Cloning, sequencing and bacterial expression of human glycine tRNA synthetase

Williams, James H.; Osvath, Sarah R.; Khong, Tee Fern; Pearse, Martin J.; Power, D.M.

doi:10.1093/nar/23.8.1307

Cited by 13 publications

(10 citation statements)

References 12 publications

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“…1D). Factoring in the size of the 6XHis tag, these data are consistent with the previously predicted molecular weight of human GARS [between 77 and 80 kDa (Targoff, 1990;Hirakata et al, 1992;Williams et al, 1995)]. The 673-685 peptide abolishes detection of this band with anti-GARS 673 .…”

Section: The G240r Gars Mutation Does Not Impair Transcription or Trasupporting

confidence: 88%

Functional Analyses of Glycyl-tRNA Synthetase Mutations Suggest a Key Role for tRNA-Charging Enzymes in Peripheral Axons

Antonellis¹,

Lee-Lin²,

Wasterlain³

et al. 2006

J. Neurosci.

115

160

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Charcot-Marie-Tooth disease type 2D (CMT2D) and distal spinal muscular atrophy type V (dSMA-V) are axonal neuropathies characterized by a phenotype that is more severe in the upper extremities. We previously implicated mutations in the gene encoding glycyl-tRNA synthetase (GARS) as the cause of CMT2D and dSMA-V. GARS is a member of the family of aminoacyl-tRNA synthetases responsible for charging tRNA with cognate amino acids; GARS ligates glycine to tRNA Gly . Here, we present functional analyses of disease-associated GARS mutations and show that there are not any significant mutation-associated changes in GARS expression levels; that the majority of identified GARS mutations modeled in yeast severely impair viability; and that, in most cases, mutant GARS protein mislocalizes in neuronal cells. Indeed, four of the five mutations studied show loss-of-function features in at least one assay, suggesting that tRNAcharging deficits play a role in disease pathogenesis. Finally, we detected endogenous GARS-associated granules in the neurite projections of cultured neurons and in the peripheral nerve axons of normal human tissue. These data are particularly important in light of the recent identification of CMT-associated mutations in another tRNA synthetase gene [YARS (tyrosyl-tRNA synthetase gene)]. Together, these findings suggest that tRNA-charging enzymes play a key role in maintaining peripheral axons.

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Section: The G240r Gars Mutation Does Not Impair Transcription or Trasupporting

confidence: 88%

Functional Analyses of Glycyl-tRNA Synthetase Mutations Suggest a Key Role for tRNA-Charging Enzymes in Peripheral Axons

Antonellis¹,

Lee-Lin²,

Wasterlain³

et al. 2006

J. Neurosci.

115

160

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“…GARS is an aminoacyl-tRNA synthetase: a group of evolutionary conserved enzymes that are essential for translation because they catalyse the addition of an amino acid to its cognate tRNA for protein synthesis. GARS is expressed ubiquitously and functions as a homodimer (Shiba et al, 1994;Williams et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot-Marie-Tooth type 2D peripheral neuropathy

Achilli

Bros-Facer

Williams

et al. 2009

Disease Models &Amp; Mechanisms

167

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C201R, with a point mutation that leads to a non-conservative substitution within GARS. Heterozygous mice with a C3H genetic background have loss of grip strength, decreased motor flexibility and disruption of fine motor control; this relatively mild phenotype is more severe on a C57BL/6 background. Homozygous mutants have a highly deleterious set of features, including movement difficulties and death before weaning. Heterozygous animals have a reduction in axon diameter in peripheral nerves, slowing of nerve conduction and an alteration in the recovery cycle of myelinated axons, as well as innervation defects. An assessment of GARS levels showed increased protein in 15-day-old mice compared with controls; however, this increase was not observed in 3-month-old animals, indicating that GARS function may be more crucial in younger animals. We found that enzyme activity was not reduced detectably in heterozygotes at any age, but was diminished greatly in homozygous mice compared with controls; thus, homozygous animals may suffer from a partial loss of function. The Gars C201R mutation described here is a contribution to our understanding of the mechanism by which mutations in tRNA synthetases, which are fundamentally important, ubiquitously expressed enzymes, cause axonopathy in specific sets of neurons.

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“…[15][16][17] This is in contrast to other aaRSs for which cytoplasmic and mitochondrial tRNA aminoacylation is achieved by the products of 2 separate genes (the only other exception being lysyl-tRNA synthetase). The GARS gene was predicted to produce both cytoplasmic and mitochondrial enzymes via alternative translational start sites.…”

Section: Introductionmentioning

confidence: 99%

“…The GARS gene was predicted to produce both cytoplasmic and mitochondrial enzymes via alternative translational start sites. 15 Analysis of fetal liver cDNA encoding human GARS, 16 as well as primer extension experiments on transfected cells. 17 identified a single mRNA characterized by a long 5 0 -UTR with 5 putative initiation codons (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, the presence of 3 other upstream initiation codons could indicate the existence of some regulatory mechanism(s); for instance one of these codons would lead to the synthesis of a short upstream Open Reading Frame (uORF), encoding 47 amino acids and potentially controlling GARS expression. 16 This study focuses on the control of human GARS expression. Indeed, many aspects of the regulation of this enzyme remain unclear, especially its tissue specific expression and the control of both mitochondrial and cytosolic forms of the enzyme.…”

Section: Introductionmentioning

confidence: 99%

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Elaborate uORF/IRES features control expression and localization of human glycyl-tRNA synthetase

Alexandrova

Paulus²,

Rudinger-Thirion

et al. 2015

RNA Biology

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The canonical activity of glycyl-tRNA synthetase (GARS) is to charge glycine onto its cognate tRNAs. However, outside translation, GARS also participates in many other functions. A single gene encodes both the cytosolic and mitochondrial forms of GARS but 2 mRNA isoforms were identified. Using immunolocalization assays, in vitro translation assays and bicistronic constructs we provide experimental evidence that one of these mRNAs tightly controls expression and localization of human GARS. An intricate regulatory domain was found in its 5 0 -UTR which displays a functional Internal Ribosome Entry Site and an upstream Open Reading Frame. Together, these elements hinder the synthesis of the mitochondrial GARS and target the translation of the cytosolic enzyme to ER-bound ribosomes. This finding reveals a complex picture of GARS translation and localization in mammals. In this context, we discuss how human GARS expression could influence its moonlighting activities and its involvement in diseases.

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Cloning, sequencing and bacterial expression of human glycine tRNA synthetase

Abstract: The human glycine tRNA synthetase gene (GlyRS)

Cited by 13 publications

References 12 publications

Functional Analyses of Glycyl-tRNA Synthetase Mutations Suggest a Key Role for tRNA-Charging Enzymes in Peripheral Axons

Functional Analyses of Glycyl-tRNA Synthetase Mutations Suggest a Key Role for tRNA-Charging Enzymes in Peripheral Axons

An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot-Marie-Tooth type 2D peripheral neuropathy

Elaborate uORF/IRES features control expression and localization of human glycyl-tRNA synthetase

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