A Human Disease-causing Point Mutation in Mitochondrial Threonyl-tRNA Synthetase Induces Both Structural and Functional Defects

Abstract

Mitochondria require all translational components, including aminoacyl-tRNA synthetases (aaRSs), to complete organelle protein synthesis. Some aaRS mutations cause mitochondrial disorders, including human mitochondrial threonyl-tRNA synthetase (hmtThrRS) (encoded by TARS2), the P282L mutation of which causes mitochondrial encephalomyopathies. However, its catalytic and structural consequences remain unclear. Herein, we cloned TARS2 and purified the wild-type and P282L mutant hmtThrRS. hmtThrRS misactivates non-cognate Ser and uses post-transfer editing to clear erroneously synthesized products. In vitro and in vivo analyses revealed that the mutation induces a decrease in Thr activation, aminoacylation, and proofreading activities and a change in the protein structure and/or stability, which might cause reduced catalytic efficiency. We also identified a splicing variant of TARS2 mRNA lacking exons 8 and 9, the protein product of which is targeted into mitochondria. In HEK293T cells, the variant does not dimerize and cannot complement the ThrRS knock-out strain in yeast, suggesting that the truncated protein is inactive and might have a noncanonical function, as observed for other aaRS fragments. The present study describes the aminoacylation and editing properties of hmtThrRS, clarifies the molecular consequences of the P282L mutation, and shows that the yeast ThrRS-deletion model is suitable to test pathology-associated point mutations or alternative splicing variants of mammalian aaRS mRNAs.Aminoacyl-tRNA synthetases (aaRSs) 4 supply the ribosome with aminoacyl-tRNA substrates for protein synthesis (1, 2). This process starts with amino acid activation by condensation with ATP to form the aminoacyl adenylate aa-AMP and pyrophosphate. The activated amino acid then reacts with the 3Ј-end of the cognate tRNA to yield the aminoacyl-tRNA (aatRNA), which is transferred by EF-Tu to the protein biosynthesis machinery as a building block. Highly accurate protein synthesis is indispensable for cell growth. To maintain fidelity during protein synthesis, aaRS needs to select the correct amino acid and tRNA substrates from a large number of structurally similar molecules in the cells. The specificity of aaRS is greatly challenged by the presence of the 20 amino acids in which the physicochemical properties are sometimes very similar and by non-proteogenous amino acids and diverse metabolites produced by cell metabolism. aaRSs that do not show an overall selectivity above 1 in 3000 are predicted to require proofreading (editing) activity to maintain sufficient accuracy during aa-tRNA synthesis (3). In fact, editing activity has evolved in half of the currently identified aaRS to remove aberrantly produced aa-AMP (pretransfer editing) and/or aa-tRNA (posttransfer editing) (4). tRNA mischarging may be toxic and deleterious for cells. Even a slight decrease in aminoacylation accuracy could cause an intracellular accumulation of misfolded proteins and up-regulation of cytoplasmic protein chaperones in neurons, leading to severe...

Human aminoacyl-tRNA synthetases in diseases of the nervous system

“…It was also shown that the pathogenic mutation P282L decreases both aminoacylation and proofreading activities of mt-ThrRS. 92 Taken together, fatal infantile syndromes seem to be caused primarily by the loss-of-function variants of AaRSs. Such deficiency is likely to cause the major disturbance of proteostasis, which ultimately leads to systemic failures and death.…”

Homozygosity for FARSB mutation leads to Phe-tRNA synthetase-related disease of growth restriction, brain calcification, and interstitial lung disease

“…These enzymes are also involved in cellular signaling and metabolism which are linked to the survival of the cell (Kim et al, 2003;Wakasugi & Schimmel, 1999). Therefore, dysfunction or absence of ARSs results in severe or lethal conditions in multiple species (Berg, Rogers, Muralla, & Meinke, 2005;Ni & Luo, 2018;Wang et al, 2016). Mutations in ARS genes implicated in a wide array of monogenic diseases in human with autosomal recessive and dominant inheritance (Oprescu, Griffin, Beg, & Antonellis, 2017).…”

Mutations in the mitochondrial tryptophanyl‐tRNA synthetase cause growth retardation and progressive leukoencephalopathy

“…Whether this is attributable to the WARS2 variants is unclear. Varying responses to different ARS2 mutations might be explained by different tissue‐specific responses to ARS2 mutations as exemplified by a mouse model for DARS2 (Dogan et al, ), or by differences in the compatibility between the ARS2 variants and their corresponding tRNAs (Meiklejohn et al, ; Perli et al, ; Wang et al, ). Additionally, besides the primary defect of reduced aminoacylation, secondary metabolic defects, such as the accumulation of aminoacyl intermediates, such as tryptophan or tryptamine, an intermediate of tryptophan metabolism and potentially a neurotransmitter, may further affect brain function.…”