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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Ribo-seq of HEK293T overexpressing WT and G240R GARS

ABSTRACT: Charcot-Marie-Tooth (CMT) disease can be caused by mutations in Aminoacyl-tRNA-Synthetases, including G240R mutation in Glycyl-tRNA-Synthetase (GARS). Ribo-seq generates snapshots of translating ribosomes on mRNA and therefore allows analysis of ribosome pausing mRNA. Here we performed Ribo-seq on lysates of HEK293T cells overexpressing GARS, WT or G240R, to dissect mechanism of CMT linked with translation. We found that GARS G240R causes pausing of ribosomes with glycine codons in A-site. The effect is specific for 21 nt ribosome-protected fragments, produced by ribosomes with empty A-sites, suggestive of the deficit of charged Glycyl-tRNA in GARS G240R-CMT.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Marina Chekulaeva

PROVIDER: E-MTAB-10342 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Charcot-Marie-Tooth mutation in glycyl-tRNA synthetase stalls ribosomes in a pre-accommodation state and activates integrated stress response.

Mendonsa Samantha S von Kuegelgen Nicolai N Bujanic Lucija L Chekulaeva Marina M

Nucleic acids research 20210901 17

Toxic gain-of-function mutations in aminoacyl-tRNA synthetases cause a degeneration of peripheral motor and sensory axons, known as Charcot-Marie-Tooth (CMT) disease. While these mutations do not disrupt overall aminoacylation activity, they interfere with translation via an unknown mechanism. Here, we dissect the mechanism of function of CMT mutant glycyl-tRNA synthetase (CMT-GARS), using high-resolution ribosome profiling and reporter assays. We find that CMT-GARS mutants deplete the pool of g ...[more]

PMID: 34403468

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tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase

Project description:Heterozygous mutations in six tRNA synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT-mutant glycyl- or tyrosyl-tRNA synthetases inhibit global protein synthesis by an unknown mechanism, independent of aminoacylation activity. We report that tRNAGly overexpression rescues protein synthesis and peripheral neuropathy phenotypes in Drosophila and mouse models of CMT caused by glycyl-tRNA synthetase (GlyRS) mutations (CMT2D). Kinetic experiments revealed that CMT-mutant GlyRS bind tRNAGly, but display markedly slow release rates. This tRNAGly sequestration may deplete the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly supply to the ribosome and translation deficit.

2021-09-07 | GSE160584 | GEO

Transcriptional dysregulation by a nucleus-localized aminoacyl-tRNA synthetase is associated with Charcot-Marie-Tooth neuropathy

Project description:Charcot-Marie-Tooth disease (CMT) is a length-dependent peripheral neuropathy. The aminoacyl-tRNA synthetases constitute the largest protein family implicated in CMT. Aminoacyl-tRNA synthetases are predominantly cytoplasmic, but are also present in the nucleus. Here we show that a nuclear function of tyrosyl-tRNA synthetase (TyrRS) is implicated in a Drosophila model of CMT. CMT-causing mutations in TyrRS induce unique conformational changes, which confer capacity for aberrant interactions with transcriptional regulators in the nucleus, leading to transcription factor E2F1 hyperactivation. Using neuronal tissues, we reveal a broad transcriptional regulation network associated with wild-type TyrRS expression, which is disturbed when a CMT-mutant is expressed. Pharmacological inhibition of TyrRS nuclear entry with embelin reduces, whereas genetic nuclear exclusion of mutant TyrRS prevents hallmark phenotypes of CMT in the Drosophila model. These data highlight that this translation factor may contribute to transcriptional regulation in neurons, and suggest a therapeutic target for CMT.

2019-10-01 | GSE125311 | GEO

A non-canonical function of tyrosyl-tRNA synthetase in actin bundling

Project description:Aminoacyl-tRNA synthetases are the largest protein family implicated in Charcot-Marie-Tooth disease (CMT), the most common inherited peripheral neuropathy that is currently incurable. These essential enzymes catalyze the attachment of amino acids to their cognate tRNAs, thereby enabling the protein biosynthesis in every cell. Surprisingly, loss of aminoacylation is not a prerequisite for CMT to occur, suggesting a disease mechanism associated with a gain of neurotoxic function. Via an unbiased genetic modifier screen in a Drosophila model of tyrosyl-tRNA synthetase (YARS) -induced CMT, we established a link between the tRNA-ligase and regulators of actin cytoskeleton. By investigating the interactome of YARS in cellulo we found this synthetase to be enriched in protein complexes containing actin and actin-modifying proteins. Follow up in vitro and in vivo studies demonstrated that YARS itself has not only actin binding- but also actin-bundling properties independent of its aminoacylation function. These non-canonical activities are evolutionary conserved and contribute to the organization of actin cytoskeleton in Drosophila neurons and in patient-derived cells. An enzymatically active YARSCMT mutant caused stronger actin bundling in vitro, disorganization of actin bundle-rich stress fibers in patient-derived cells and impaired multiple actin-based steps of the synaptic vesicle cycle in fly neurons. Genetic modulation of the F-actin organization state restored synaptic vesicle mobility and rescued hallmark electrophysiological and morphological features in the neurons of fruit flies expressing different YARSCMT mutations. Thus, we uncover a role of tyrosyl-tRNA synthetase in actin organization that is implicated in the CMT neuropathy.

2023-01-09 | PXD037630 | Pride

RNA sequencing of MShef11 WT, MFN2R94Q/+, MFN2 R94Q/R94Q differentiated to limb innervating motor neurons

Project description:We have generated CRISPR edited versions of hESC line MShef11 to produce MFN2 R94Q/+ and MFN2 R94Q/R94Q lines as a model for Charcot Marie Tooth Disease (CMT) 2A. This were differentiated to limb innervating motor neurons, the predominantly affected cell time in CMT2A and RNA was examined to investigate differences in cell lines.

2022-07-28 | E-MTAB-10831 | biostudies-arrayexpress

Determining ribosome-associated mRNA in motor neurons after nerve crush [15NGS-001-rwb]

Project description:Determining ribosome-associated mRNA in motor neurons of wild-type adult mice 4 days after unilateral sciatic nerve crush How mutations in broadly expressed housekeeping genes lead to neurodegeneration in specific cell types remains unclear. Mutations in ubiquitously expressed tRNA synthetase genes cause axonal peripheral neuropathy, accounting for at least six forms of Charcot-Marie-Tooth disease. Genetic evidence in mouse and Drosophila models suggests a neomorphic gain-of-function mechanism. Here, we use in vivo, cell-type-specific transcriptional and translational profiling of affected peripheral neurons to show that mutant tRNA synthetases impair translation and activate the integrated stress response (ISR) through the sensor kinase, GCN2. The chronic activation of the ISR contributes to the pathophysiology, and genetic deletion of Gcn2 alleviates the peripheral neuropathy. The activation of GCN2 by tRNA synthetase mutations indicates their neomorphic activity is still related to translation and suggests inhibiting GCN2 or the ISR as a therapeutic strategy.

2020-02-11 | GSE144996 | GEO

McFarland2020 - Totally Asymmetric Simple Exclusion Process (TASEP) model describing the movement of particles (ribosomes) along a lattice (the mRNA)

Project description:A stochastic simulation of yeast translation using Total Asymmetric Simple Exclusion Principle (TASEP) principles, representing ribosomes, tRNAs, mRNAs, and a tRNA re-charging process involving aminoacyl tRNA synthetases

2020-02-05 | MODEL2001080004 | BioModels

Expression data from Gdap1 knock-out (deletion of exon 5) mice

Project description:GDAP1 is a mitochondrial fission factor and mutations in GDAP1 cause Charcot-Marie-Tooth disease. Gdap1 knockout mice, mimicking genetic alterations of patients suffering from severe CMT forms, develop an age-related, hypomyelinating peripheral neuropathy. We used microarrays to determine changes in the expression profiles in the peripheral nervous system before a phenotype was detectable in the animal model (2 month of age).

2014-01-03 | GSE51650 | GEO

Regulation of Ex-Translational Activities Is Primary Function of Multi-tRNA Synthetase Complex

Project description:During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases (aaRS) and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has long been proposed to increase protein synthesis efficiency by passing charged tRNAs directly to ribosomes. An alternative is that the MSC repurposes specific synthetases for ex-translational functions that are activated by cues that direct specific enzymes to novel targets. To explore this question, we generated mammalian cell clones in which ArgRS and GlnRS were absent from the MSC to give a stable complex lacking the two enzymes (MSCΔRQ). Protein synthesis, under a variety of stress conditions, was unchanged in MSCΔRQ cells. Most strikingly, levels of charged tRNAGln and tRNAArg remained unchanged and no ribosome pausing was observed at codons for Arg and Gln. Thus, increasing or regulating protein synthesis efficiency is not dependent on ArgRS and GlnRS in the MSC. Alternatively, and consistent with previously reported ex-translational roles, we found manipulations that do not affect protein synthesis but instead MSC cellular localization.

2020-11-18 | GSE158212 | GEO

Histidine supplementation can escalate or rescue HARS deficiency in a Charcot Marie Tooth Disease model

Project description:Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging amino acids onto cognate tRNAs during protein synthesis¬. In histidyl-tRNA synthetase (HARS), autosomal dominant mutations in the HARS catalytic domain are associated with Charcot Marie Tooth Disease Type 2W (CMT2W), while anticodon-binding domain mutations cause Usher Syndrome Type IIIB (USH3B). We use yeast as a model system to study disease-causing HARS mutations (V133F, V155G, Y330C, S356N) associated with CMT2W, and Y454S, associated with USH3B. All human HARS variants complemented genomic deletion of the yeast ortholog HTS1 at high expression levels. CMT2W associated mutations, but not Y454S, result in reduced growth. HARS V155G and S356N cause accumulation of insoluble proteins and mistranslation in yeast, and the growth defect of these mutants was rescued by histidine addition to the growth media, restoring the soluble proteome. V133F and Y330C, on the other hand, lead to decreased HARS abundance. Histidine supplementation further reduced viability in yeast expressing V133F and Y330C, and lead to insoluble protein accumulation, indicating histidine toxicity associated with these mutants. Because histidine is in clinical trials as treatment for USH3B, our data will inform future treatment options for these as well as CMT patients, where histidine supplementation may either have a toxic or compensating effect dependingon the nature of the causative HARS variant.

2023-03-10 | PXD034865 | Pride

Methionyl-tRNA synthetase interactome

2020-12-23 | PXD021527 | Pride

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