WHEP Domains Direct Noncanonical Function of Glutamyl-Prolyl tRNA Synthetase in Translational Control of Gene Expression

Jia, Jinsheng; Arif, Abul; Ray, Partho Sarothi; Fox, Paul L.

doi:10.1016/j.molcel.2008.01.010

Cited by 115 publications

(148 citation statements)

References 48 publications

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“…As an illustrative example, glutamyl-prolyl tRNA synthetase (EPRS) is phosphorylated in macrophages in response to interferon-γ treatment, triggering its release from the multi-synthetase complex and its subsequent assembly into the IFN-γ-activated inhibitor of translation (GAIT) complex ). Phosphorylation also promotes conformational changes in the WHEP domains of EPRS that activate its mRNA-binding activity, enabling translational repression of proinflammatory genes (Jia et al 2008;Arif et al 2009Arif et al , 2011. The REM (RNA, enzyme, and metabolite) network hypothesis (Hentze and Preiss 2010) proposes an additional layer of RNA-binding regulation, where substrate (or cofactor) and RNA compete for the metabolitebinding pocket of an enzyme.…”

Section: Discussionmentioning

confidence: 99%

A versatile assay for RNA-binding proteins in living cells

Strein

Alleaume

Rothbauer

et al. 2014

RNA

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RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein-mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology.

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Section: Discussionmentioning

confidence: 99%

A versatile assay for RNA-binding proteins in living cells

Strein

Alleaume

Rothbauer

et al. 2014

RNA

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“…However, although aminoacylation is the only known function for GARS, a wide range of non-canonical functions has been shown for other aminoacyl-tRNA synthetases, including an involvement in splicing, apoptosis, transcription, protein folding, and trafficking (Park et al, 2005;Park et al, 2008;Jia et al, 2008;Ribas de Pouplana and Geslain, 2008). Thus, it is possible that heterozygous and/or homozygous phenotypes do not arise from defects in translation, but arise because of aberrations in other, unknown, functions of GARS.…”

Section: Discussionmentioning

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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“…Recent investigations have begun to illuminate these roles. These include endocytosis and membrane fusion, 17 translational control, 18 nuclear tRNA transport, 19 signaling, 20 pathogen virulence, 21 and cell death. 22 It is not clear how GAPDH can be targeted for these specific functions in the cell, but is believed to involve posttranslational modification of the protein.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of glyceraldehyde‐3‐phosphate dehydrogenase inactivation by tyrosine nitration

Palamalai¹,

Miyagi²

2010

Protein Science

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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifaceted protein that is involved in numerous processes including glycolysis, translational silencing, transcriptional regulation of specific genes, and acting as a nitric oxide sensor. The precise mechanism on how GAPDH is targeted to these different roles is unclear but believed to involve specific posttranslational modification to the protein. Numerous studies have demonstrated that GAPDH is a target for tyrosine nitration. However, the site of modification and the molecular consequence have not been defined. Rabbit GAPDH with a reversibly protected catalytic cysteine was nitrated in vitro with tetranitromethane, resulting in complete loss of GAPDH catalytic activity. Nitration was estimated as 0.32 mol of nitrotyrosine residue per mole of GAPDH. Mass spectrometry analysis of nitrated GAPDH indicated that Tyr311 and Tyr317 were the sole sites of nitration. The X-ray crystal structure revealed that the distances between Tyr311 and Tyr317 and the cofactor nicotinamide adenine dinucleotide (NAD 1 ) were less than 7.2 and 3.7 Å , respectively, implying that nitration of these two residues may affect NAD 1 binding. This possibility was assessed using an NAD 1 binding assay, which showed that nitrated GAPDH was incapable of binding NAD 1 . Thus, these results strongly suggest that Tyr311 and Tyr317 nitration prohibits NAD 1 binding, leading to the loss of catalytic activity.

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WHEP Domains Direct Noncanonical Function of Glutamyl-Prolyl tRNA Synthetase in Translational Control of Gene Expression

Cited by 115 publications

References 48 publications

A versatile assay for RNA-binding proteins in living cells

A versatile assay for RNA-binding proteins in living cells

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

Mechanism of glyceraldehyde‐3‐phosphate dehydrogenase inactivation by tyrosine nitration

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