The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells.

Heather P Harding,David Ron,Adriana Ordonez,Alison J Inglis,Leopold Parts,Roger L Williams,Felicity Allen

doi:10.7554/elife.50149

Abstract

The eukaryotic translation initiation factor 2α (eIF2α) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2α kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2α phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation.

Highlights

Phosphorylation of translation initiation factor 2 on serine 51 of its alpha subunit is a potent mechanism for translational regulation in eukaryotes
The CHOP::GFP reporter remained responsive to the glycosylation inhibitor tunicamycin, a toxin that activates the Intea grated Stress Response (ISR) orthogonally, through an ER stress inducible eukaryotic translation initiation factor 2a (eIF2a) kinase, PERK (Figure 1A and B). (Harding et al, 1999; Harding et al, 2000)
The reporter cell lines were deemed suitable tools to search for additional components that may contribute to General Control Non-depressible 2 (GCN2)-dependent ISR activation

Summary

Introduction

Phosphorylation of translation initiation factor 2 on serine 51 of its alpha subunit (eIF2a) is a potent mechanism for translational regulation in eukaryotes. The consequences to rates of translation initiation are mRNA-specific. By way of this direct effect on protein synthesis and its indirect consequences to the abundance of downstream effector proteins, levels of eIF2a phosphorylation modulate gene expression translationally and transcriptionally (Hinnebusch, 2014). In animals four different kinases couple unrelated stress signals to eIF2 phosphorylation (eIF2 P). A a eIF2 P effectively integrates these into a stereotypical downstream response referred to as the Intea grated Stress Response (ISR) (Harding et al, 2003). The four eIF2a kinases, GCN2, PERK, PKR and HRI, share a similar kinase effector domain, but diverge in the molecular mechanisms and nature of the upstream signals that regulate their kinase activities

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