The molecular aetiology of tRNA synthetase depletion

induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels

Matthew R. McFarland, Corina D. Keller, Brandon M. Childers, Holly Corrigall, Adélaïde Raguin, M. Carmen Romano, Ian Stansfield

Research output: Working paper

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Abstract

During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.
Original languageEnglish
PublisherbioRxiv
DOIs
Publication statusPublished - 17 Apr 2019

Fingerprint

RNA, Transfer, Gln
Amino Acyl-tRNA Synthetases
Starvation
Transfer RNA
Maintenance
Amino Acids
Amino Acid-Specific Transfer RNA
Phosphotransferases
Transfer RNA Aminoacylation
Doxycycline
Nervous System Diseases
Glutamine
Ribosomes
Northern Blotting
Yeasts
Growth
Population
Genes
Proteins

Keywords

  • translation
  • tRNA synthetase
  • Saccharomyces cerevisiae
  • GCN4
  • Totally Asymmetric Simple Exclusion Process

Cite this

The molecular aetiology of tRNA synthetase depletion : induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels. / McFarland, Matthew R.; Keller, Corina D.; Childers, Brandon M.; Corrigall, Holly; Raguin, Adélaïde; Romano, M. Carmen; Stansfield, Ian.

bioRxiv, 2019.

Research output: Working paper

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abstract = "During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.",
keywords = "translation, tRNA synthetase, Saccharomyces cerevisiae, GCN4, Totally Asymmetric Simple Exclusion Process",
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note = "ACKNOWLEDGEMENTS The authors gratefully acknowledge the RNA sequencing and mass spectrometry support provided by the University of Aberdeen’s Centre for Genome Enabled Biology and Medicine, and Proteomics respectively. MMcF carried out strain construction and characterisation, flow cytometry, GCN4 assays, RNA sequencing and mass spectrometry data analysis. IS conducted GCN4 assays. HC carried out vector and strain construction and tRNA charging assays. Polysome profiling analysis was carried out by BC. AR and MCR coded the original global translation model. CK and MCR extended the global translation model, CK coded and analysed the synthetase sequestration model, carried out the global translation model simulations and analysis. MCR and IS conceived the study and guided the research. IS, MCR, CK and MMcF co-wrote the manuscript. FUNDING This work was supported by the Biotechnology and Biological Sciences Research Council [BBSRC grant numbers BB/I020926/1 to IS and BB/N017161/1 to IS and MCR], and BBSRC PhD studentship awards to IS and MCR [M108703G and C103817D].",
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N1 - ACKNOWLEDGEMENTS The authors gratefully acknowledge the RNA sequencing and mass spectrometry support provided by the University of Aberdeen’s Centre for Genome Enabled Biology and Medicine, and Proteomics respectively. MMcF carried out strain construction and characterisation, flow cytometry, GCN4 assays, RNA sequencing and mass spectrometry data analysis. IS conducted GCN4 assays. HC carried out vector and strain construction and tRNA charging assays. Polysome profiling analysis was carried out by BC. AR and MCR coded the original global translation model. CK and MCR extended the global translation model, CK coded and analysed the synthetase sequestration model, carried out the global translation model simulations and analysis. MCR and IS conceived the study and guided the research. IS, MCR, CK and MMcF co-wrote the manuscript. FUNDING This work was supported by the Biotechnology and Biological Sciences Research Council [BBSRC grant numbers BB/I020926/1 to IS and BB/N017161/1 to IS and MCR], and BBSRC PhD studentship awards to IS and MCR [M108703G and C103817D].

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N2 - During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.

AB - During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.

KW - translation

KW - tRNA synthetase

KW - Saccharomyces cerevisiae

KW - GCN4

KW - Totally Asymmetric Simple Exclusion Process

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