title: 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 non-canonical 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.
authors.affiliation: From the State Key Laboratory of Molecular Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China, the School of Life Science and Technology, ShanghaiTech University, 319 Yue Yang Road, Shanghai 200031, China, and.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.affiliation: From the State Key Laboratory of Molecular Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China, xlzhou@sibcb.ac.cn.
authors.forename: Xiao-Long
authors.initials: XL
authors.lastname: Zhou
authors.affiliation: From the State Key Laboratory of Molecular Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Zhi-Rong
authors.initials: ZR
authors.lastname: Ruan
authors.affiliation: From the State Key Laboratory of Molecular Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Ru-Juan
authors.initials: RJ
authors.lastname: Liu
authors.affiliation: the Architecture et Réactivité de l'ARN, UPR9002 CNRS, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, 15 rue René Descartes, 67084 Strasbourg, France.
authors.forename: Gilbert
authors.initials: G
authors.lastname: Eriani
authors.affiliation: From the State Key Laboratory of Molecular Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China, the School of Life Science and Technology, ShanghaiTech University, 319 Yue Yang Road, Shanghai 200031, China, and edwang@sibcb.ac.cn.
authors.forename: En-Duo
authors.initials: ED
authors.lastname: Wang
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D004789:Enzyme Activation; D004795:Enzyme Stability; D005816:Genetic Complementation Test; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D017237:Mitochondrial Encephalomyopathies / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D055503:Protein Multimerization; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013914:Threonine-tRNA Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D046249:Transfer RNA Aminoacylation
chemical_list: D007527:Isoenzymes; D013912:Threonine; D000249:Adenosine Monophosphate; D012694:Serine; D013914:Threonine-tRNA Ligase
keywords: alternative splicing; aminoacyl-tRNA synthetase; enzyme kinetics; mitochondria; mitochondrial disease; threonyl-tRNA synthetase
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