Abstract
| Original language | English |
|---|---|
| Journal | Nucleic Acids Research |
| DOIs | |
| Publication status | Accepted/In press - 16 Jan 2020 |
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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.; Adeniyi, Stephen A.; Corrigall, Holly; Raguin, Adélaïde; Romano, M. Carmen; Stansfield, Ian.
In: Nucleic Acids Research, 16.01.2020.Research output: Contribution to journal › Article
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TY - JOUR
T1 - The molecular aetiology of tRNA synthetase depletion
T2 - induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels
AU - McFarland, Matthew R.
AU - Keller, Corina D.
AU - Childers, Brandon M.
AU - Adeniyi, Stephen A.
AU - Corrigall, Holly
AU - Raguin, Adélaïde
AU - Romano, M. Carmen
AU - Stansfield, Ian
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. The Global Translation Model is deposited in the Biomodels database (76) with reference MODEL2001080004. MMcF carried out strain construction and characterisation, 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. SA carried out the ribosome quantification experiments. 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].
PY - 2020/1/16
Y1 - 2020/1/16
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
U2 - 10.1101/610790
DO - 10.1101/610790
M3 - Article
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
ER -