Yeast-Based Functional Investigation of ELAC2 Variants in Mitochondrial Dysfunction

Background and goals: ELAC2 encodes the mitochondrial RNase Z, which is crucial for removing 3′ extensions from pre-tRNAs, a key step in tRNA biogenesis (Brzezniak et al, 2011; Rossmanith, 2011). Pathogenic ELAC2 variants affect mitochondrial gene expression, leading to respiratory chain deficiencies, hypertrophic cardiomyopathy (HCM), and lactic acidosis (Haack et al, 2013; Saoura et al, 2019). Compound heterozygous ELAC2 mutations were identified in a Chilean patient with epilepsy, hypotonia, and mild HCM. To elucidate the functional consequence of these variants and investigate previously reported variants (p.Lys660Ile; p.Ala680Val), associated with lactic acidosis, HMC and reduced Complex I and IV activity (Saoura et al, 2019), we analyzed patient fibroblasts. and modelled the mutations in the yeast Saccharomyces cerevisiae. Material and Methods Whole exome sequencing revealed two heterozygous variants p.Val348Met and p.Thr298Ile. Western blot analyses were performed on Chilean patient fibroblasts. For yeast modelling, mutations were introduced into the homologous TRZ1 gene. The trz1Δ strain, harbouring wild type TRZ1 for viability (Haack et al, 2013), was transformed with mutant alleles and the functional consequences were assessed using plasmid shuffling methods (Baruffini et al., 2010). We evaluated viability, oxidative growth, oxidative consumption rate (OCR), mtDNA stability, and mitochondrial protein synthesis (MPS). Results Western blot analysis performed in fibroblasts showed a 50% reduction of ELAC2 protein level, with no defects of respiratory chain enzymes subunits, according to previous reports where oxidative defects were noted only in muscle and heart tissues. All mutant alleles complemented the trz1Δ strain except the allele expressing the mutation equivalent to Ala680Val, which resulted in a complete loss of function. The Val348Met equivalent mutation, impaired growth at 37°C, while Thr298Ile and Lys660Ile equivalents caused mild oxidative defect at 28°C, exacerbated at 37°C. OCR analysis parallelized with oxidative growth defects; increased mitochondrial mutability (15% for V348M, 38% and 48% for T298I and K660I, respectively) was also observed indicating defects in protein synthesis, confirmed by reduced in vivo MPS, especially of CIV subunit Cox1. Conclusions Modelling of the ELAC2 variants in yeast confirmed the pathogenicity of all the mutations as causative of the pathological phenotype observed in patients.