Bioenergetics: Open Access

Defects in Mitochondrial Tyrosyl-Trna Synthetases Result in Decreased Retinal Oxidative Phosphorylation, According to an Animal Model

wmmbeg^* Department of Bioengertics, Equine Clinic, Faculty of Veterinary Medicine B41, University of Liege, , Belgium
^*Correspondence: wmmbeg, Department of Bioengertics, Equine Clinic, Faculty of Veterinary Medicine B41, University of Liege, , Belgium, Email:

Published: 26-Nov-2021

Introduction

Mitochondria maintain a distinct pool of ribosomal machinery, including tRNAs and tRNAs activating enzymes, such as mitochondrial tyrosyl-tRNA synthetase (YARS2). Mutations in YARS2, which typically lead to the impairment of mitochondrial protein synthesis, have been linked to an array of human diseases including optic neuropathy. However, the lack of YARS2 mutation animal model makes us difficult to elucidate the pathophysiology underlying YARS2 deficiency. To explore this system, we generated YARS2 knockout (KO) HeLa cells and zebrafish using CRISPR/Cas9 technology. We observed the aberrant tRNATyr aminoacylation overall and reductions in the levels in mitochondrion- and nucleus-encoding subunits of oxidative phosphorylation system (OXPHOS), which were especially pronounced effects in the subunits of complex I and complex IV. These deficiencies manifested the decreased levels of intact supercomplexes overall. Immunoprecipitation assays showed that YARS2 bound to specific subunits of complex I and complex IV, suggesting the posttranslational stabilization of OXPHOS. Furthermore, YARS2 ablation caused defects in the stability and activities of OXPHOS complexes. These biochemical defects could be rescued by the overexpression of YARS2 cDNA in the YARS2KO cells. In zebrafish, the yars2KO larva conferred deficient COX activities in the retina, abnormal mitochondrial morphology, and numbers in the photoreceptor and retinal ganglion cells. The zebrafish further exhibited the retinal defects affecting both rods and cones. Vision defects in yars2KO zebrafish recapitulated the clinical phenotypes in the optic neuropathy patients carrying the YARS2 mutations. Our findings highlighted the critical role of YARS2 in the stability and activity of OXPHOS and its pathological consequence in vision impairments.

Defects in mitochondrial protein synthesis are associated with a wide spectrum of human diseases, with a diversity of etiologies, ages of onset, involved organ systems and clinical presentations. Mitochondrial translation deficiencies resulted from defects in two rRNAs or 22 tRNAs, encoded by mitochondrial DNA (mtDNA), or alterations in the components of mitochondrial translation machinery (ribosomal proteins, ribosomal assembly proteins, aminoacyl-tRNA synthetases, tRNA-modifying enzymes, tRNA methylating enzymes, initiation, elongation, and termination factors), encoded by nuclear genes. In particular, all 19 mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) were synthesized in the cytosol and subsequently imported into mitochondria. These mt-aaRSs are a group of enzymes that catalyze a two-step reaction where they first activate the cognate amino acid with ATP to form an aminoacyl-adenylate, then subsequently transfer the aminoacyl group of the aminoacyl-adenylate to the bound tRNA for protein synthesis. In addition to their central roles in the translation, these aminoacyl-tRNA synthetases may have other functions, as in the case of cytoplasmic aminoacyl-tRNA synthetases. Defects in nuclear genes encoding mt-aaRSs have emerged as an important cause of mitochondrial disorders linked to diverse clinical presentations, usually with an early onset and transmitted as autosomal recessive traits. Remarkably, the pathologies linked to defects in mt-aaRSs display a marked bias for the central nervous system, including the leukodystrophy, encephalopathy, Perrault syndrome, sensorineural deafness, and visual impairment. However, the pathophysiology of these disease-linked mt-aaRS mutations, especially the pleiotropic and tissue-specific effects, remains poorly understood.

Mutations of YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase were responsible for the myopathy, lactic acidosis, and sideroblastic anemia (MLASA) with mitochondrial respiratory chain complex deficiencies. Recently, we demonstrated that the YARS2, mutation contributed to the phenotypic expression of Leber's hereditary optic neuropathy (LHON)-associated mtDNA mutations or deafness-associated tRNASer mutation. The primary defects in these YARS2 mutations were the deficient aminoacylation. The aberrant tRNATyr metabolism impaired mitochondrial translation, especially for the synthesis of polypeptides with high content of tyrosine codon such as ND4, ND5, ND6, and CO2 in cell lines carrying YARS2 mutations. Strikingly, cell lines carrying the YARS2 mutations exhibited marked reductions in the nucleus-encoding mitochondrial proteins: NDUFS3 and NDUFB8 [subunits of NADH:ubiquinone oxidoreductase (complex I)] and COX10 [subunit of cytochrome c oxidase. These implied that YARS2 may play an important role in the biogenesis of oxidative phosphorylation (OXPHOS) system. To test this hypothesis, we used CRISPR/Cas9 genomic editing approach in HeLa cells to produce the targeted deletion in YARS2 gene. To further confirm the defects of YARS2 knockout (KO) (YARS2KO) in the HeLa cells, we transferred a plasmid carrying the full-length YARS2 cDNA into the YARS2KO HeLa cells. The YARS2KO cell lines were assessed for the effects of YARS2 mutations on the biogenesis of OXPHOS, by western blot analysis using the subunits of OXPHOS and activity of respiratory chain complexes. To examine whether YARS2 interacts with the OXPHOS, we performed the immunoprecipitation assay using FLAG and HA antibodies in mitochondria of HeLa cell lines. To investigate whether defects in yars2 caused the visual impairment in vivo, we investigated the yars2 knockout zebrafish produced by genome editing using the CRISPR/Cas9 system.