title: Identification of lethal mutations in yeast threonyl-tRNA synthetase revealing critical residues in its human homolog.
abstract: Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs.
authors.affiliation: From the Center for RNA Research, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.
authors.forename: Zhi-Rong
authors.initials: ZR
authors.lastname: Ruan
authors.affiliation: From the Center for RNA Research, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.
authors.forename: Zhi-Peng
authors.initials: ZP
authors.lastname: Fang
authors.affiliation: From the Center for RNA Research, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Ye
authors.affiliation: From the Center for RNA Research, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.
authors.forename: Hui-Yan
authors.initials: HY
authors.lastname: Lei
authors.affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 Rue René Descartes, 67084 Strasbourg, France.
authors.forename: Gilbert
authors.initials: G
authors.lastname: Eriani
authors.affiliation: From the Center for RNA Research, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China, xlzhou@sibcb.ac.cn.
authors.forename: Xiao-Long
authors.initials: XL
authors.lastname: Zhou
authors.affiliation: From the Center for RNA Research, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China, the School of Life Science and Technology, ShanghaiTech University, 320 Yue Yang Road, Shanghai 200031, China, and edwang@sibcb.ac.cn.
authors.forename: En-Duo
authors.initials: ED
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D015723:Gene Library; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013914:Threonine-tRNA Ligase / Q000235:genetics*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D000604:Amino Acyl-tRNA Synthetases; D013914:Threonine-tRNA Ligase
keywords: Aminoacyl tRNA Synthetase; Aminoacylation, Editing; Mutagenesis; Protein Structure; Transfer RNA (tRNA); Translation