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

Mendonsa, Samantha; Kuegelgen, Nicolai von; Bujanic, Lucija; Chekulaeva, Marina

doi:10.1093/nar/gkab730

Cited by 21 publications

(29 citation statements)

References 47 publications

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“…The mutant forms of GlyRS and TyrRS bind their cognate tRNAs, but have a much slower off-rate, effectively resulting in the sequestration of the tRNAs and precluding their transfer to the ribosome to participate in translation. Consistent with tRNA sequestration being considered a toxic, gain-of-function activity, overexpression of mutant forms of GARS in HEK293 cells resulted in ribosome stalling at Glycine codons, as expected if the mutant enzymes were expressed at high enough levels to sequester tRNAs in cell types where the tRNAs are not typically limiting ( Mendonsa et al, 2021 ). In addition to these biochemical findings, this mechanism is also supported genetically for GARS .…”

Section: Cellular and Biochemical Mechanisms Suggest Therapeutic Strategiessupporting

confidence: 52%

An Integrated Approach to Studying Rare Neuromuscular Diseases Using Animal and Human Cell-Based Models

Hines

Lutz

Murray

et al. 2022

Front. Cell Dev. Biol.

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As sequencing technology improves, the identification of new disease-associated genes and new alleles of known genes is rapidly increasing our understanding of the genetic underpinnings of rare diseases, including neuromuscular diseases. However, precisely because these disorders are rare and often heterogeneous, they are difficult to study in patient populations. In parallel, our ability to engineer the genomes of model organisms, such as mice or rats, has gotten increasingly efficient through techniques such as CRISPR/Cas9 genome editing, allowing the creation of precision human disease models. Such in vivo model systems provide an efficient means for exploring disease mechanisms and identifying therapeutic strategies. Furthermore, animal models provide a platform for preclinical studies to test the efficacy of those strategies. Determining whether the same mechanisms are involved in the human disease and confirming relevant parameters for treatment ideally involves a human experimental system. One system currently being used is induced pluripotent stem cells (iPSCs), which can then be differentiated into the relevant cell type(s) for in vitro confirmation of disease mechanisms and variables such as target engagement. Here we provide a demonstration of these approaches using the example of tRNA-synthetase-associated inherited peripheral neuropathies, rare forms of Charcot-Marie-Tooth disease (CMT). Mouse models have led to a better understanding of both the genetic and cellular mechanisms underlying the disease. To determine if the mechanisms are similar in human cells, we will use genetically engineered iPSC-based models. This will allow comparisons of different CMT-associated GARS alleles in the same genetic background, reducing the variability found between patient samples and simplifying the availability of cell-based models for a rare disease. The necessity of integrating mouse and human models, strategies for accomplishing this integration, and the challenges of doing it at scale are discussed using recently published work detailing the cellular mechanisms underlying GARS-associated CMT as a framework.

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Section: Cellular and Biochemical Mechanisms Suggest Therapeutic Strategiessupporting

confidence: 52%

An Integrated Approach to Studying Rare Neuromuscular Diseases Using Animal and Human Cell-Based Models

Hines

Lutz

Murray

et al. 2022

Front. Cell Dev. Biol.

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“…3xflag-SARS-CoV-2 NSP1-encoding plasmid and pEBG-3xflag, used as a vector, have been described previously ( Mendonsa et al 2021 ). Analogous plasmid expressing SARS-CoV-1 NSP1 was generated using a similar strategy: CDS of NSP1 was PCR amplified, using SARS-CoV-1 cDNA as a template, and cloned between the SbfI and NotI sites of pEBG-3xflag.…”

Section: Methodsmentioning

confidence: 99%

The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression

Bujanic

Shevchuk

Kügelgen

et al. 2022

RNA

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SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem-loop (SL1) in viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19 and 20) and another within NSP1 (R124) which are required for viral evasion, and thus might present promising drug targets. We target SL1 with the anti-sense oligo (ASO) to efficiently and specifically downregulate SARS-CoV-2 mRNA. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of anti-viral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing.

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“…A similar result was observed in a mouse model of Charcot-Marie-Tooth disease where loss of GTPBP2 aggravated neuropathy phenotypes and increased ribosome stalling. [122,123] In addition, loss of PELO or HBS1L generally results in severe defects in development of the cerebellum in mice, pointing to the importance of the rescue of stalled ribosomes in neuronal development. [119] Stalling of ribosomes due to poor codon optimality, inherently stallinducing amino acids (often containing proline or polybasic repeats), mRNA secondary structure, or damaged mRNAs results in ribosome collisions made up of disomes and trisomes.…”

Section: The Mechanism Of Ribosome Recycling During Ribosome Rescuementioning

confidence: 99%

Rebirth of the translational machinery: The importance of recycling ribosomes

Young

Guydosh

2022

BioEssays

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Translation of the genetic code occurs in a cycle where ribosomes engage mRNAs, synthesize protein, and then disengage in order to repeat the process again. The final part of this process-ribosome recycling, where ribosomes dissociate from mRNAsinvolves a complex molecular choreography of specific protein factors to remove the large and small subunits of the ribosome in a coordinated fashion. Errors in this process can lead to the accumulation of ribosomes at stop codons or translation of downstream open reading frames (ORFs). Ribosome recycling is also critical when a ribosome stalls during the elongation phase of translation and must be rescued to allow continued translation of the mRNA. Here we discuss the molecular interactions that drive ribosome recycling, and their regulation in the cell. We also examine the consequences of inefficient recycling with regards to disease, and its functional roles in synthesis of novel peptides, regulation of gene expression, and control of mRNA-associated proteins. Alterations in ribosome recycling efficiency have the potential to impact many cellular functions but additional work is needed to understand how this regulatory power is utilized.

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

Cited by 21 publications

References 47 publications

An Integrated Approach to Studying Rare Neuromuscular Diseases Using Animal and Human Cell-Based Models

An Integrated Approach to Studying Rare Neuromuscular Diseases Using Animal and Human Cell-Based Models

The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression

Rebirth of the translational machinery: The importance of recycling ribosomes

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