The molecular aetiology of tRNA synthetase depletion: induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels.

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

doi:10.1093/nar/gkaa055

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.

Highlights

During gene expression, the translation of mRNA transcripts to produce a polypeptide chain involves the coordinated activities of an estimated repertoire of 200,000 ribosomes and 3 million tRNAs with associated translation factors, together representing a cellular production line that manufactures 70% of the dry weight of the cell in the form of protein (1)
We show that the primary response of the cell to restriction of tRNA synthetase activity is a simultaneous slow-down of translation and growth rate, which homeostatically limits the accumulation of uncharged tRNA species, and prevents the formation of ribosome queues on the mRNA
Previous research has shown that the S.cerevisiae translation system is robust to reductions in the abundance of tRNA synthetases, since for the majority of the 20 synthetases, heterozygous deletions are tolerated (57)

Summary

Introduction

The translation of mRNA transcripts to produce a polypeptide chain involves the coordinated activities of an estimated repertoire of 200,000 ribosomes and 3 million tRNAs with associated translation factors, together representing a cellular production line that manufactures 70% of the dry weight of the cell in the form of protein (1). The delivery of amino acids to the ribosome by aminoacyl tRNAs, bound to GTP-charged elongation factor 1 (eEF1A in eukaryotes) is central to the translation process (2). Following charging of the tRNA with amino acid, it can again bind to eEF1A for delivery to the ribosomal A site. TRNAs cycle between charged and uncharged states, driven by the respective activities of tRNA synthetase and translating ribosome populations. Balancing of the demand of the translating ribosomes for aminoacyl tRNAs, with the capacity of the tRNA synthetase population to supply them, is vital to ensure translation can proceed without pausing. A mismatch between charged tRNA supply, and translational demand can cause accumulation of uncharged tRNA, with consequent activation of the Gcn[2] kinase, triggering a GCN4 amino acid starvation response (4)

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Results

Conclusion