Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype

Abstract

The m.3243A>G mutation within the 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 a 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 deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.

Molecular Investigation of Mitochondrial RNA19 Role in the Pathogenesis of MELAS Disease

“…The construct to facilitate inducible expression of the isolated carboxy-terminal domain of LARS2 (Cterm), fused with Cox8 mitochondrial presequence at the N-terminus and FLAG-tag at the C-terminus, was prepared from the plasmid pcDNA6.2/Cox8-Cterm-FLAG [ 17 ]. The plasmid was NotI-/ApaI-digested, and the obtained insert was gel-eluted and ligated into the tetracycline-inducible pcDNA5/FRT/TO ® vector (Invitrogen, Waltham, MA, USA), according to standard cloning techniques.…”

“…Recent research from our laboratory showed that RNA19 can in cellulo bind the carboxy-terminal domain of the human mitochondrial leucyl-tRNA synthetase, LARS2 [ 17 ]. Overexpression of this isolated domain, concisely named Cterm, has proven effective in rescuing the defective phenotype associated with the 3243A>G transition in human cells [ 18 , 19 ].…”

“…Overexpression of this isolated domain, concisely named Cterm, has proven effective in rescuing the defective phenotype associated with the 3243A>G transition in human cells [ 18 , 19 ]. Importantly, exogenous expression of the Cterm was able to recover de novo mitochondrial protein synthesis, ameliorating the viability of MELAS cybrids [ 17 ]. However, to date, the molecular mechanisms of Cterm-mediated rescue are still unknown.…”

Mitochondrial DNA abnormalities and metabolic syndrome

“…β Diabetes caused by oxidative phosphorylation is a rare type of diabetes. Mitochondrial tRNALeu (UUR) 3243A > G-spot mutation is the most common cause of mitochondrial diabetes (Capriglia et al, 2021). Kearns-Sayre syndrome, which is caused by a large deletion in the mitochondrial DNA, can also be associated with diabetes (Kamal et al, 2015) (Table 1).…”

Mitochondria: From Physiology to Pathology

“…Several papers have reported that overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) or its C-terminal domain (Cterm) has proven effective in rescuing the pathological phenotype in cellular models. In their research article, Capriglia et al [ 10 ] investigated the molecular basis underlying the ability of the Cterm domain in rescuing the MELAS phenotype. The cellular model employed, consisting of a trans-mitochondrial cybrid line with a >95% mutation load, confirmed the therapeutic potential of the Cterm peptide but also showed that its rescuing ability was independent of the mitochondrial bioenergetics, unlike what has previously been observed in other cybrid lines.…”