Upstream Open Reading Frames Differentially Regulate Gene-specific Translation in the Integrated Stress Response

Abstract

Translation regulation largely occurs during initiation, which features ribosome assembly onto mRNAs and selection of the translation start site. Short, upstream ORFs (uORFs) located in the 5'-leader of the mRNA can be selected for translation. Multiple transcripts associated with stress amelioration are preferentially translated through uORF-mediated mechanisms during activation of the integrated stress response (ISR) in which phosphorylation of the α subunit of eIF2 results in a coincident global reduction in translation initiation. This review presents key features of uORFs that serve to optimize translational control that is essential for regulation of cell fate in response to environmental stresses.

Keywords: eukaryotic translation initiation; stress response; translation; translation control; translation regulation.

FIGURE 1.

uORFs regulate downstream CDS translation. uORFs can have multiple core properties, including promoting ribosome reinitiation after uORF translation, ribosome elongation stalling while translating the uORF, ribosome dissociation from the mRNA, ribosome translation of uORFs past the CDS start codon, or ribosome bypass of the uORF. CDSs are indicated by the blue bar; positive-acting uORFs are indicated by a green bar; negative-acting uORFs are indicated by a red bar; and uORFs that have no effect on downstream translation are indicated by a yellow bar. Scanning and elongating ribosomes are illustrated by the gray ovals.

FIGURE 2.

The integrated stress response features a global reduction in translation initiation concomitant with the preferential translation of stress remediation transcripts. A, depiction of polysome profiles as measured by sucrose gradient analyses of lysates prepared from mouse embryonic fibroblast cells that were left untreated (black line) or subjected to the ER stress inducer thapsigargin (red line). Basal polysome profiles feature distinctive peaks for the 40S and 60S ribosomal subunits and the 80S monosome, with large peaks observed for heavy polysomes, indicative of high levels of global translation. Polysome profiles from cells subjected to ER stress feature decreased heavy polysomes and an elevated 80S monosome peak that is indicative of inhibition of global translation initiation during eIF2α-P. Those mRNAs that are preferentially translated during cellular stress are largely associated with heavy polysomes, whereas those mRNAs that are repressed during cellular stress are largely associated with 80S monosomes and light polysomes. The mRNAs that are translated constitutively are associated with polysomes independent of stress. B, depiction of the preferentially translated mRNAs and their function in stress remediation. Multiple preferentially translated mRNAs encode transcription factors that promote stress alleviation (ATF4, C/EBPα, and C/EBPβ). If the cellular stress is too great to overcome, a subset of transcription factors promotes a pro-apoptotic signaling cascade (CHOP and ATF5). Feedback dephosphorylation occurs through the activity of the preferentially translated GADD34. Priming of the cell for resumption of global translation occurs through the activity of the preferentially translated nutrient transporters SLC35A4 and CAT1, as well as the glutamyl-prolyl tRNA synthetase EPRS. Cell fate regulator IBTKα is also preferentially translated through an uORF-mediated mechanism.

FIGURE 3.

uORFs regulate mRNA translation through diverse mechanisms. A, depiction of the ATF4 mechanism of preferential translation. During nonstressed conditions, there are low levels of eIF2α-P and high levels of eIF2-GTP. Ribosomes scanning the ATF4 mRNA initiate at the 5′-proximal uORF1, and following termination, quickly reacquire a new eIF2 ternary complex. Competent 40S scanning ribosomes (dark gray oval) then reinitiate translation at uORF2, which overlaps out-of-frame with the ATF4 CDS. Translation of uORF2 results in ribosome termination and dissociation 3′ of the ATF4 initiation codon, resulting in low ATF4 expression. During cellular stress, elevated eIF2α-P results in low levels of eIF2-GTP. Ribosomes scanning the ATF4 mRNA initiate at uORF1 and post-uORF translation resume scanning. Due to the low levels of eIF2 ternary complex, the 40S ribosome (light gray oval) scans pass the initiation codon of the inhibitory uORF2 before reacquiring a new ternary complex (dark gray oval). Delayed acquisition of the eIF2 ternary complex results in translation initiation at the ATF4 CDS and an increase in ATF4 expression during cellular stress. B, depiction of the GADD34 mechanism of preferential translation. During nonstressed conditions, scanning ribosomes bypass the GADD34 uORF1 due to its poor start codon context and initiate translation at uORF2. Translation of a Pro-Pro-Gly peptide sequence juxtaposed to the uORF2 stop codon results in an inefficient ribosome termination event that increases ribosome release from the mRNA and causes low levels of basal GADD34 expression. During cellular stress, elevated eIF2α-P results in a ribosomal bypass of uORF1 due to its poor start codon context and uORF2 due to its moderate Kozak consensus sequence. Bypass of the inhibitory uORF2 by a portion of scanning ribosomes results in increased translation initiation at the GADD34 CDS and an increase in GADD34 expression.

FIGURE 4.

uORF mechanisms of translation control are evolutionarily conserved. A, illustration of the IBTKα 5′-leader in multiple species including: H. sapiens, Callithrix jacchus, and Sus scrofa. Translation of IBTKα mRNA is regulated by a bypass mechanism. The inhibitory uORFs 1 and 2 (red bars) repress IBTKα CDS translation during nonstressed conditions. The inhibitory effects of uORF1 and 2 are overcome during eIF2α-P, facilitating the preferential translation of IBTKα (blue bar). uORF 3 and 4 (yellow bars) are considered to be dispensable for IBTKα translation control and are not conserved between species. B, depiction of the 5′-leaders for D. melanogaster and M. musculus GADD34 mRNAs. The 5′-leader of GADD34 mRNA in both species contains a dispensable uORF1 (yellow bar) that is largely bypassed independent of cellular stress. uORF2 (red bar) in both mRNAs is translated during basal conditions and is inhibitory to downstream GADD34 CDS translation. uORF2 in D. melanogaster overlaps out-of-frame with the GADD34 CDS (blue bar) and promotes ribosome dissociation from the mRNA 3′ of the initiation codon of the GADD34 CDS. The M. musculus uORF contains an inhibitory Pro-Pro-Gly sequence juxtaposed to the uORF2 termination codon that promotes inefficient termination that increases ribosome dissociation from the mRNA. During cellular stress, the inhibitory uORF2 in either D. melanogaster or M. musculus is bypassed, resulting in increased translation initiation at the GADD34 CDS and increased GADD34 protein synthesis. Bypass of M. musculus uORF2 relies upon its moderate start codon context, whereas bypass of D. melanogaster uORF2 may rely upon additional factors. C, illustration of the 5′-leader of GCN4 in fungal species S. cerevisiae and C. albicans. Translation control of S. cerevisiae GCN4 relies on a delayed translation reinitiation model in which translation of the positive acting uORF1 (green bar) promotes translation reinitiation at downstream uORFs. Translation of the following uORFs 2, 3, and 4 (red bars) in the S. cerevisiae GCN4 5′-leader are inhibitory to downstream translation by promoting ribosome dissociation from the mRNA in nonstressed conditions. During cellular stress, low levels of eIF2 ternary complex levels allow the scanning 40S ribosome to scan through the inhibitory uORFs in GCN4 post-uORF1 translation, resulting in translation initiation at the GCN4 CDS (blue bar). C. albicans translation control relies on a bypass mechanism in which only uORF3 (red bar) is required for regulation of GCN4 expression. In nonstressed conditions, translation of uORF3 precludes the ribosome from initiating translation at the C. albicans GCN4 CDS, presumably through ribosome dissociation from the mRNA. During cellular stress, eIF2α-P promotes bypass of the inhibitory uORF3, thereby facilitating an increase in translation of the GCN4 coding region (blue bar).