by lactate measurement. Finally, mitochondrial mass and mitophagy also did not change. We suggest that in MELAS cells the beneficial effect of Cterm on cell viability may be mediated by factors that are independent of the mitochondrial bioenergetics.