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

  • Sara K. Young
    Affiliations
    Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5126
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  • Ronald C. Wek
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 635 Barnhill Dr., Indiana University School of Medicine, Indianapolis, Indiana 46202-5126; Tel.: 317-274-0549; Fax: 317-274-4686
    Affiliations
    Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5126
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant GM049164 (to R. C. W.) and the Ralph W. and Grace M. Showalter Research Trust Fund. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Open AccessPublished:June 29, 2016DOI:https://doi.org/10.1074/jbc.R116.733899
      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.

      Introduction

      Multiple genome-wide analyses, including those utilizing ribosome and polysome profiling and mass spectrometry approaches, have provided evidence demonstrating that translation is a major regulator of gene expression (
      • Schwanhäusser B.
      • Gossen M.
      • Dittmar G.
      • Selbach M.
      Global analysis of cellular protein translation by pulsed SILAC.
      ,
      • Aviner R.
      • Geiger T.
      • Elroy-Stein O.
      Novel proteomic approach (PUNCH-P) reveals cell cycle-specific fluctuations in mRNA translation.
      ,
      • Ingolia N.T.
      • Ghaemmaghami S.
      • Newman J.R.
      • Weissman J.S.
      Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.
      ,
      • Gao X.
      • Wan J.
      • Liu B.
      • Ma M.
      • Shen B.
      • Qian S.B.
      Quantitative profiling of initiating ribosomes in vivo.
      ,
      • Baird T.D.
      • Palam L.R.
      • Fusakio M.E.
      • Willy J.A.
      • Davis C.M.
      • McClintick J.N.
      • Anthony T.G.
      • Wek R.C.
      Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα.
      ). In addition to protein coding sequences (CDSs),
      The abbreviations used are: CDS
      coding sequence
      uORF
      upstream open reading frame
      ISR
      integrated stress response
      LAP
      liver-enriched activating protein
      LIP
      liver-enriched inhibitory protein
      IRES
      internal ribosome entry sequence
      ER
      endoplasmic reticulum.
      another class of ORFs suggested to be translated at high frequency consists of short, upstream ORFs (uORFs) that are located within the 5′-leader of mRNAs (
      • Ingolia N.T.
      • Ghaemmaghami S.
      • Newman J.R.
      • Weissman J.S.
      Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.
      ,
      • Gao X.
      • Wan J.
      • Liu B.
      • Ma M.
      • Shen B.
      • Qian S.B.
      Quantitative profiling of initiating ribosomes in vivo.
      ,
      • Baird T.D.
      • Palam L.R.
      • Fusakio M.E.
      • Willy J.A.
      • Davis C.M.
      • McClintick J.N.
      • Anthony T.G.
      • Wek R.C.
      Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα.
      ,
      • Fritsch C.
      • Herrmann A.
      • Nothnagel M.
      • Szafranski K.
      • Huse K.
      • Schumann F.
      • Schreiber S.
      • Platzer M.
      • Krawczak M.
      • Hampe J.
      • Brosch M.
      Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting.
      ). Over 40% of mammalian mRNAs contain uORFs, illustrating that uORFs are prevalent genome-wide and can serve as major regulators of translation (
      • Baird T.D.
      • Palam L.R.
      • Fusakio M.E.
      • Willy J.A.
      • Davis C.M.
      • McClintick J.N.
      • Anthony T.G.
      • Wek R.C.
      Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα.
      ,
      • Iacono M.
      • Mignone F.
      • Pesole G.
      uAUG and uORFs in human and rodent 5′untranslated mRNAs.
      ,
      • Calvo S.E.
      • Pagliarini D.J.
      • Mootha V.K.
      Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans.
      ). Approximation of uORF prevalence has relied upon the use of an AUG to denote the uORF start codon; however, recent ribosome profiling studies indicate that non-canonical initiation codons (e.g. CUG, UUG, and GUG) can also serve as competent sites of translation initiation (
      • Ingolia N.T.
      • Ghaemmaghami S.
      • Newman J.R.
      • Weissman J.S.
      Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.
      ,
      • Gao X.
      • Wan J.
      • Liu B.
      • Ma M.
      • Shen B.
      • Qian S.B.
      Quantitative profiling of initiating ribosomes in vivo.
      ,
      • Fritsch C.
      • Herrmann A.
      • Nothnagel M.
      • Szafranski K.
      • Huse K.
      • Schumann F.
      • Schreiber S.
      • Platzer M.
      • Krawczak M.
      • Hampe J.
      • Brosch M.
      Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting.
      ). These findings suggest that the magnitude of uORF prevalence and the contribution of uORF translation in the regulation of gene expression have likely been underestimated.
      Typically, uORFs are considered to be inhibitors of downstream translation initiation at CDSs. The inhibitory effect of uORFs is attributed to the fact that in eukaryotes the 43S preinitiation complex binds to the 5′-cap structure of the mRNA, then scans processively 5′ to 3′ and initiates translation at the first encountered initiation codon that is in an optimal context (
      • Kozak M.
      The scanning model for translation: an update.
      ). The 43S preinitiation complex is composed of multiple factors including eIF3, eIF1, eIF1A, the eIF2·GTP·Met-tRNAiMet ternary complex, and the small 40S ribosomal subunit (
      • Jackson R.J.
      • Hellen C.U.
      • Pestova T.V.
      The mechanism of eukaryotic translation initiation and principles of its regulation.
      ). Disassociation of the eIF2 ternary complex and other critical initiation factors during translation of constitutively repressing uORFs is suggested to be the cause of the low levels of subsequent translation reinitiation at downstream coding sequences.
      Although uORFs can serve as repressors of CDS translation in a constitutive manner, there are also examples of uORFs that serve as dampeners in a controlled fashion or even promote translation initiation at the CDS in response to environmental stresses (
      • Gao X.
      • Wan J.
      • Liu B.
      • Ma M.
      • Shen B.
      • Qian S.B.
      Quantitative profiling of initiating ribosomes in vivo.
      ,
      • Baird T.D.
      • Palam L.R.
      • Fusakio M.E.
      • Willy J.A.
      • Davis C.M.
      • McClintick J.N.
      • Anthony T.G.
      • Wek R.C.
      Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα.
      ,
      • Andreev D.E.
      • O'Connor P.B.
      • Fahey C.
      • Kenny E.M.
      • Terenin I.M.
      • Dmitriev S.E.
      • Cormican P.
      • Morris D.W.
      • Shatsky I.N.
      • Baranov P.V.
      Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression.
      ). Based on these studies, uORFs can have the following core properties that are critical for translational control (Fig. 1). 1) They enhance reinitiation after uORF translation, allowing for degrees of translation initiation at the downstream CDS. For example, translation of short uORFs can allow for retention of critical initiation factors, such as eIF3, which facilitate efficient translation reinitiation (
      • Kozak M.
      Constraints on reinitiation of translation in mammals.
      ,
      • Szamecz B.
      • Rutkai E.
      • Cuchalová L.
      • Munzarová V.
      • Herrmannová A.
      • Nielsen K.H.
      • Burela L.
      • Hinnebusch A.G.
      • Valásek L.
      eIF3a cooperates with sequences 5′ of uORF1 to promote resumption of scanning by post-termination ribosomes for reinitiation on GCN4 mRNA.
      ). 2) They demonstrate direct ribosome elongation stalling during translation of the uORF and, as a consequence, thwart translation at the downstream CDS. Pauses in elongation can be reliant upon RNA secondary structure, codon usage bias, or polypeptide sequences encoded in the uORF, as well as interaction of trans-acting factors (
      • Kozak M.
      Constraints on reinitiation of translation in mammals.
      ,
      • Young S.K.
      • Palam L.R.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome elongation stall directs gene-specific translation in the integrated stress response.
      ,
      • Law G.L.
      • Raney A.
      • Heusner C.
      • Morris D.R.
      Polyamine regulation of ribosome pausing at the upstream open reading frame of S-adenosylmethionine decarboxylase.
      ,
      • Col B.
      • Oltean S.
      • Banerjee R.
      Translational regulation of human methionine synthase by upstream open reading frames.
      ,
      • Wei J.
      • Wu C.
      • Sachs M.S.
      The arginine attenuator peptide interferes with the ribosome peptidyl transferase center.
      ). 3) They promote ribosome dissociation from the mRNA and therefore diminish subsequent CDS translation. Ribosome dissociation from the mRNA post-uORF translation can also be regulated by nucleotide sequences or the polypeptide encoded in the uORF (
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ,
      • Hinnebusch A.G.
      Translational regulation of GCN4 and the general amino acid control of yeast.
      ,
      • Grant C.M.
      • Hinnebusch A.G.
      Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control.
      ,
      • Abastado J.P.
      • Miller P.F.
      • Jackson B.M.
      • Hinnebusch A.G.
      Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis of GCN4 translational control.
      ,
      • Pöyry T.A.
      • Kaminski A.
      • Connell E.J.
      • Fraser C.S.
      • Jackson R.J.
      The mechanism of an exceptional case of reinitiation after translation of a long ORF reveals why such events do not generally occur in mammalian mRNA translation.
      ,
      • Zinoviev A.
      • Hellen C.U.
      • Pestova T.V.
      Multiple mechanisms of reinitiation on bicistronic calicivirus mRNAs.
      ). 4) They position uORFs out-of-frame with CDSs, resulting in ribosome termination downstream of CDS start codons. Translating ribosomes are suggested to only “back up” in a 3′ to 5′ fashion for a small number of nucleotides, resulting in low translation of the CDS (
      • Vattem K.M.
      • Wek R.C.
      Reinitiation involving upstream open reading frames regulates ATF4 mRNA translation in mammalian cells.
      ,
      • Sundaram A.
      • Grant C.M.
      A single inhibitory upstream open reading frame (uORF) is sufficient to regulate Candida albicans GCN4 translation in response to amino acid starvation conditions.
      ,
      • Young S.K.
      • Baird T.D.
      • Wek R.C.
      Translation regulation of the glutamyl-prolyl-tRNA synthetase gene EPRS through bypass of upstream open reading frames with noncanonical initiation codons.
      ). 5) They allow for scanning ribosomes to bypass the uORF either in a largely constitutive fashion or upon induction of physiological signals. Ribosome bypass is thought to occur at least in part due to the nucleotide sequences flanking the initiation codon (
      • Kozak M.
      The scanning model for translation: an update.
      ,
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ,
      • Palam L.R.
      • Baird T.D.
      • Wek R.C.
      Phosphorylation of eIF2 facilitates ribosomal bypass of an inhibitory upstream ORF to enhance CHOP translation.
      ,
      • Calkhoven C.F.
      • Bouwman P.R.
      • Snippe L.
      • Ab G.
      Translation start site multiplicity of the CCAAT/enhancer binding protein α mRNA is dictated by a small 5′ open reading frame.
      ). These uORF properties, which can be integrated individually or in combination within mRNAs, help determine the specific mechanism of translational control of a given gene (Fig. 1). This review highlights the mechanisms by which uORFs can modulate translation at CDSs, and the processes by which uORFs with these diverse properties can be integrated individually or in combination into mRNAs to facilitate differential regulation of translation.
      Figure thumbnail gr1
      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.

      Translation Regulation during the Integrated Stress Response

      Phosphorylation of eIF2 on its α subunit at serine 51 (eIF2α-P) inhibits the activity of the guanine nucleotide exchange factor eIF2B that results in a decrease in the exchange of GDP for GTP, thus lowering formation of the 43S preinitiation complex that triggers a global reduction in translation initiation (Fig. 2A) (
      • Rowlands A.G.
      • Panniers R.
      • Henshaw E.C.
      The catalytic mechanism of guanine nucleotide exchange factor action and competitive inhibition by phosphorylated eukaryotic initiation factor 2.
      ,
      • Jennings M.D.
      • Pavitt G.D.
      A new function and complexity for protein translation initiation factor eIF2B.
      ,
      • Jennings M.D.
      • Zhou Y.
      • Mohammad-Qureshi S.S.
      • Bennett D.
      • Pavitt G.D.
      eIF2B promotes eIF5 dissociation from eIF2·GDP to facilitate guanine nucleotide exchange for translation initiation.
      ). Because eIF2α-P can direct translational control in response to a range of different environmental stresses, this pathway is often referred to as the integrated stress response (ISR) (
      • Harding H.P.
      • Zhang Y.
      • Zeng H.
      • Novoa I.
      • Lu P.D.
      • Calfon M.
      • Sadri N.
      • Yun C.
      • Popko B.
      • Paules R.
      • Stojdl D.F.
      • Bell J.C.
      • Hettmann T.
      • Leiden J.M.
      • Ron D.
      An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.
      ). Lowered protein synthesis would allow cells to conserve nutrients and energy and facilitate reprogramming of gene expression to alleviate stress damage. To aid in reprogramming of gene expression during eIF2α-P, a subset of mRNAs is preferentially translated via uORF-mediated mechanisms. uORFs are similarly distributed among mRNAs that are repressed, resistant, or preferentially translated during cellular stress and eIF2α-P, emphasizing that it is the specific properties of the uORFs that are critical for their regulatory capabilities in translation (Fig. 2A) (
      • Baird T.D.
      • Palam L.R.
      • Fusakio M.E.
      • Willy J.A.
      • Davis C.M.
      • McClintick J.N.
      • Anthony T.G.
      • Wek R.C.
      Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα.
      ). Furthermore, the proper mixing and matching of these uORF features are critical for uORF-mediated translational control mechanisms that appropriately regulate gene expression.
      Figure thumbnail gr2
      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.
      Encoded CDS products of mRNAs that are preferentially translated through uORF-mediated mechanisms play diverse roles in remediation of cellular stress (Fig. 2B). Included among the ISR preferentially translated gene transcripts are ATF4 (CREB2), CHOP (DDIT3/GADD153), ATF5, and C/EBPα and C/EBPβ that each encode basic leucine zipper transcription factors that modify gene expression programs to address cellular stress (
      • Vattem K.M.
      • Wek R.C.
      Reinitiation involving upstream open reading frames regulates ATF4 mRNA translation in mammalian cells.
      ,
      • Palam L.R.
      • Baird T.D.
      • Wek R.C.
      Phosphorylation of eIF2 facilitates ribosomal bypass of an inhibitory upstream ORF to enhance CHOP translation.
      ,
      • Harding H.P.
      • Novoa I.
      • Zhang Y.
      • Zeng H.
      • Wek R.
      • Schapira M.
      • Ron D.
      Regulated translation initiation controls stress-induced gene expression in mammalian cells.
      ,
      • Lu P.D.
      • Harding H.P.
      • Ron D.
      Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response.
      ,
      • Jousse C.
      • Bruhat A.
      • Carraro V.
      • Urano F.
      • Ferrara M.
      • Ron D.
      • Fafournoux P.
      Inhibition of CHOP translation by a peptide encoded by an open reading frame localized in the chop 5′UTR.
      ,
      • Zhou D.
      • Palam L.R.
      • Jiang L.
      • Narasimhan J.
      • Staschke K.A.
      • Wek R.C.
      Phosphorylation of eIF2 directs ATF5 translational control in response to diverse stress conditions.
      ,
      • Calkhoven C.F.
      • Müller C.
      • Leutz A.
      Translational control of C/EBPα and C/EBPβ isoform expression.
      ). GADD34 (PPP1R15A) combines with the catalytic subunit of protein phosphatase 1 (PP1c) to regulate dephosphorylation of eIF2α-P and restore protein synthesis (Fig. 2B) (
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ,
      • Lee Y.Y.
      • Cevallos R.C.
      • Jan E.
      An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2α phosphorylation.
      ,
      • Harding H.P.
      • Zhang Y.
      • Scheuner D.
      • Chen J.J.
      • Kaufman R.J.
      • Ron D.
      Ppp1r15 gene knockout reveals an essential role for translation initiation factor 2 α (eIF2α) dephosphorylation in mammalian development.
      ,
      • Novoa I.
      • Zeng H.
      • Harding H.P.
      • Ron D.
      Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2α.
      ,
      • Connor J.H.
      • Weiser D.C.
      • Li S.
      • Hallenbeck J.M.
      • Shenolikar S.
      Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1.
      ). Nutrient transporters SLC35A4 and CAT1, as well as the bifunctional glutamyl-prolyl tRNA synthetase EPRS, serve to increase available nutrients and prime the cell for resumption of protein synthesis once cellular stress is remediated (Fig. 2B) (
      • Andreev D.E.
      • O'Connor P.B.
      • Fahey C.
      • Kenny E.M.
      • Terenin I.M.
      • Dmitriev S.E.
      • Cormican P.
      • Morris D.W.
      • Shatsky I.N.
      • Baranov P.V.
      Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression.
      ,
      • Young S.K.
      • Baird T.D.
      • Wek R.C.
      Translation regulation of the glutamyl-prolyl-tRNA synthetase gene EPRS through bypass of upstream open reading frames with noncanonical initiation codons.
      ). Finally, the cell fate regulator IBTKα was shown to be subject to preferential translation through a mechanism involving uORFs (Fig. 2B) (
      • Baird T.D.
      • Palam L.R.
      • Fusakio M.E.
      • Willy J.A.
      • Davis C.M.
      • McClintick J.N.
      • Anthony T.G.
      • Wek R.C.
      Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα.
      ).

      Ribosome Reinitiation in the ISR

      The capacity for the ribosome to reinitiate translation downstream has been attributed largely to the ability of the scanning ribosome to retain or reacquire critical initiation factors following uORF translation (
      • Kozak M.
      Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes.
      ). Extended distance between the uORF stop codon and CDS initiation codon allows more time for the scanning 40S to reacquire a new eIF2·GTP·Met-tRNAiMetcomplex, a significant feature of the delayed translation reinitiation model that was originally identified in the yeast Saccharomyces cerevisiae for the transcriptional activator GCN4 (
      • Hinnebusch A.G.
      Translational regulation of GCN4 and the general amino acid control of yeast.
      ,
      • Abastado J.P.
      • Miller P.F.
      • Jackson B.M.
      • Hinnebusch A.G.
      Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis of GCN4 translational control.
      ,
      • Mueller P.P.
      • Hinnebusch A.G.
      Multiple upstream AUG codons mediate translational control of GCN4.
      ) and subsequently suggested for the related mammalian ATF4 (
      • Vattem K.M.
      • Wek R.C.
      Reinitiation involving upstream open reading frames regulates ATF4 mRNA translation in mammalian cells.
      ,
      • Harding H.P.
      • Novoa I.
      • Zhang Y.
      • Zeng H.
      • Wek R.
      • Schapira M.
      • Ron D.
      Regulated translation initiation controls stress-induced gene expression in mammalian cells.
      ,
      • Lu P.D.
      • Harding H.P.
      • Ron D.
      Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response.
      ).

      Delayed Translation Reinitiation

      Both yeast GCN4 and mammalian ATF4 encode transcription factors that increase expression of genes involved in nutrient import, metabolism, and alleviation of oxidative stress (
      • Harding H.P.
      • Zhang Y.
      • Zeng H.
      • Novoa I.
      • Lu P.D.
      • Calfon M.
      • Sadri N.
      • Yun C.
      • Popko B.
      • Paules R.
      • Stojdl D.F.
      • Bell J.C.
      • Hettmann T.
      • Leiden J.M.
      • Ron D.
      An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.
      ,
      • Harding H.P.
      • Novoa I.
      • Zhang Y.
      • Zeng H.
      • Wek R.
      • Schapira M.
      • Ron D.
      Regulated translation initiation controls stress-induced gene expression in mammalian cells.
      ,
      • Jiang H.Y.
      • Wek S.A.
      • McGrath B.C.
      • Lu D.
      • Hai T.
      • Harding H.P.
      • Wang X.
      • Ron D.
      • Cavener D.R.
      • Wek R.C.
      Activating transcription factor 3 (ATF3) is integral to the eIF2 kinase stress response.
      ,
      • Mascarenhas C.
      • Edwards-Ingram L.C.
      • Zeef L.
      • Shenton D.
      • Ashe M.P.
      • Grant C.M.
      Gcn4 is required for the response to peroxide stress in the yeast Saccharomyces cerevisiae.
      ,
      • Natarajan K.
      • Meyer M.R.
      • Jackson B.M.
      • Slade D.
      • Roberts C.
      • Hinnebusch A.G.
      • Marton M.J.
      Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast.
      ,
      • Hinnebusch A.G.
      • Natarajan K.
      Gcn4p, a master regulator of gene expression, is controlled at multiple levels by diverse signals of starvation and stress.
      ,
      • Staschke K.A.
      • Dey S.
      • Zaborske J.M.
      • Palam L.R.
      • McClintick J.N.
      • Pan T.
      • Edenberg H.J.
      • Wek R.C.
      Integration of general amino acid control and target of rapamycin (TOR) regulatory pathways in nitrogen assimilation in yeast.
      ). The 5′-leader of ATF4 contains two uORFs: the 5′-proximal uORF1 that is three codons in length and a 59-codon-long uORF2 that overlaps out-of-frame with the ATF4 coding region (Fig. 3A) (
      • Vattem K.M.
      • Wek R.C.
      Reinitiation involving upstream open reading frames regulates ATF4 mRNA translation in mammalian cells.
      ,
      • Lu P.D.
      • Harding H.P.
      • Ron D.
      Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response.
      ). The GCN4 5′-leader contains four uORFs, each encoding polypeptides two to three residues in length (
      • Hinnebusch A.G.
      Translational regulation of GCN4 and the general amino acid control of yeast.
      ,
      • Abastado J.P.
      • Miller P.F.
      • Jackson B.M.
      • Hinnebusch A.G.
      Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis of GCN4 translational control.
      ). In the delayed translation reinitiation model, the 5′-proximal uORF1 in the ATF4 and GCN4 5′-leaders acts as a positive element that promotes downstream translation reinitiation (
      • Hinnebusch A.G.
      Translational regulation of GCN4 and the general amino acid control of yeast.
      ,
      • Grant C.M.
      • Hinnebusch A.G.
      Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control.
      ,
      • Vattem K.M.
      • Wek R.C.
      Reinitiation involving upstream open reading frames regulates ATF4 mRNA translation in mammalian cells.
      ). During nonstressed conditions, the 40S ribosome resumes scanning after translation of uORF1 and reacquires a new eIF2·GTP·Met-tRNAiMet complex in sufficient time to reinitiate translation at the next uORF initiation codon. In the case of ATF4, translation initiation at the overlapping out-of-frame uORF2 results in translation termination and ribosome dissociation 3′ of the ATF4 CDS, thereby reducing synthesis of ATF4 protein (Fig. 3A) (
      • Vattem K.M.
      • Wek R.C.
      Reinitiation involving upstream open reading frames regulates ATF4 mRNA translation in mammalian cells.
      ). Translation of the downstream GCN4 uORFs 3 or 4 also thwarts expression of the GCN4 coding region during nonstressed conditions (see Fig. 4C) (
      • Abastado J.P.
      • Miller P.F.
      • Jackson B.M.
      • Hinnebusch A.G.
      Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis of GCN4 translational control.
      ,
      • Miller P.F.
      • Hinnebusch A.G.
      Sequences that surround the stop codons of upstream open reading frames in GCN4 mRNA determine their distinct functions in translational control.
      ). The inhibitory property of uORF4 relies upon a 10-nucleotide sequence 3′ of the uORF4 stop codon that is suggested to interact with the 40S ribosomal subunit to promote ribosome dissociation (
      • Miller P.F.
      • Hinnebusch A.G.
      Sequences that surround the stop codons of upstream open reading frames in GCN4 mRNA determine their distinct functions in translational control.
      ).
      Figure thumbnail gr3
      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 thumbnail gr4
      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).
      During cellular stress, eIF2α-P results in lowered levels of eIF2·GTP required for delivery of Met-tRNAiMet for reinitiating ribosomes. As a consequence, after translation of uORF1, the scanning 40S ribosomal subunit takes a longer amount of time to reacquire a new eIF2 ternary complex that is required for recognition of the next translation initiation codon in the 5′-leaders of the ATF4 and GCN4 mRNAs. The delay in the acquisition of eIF2 ternary complex allows the 40S ribosomal subunit to scan through the inhibitory uORFs in the two mRNAs and instead promote translation initiation at the ATF4 or GCN4 CDS, promoting production of ATF4 and GCN4 proteins that serve to transcriptionally enhance genes important for remediation of the stress damage (Fig. 3A). Lack of appropriate GCN4 and ATF4 expression renders cells susceptible to nutrient deficiencies and oxidative damage (
      • Harding H.P.
      • Zhang Y.
      • Zeng H.
      • Novoa I.
      • Lu P.D.
      • Calfon M.
      • Sadri N.
      • Yun C.
      • Popko B.
      • Paules R.
      • Stojdl D.F.
      • Bell J.C.
      • Hettmann T.
      • Leiden J.M.
      • Ron D.
      An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.
      ,
      • Natarajan K.
      • Meyer M.R.
      • Jackson B.M.
      • Slade D.
      • Roberts C.
      • Hinnebusch A.G.
      • Marton M.J.
      Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast.
      ,
      • Fusakio M.E.
      • Willy J.A.
      • Wang Y.
      • Mirek E.T.
      • Al Baghdadi R.J.
      • Adams C.M.
      • Anthony T.G.
      • Wek R.C.
      Transcription factor ATF4 directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver.
      ).

      Translation Reinitiation and Differential CDS Translation

      Levels of eIF2α-P and the ensuing reduction of eIF2·GTP levels also play a role in start codon selection that regulates the abundance of protein isoforms encoded in the same mRNA. For example, C/EBPβ mRNA has four AUG initiation codons that encode three different protein isoforms and one uORF (
      • Calkhoven C.F.
      • Müller C.
      • Leutz A.
      Translational control of C/EBPα and C/EBPβ isoform expression.
      ). The protein isoforms encoded in C/EBPβ are transcription factors that regulate adipogenesis, immunity, and functions of bone and liver tissues (
      • Tsukada J.
      • Yoshida Y.
      • Kominato Y.
      • Auron P.E.
      The CCAAT/enhancer (C/EBP) family of basic-leucine zipper (bZIP) transcription factors is a multifaceted highly-regulated system for gene regulation.
      ,
      • Nerlov C.
      The C/EBP family of transcription factors: a paradigm for interaction between gene expression and proliferation control.
      ). All three protein isoforms contain the basic leucine zipper domain, but the two larger isoforms, liver-enriched activating proteins (LAP* and LAP), also contain an N-terminal domain that activates transcription of target genes, which is missing in the smaller isoform, liver-enriched inhibitory protein (LIP) (
      • Calkhoven C.F.
      • Müller C.
      • Leutz A.
      Translational control of C/EBPα and C/EBPβ isoform expression.
      ,
      • Wethmar K.
      • Bégay V.
      • Smink J.J.
      • Zaragoza K.
      • Wiesenthal V.
      • Dörken B.
      • Calkhoven C.F.
      • Leutz A.
      C/EBPβΔuORF mice: a genetic model for uORF-mediated translational control in mammals.
      ). As a consequence, the LAP* and LAP isoforms can dimerize and serve as activators of transcription, whereas production of LIP serves as a transcriptional repressor (
      • Nerlov C.
      The C/EBP family of transcription factors: a paradigm for interaction between gene expression and proliferation control.
      ).
      The largest protein isoform, LAP*, is translated beginning at the most 5′-proximal AUG (
      • Calkhoven C.F.
      • Müller C.
      • Leutz A.
      Translational control of C/EBPα and C/EBPβ isoform expression.
      ). The next AUG that follows the LAP* initiation site encodes an out-of-frame uORF. Importantly, the uORF termination codon is located 5′ of the initiation sites for both LAP and LIP and plays a central role in the regulation of translational expression of the LAP and LIP isoforms (
      • Calkhoven C.F.
      • Müller C.
      • Leutz A.
      Translational control of C/EBPα and C/EBPβ isoform expression.
      ). After translation of the uORF, a number of 40S ribosomes are suggested to scan past the initiation codon of the LAP isoform before reacquiring a new eIF2·GTP·Met-tRNAiMetcomplex and begin translation at the initiation codon of LIP. LIP expression is also elevated after UV irradiation, suggesting that decreased eIF2 ternary complex levels during cellular stress exacerbate this phenomenon (
      • Dey S.
      • Savant S.
      • Teske B.F.
      • Hatzoglou M.
      • Calkhoven C.F.
      • Wek R.C.
      Transcriptional repression of ATF4 gene by CCAAT/enhancer-binding protein β (C/EBPβ) differentially regulates integrated stress response.
      ). The related gene C/EBPα and its encoded protein isoforms are also translationally regulated by a 5′-proximal uORF (
      • Calkhoven C.F.
      • Bouwman P.R.
      • Snippe L.
      • Ab G.
      Translation start site multiplicity of the CCAAT/enhancer binding protein α mRNA is dictated by a small 5′ open reading frame.
      ,
      • Calkhoven C.F.
      • Müller C.
      • Leutz A.
      Translational control of C/EBPα and C/EBPβ isoform expression.
      ,
      • Wang C.
      • Chen X.
      • Wang Y.
      • Gong J.
      • Hu G.
      C/EBPαp30 plays transcriptional regulatory roles distinct from C/EBPαp42.
      ).

      Ribosome Bypass in the ISR

      Selection of the translation start site relies largely on the nucleotide context surrounding the start codon. The optimal sequence, termed the Kozak consensus sequence, is GCC(A/G)CCAUGG of which the most important residues are the purines in the −3 and +4 positions, and the initiation codon is underlined (
      • Kozak M.
      The scanning model for translation: an update.
      ,
      • Kozak M.
      Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.
      ). These two residues have been shown to interact with the eIF2α subunit and the 18S rRNA contained within the small ribosomal subunit and are proposed to promote recognition of the initiation codon (
      • Pisarev A.V.
      • Kolupaeva V.G.
      • Pisareva V.P.
      • Merrick W.C.
      • Hellen C.U.
      • Pestova T.V.
      Specific functional interactions of nucleotides at key −3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex.
      ). Additionally, poor uORF start codon context has been associated with those mRNAs that are preferentially translated, whereas mRNAs that are repressed during eIF2α-P typically contain an uORF in a strong Kozak consensus sequence (
      • Baird T.D.
      • Palam L.R.
      • Fusakio M.E.
      • Willy J.A.
      • Davis C.M.
      • McClintick J.N.
      • Anthony T.G.
      • Wek R.C.
      Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα.
      ). These findings suggest that start codon context plays a significant role in uORF-mediated translation regulation.

      Ribosome Bypass Is Dependent on Start Codon Context

      An important example in the ISR of ribosome bypass of an uORF is that of GADD34 (
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ,
      • Lee Y.Y.
      • Cevallos R.C.
      • Jan E.
      An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2α phosphorylation.
      ). GADD34 functions in feedback control of the ISR (
      • Harding H.P.
      • Zhang Y.
      • Scheuner D.
      • Chen J.J.
      • Kaufman R.J.
      • Ron D.
      Ppp1r15 gene knockout reveals an essential role for translation initiation factor 2 α (eIF2α) dephosphorylation in mammalian development.
      ,
      • Novoa I.
      • Zeng H.
      • Harding H.P.
      • Ron D.
      Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2α.
      ,
      • Connor J.H.
      • Weiser D.C.
      • Li S.
      • Hallenbeck J.M.
      • Shenolikar S.
      Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1.
      ). The GADD34 mRNA contains two uORFs (Fig. 3B). The uORF1 is considered to be a constitutive, modest dampener of downstream GADD34 expression (
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ,
      • Lee Y.Y.
      • Cevallos R.C.
      • Jan E.
      An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2α phosphorylation.
      ). uORF1 is in a poor Kozak consensus sequence and is largely bypassed both basally and during stress conditions. Translation of uORF2, however, results in a significant decrease in translation initiation at the CDS, indicating that uORF2 is largely inhibitory to downstream translation (Fig. 3B) (
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ,
      • Lee Y.Y.
      • Cevallos R.C.
      • Jan E.
      An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2α phosphorylation.
      ). The inhibitory nature of GADD34 uORF2 relies in part upon a Pro-Pro-Gly sequence juxtaposed to the stop codon (
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ). Translation of the Pro-Pro-Gly sequence in uORF2 is suggested to result in an inefficient termination that increases ribosome release from the mRNA, promoting low levels of GADD34 expression during basal conditions (Fig. 3B). This was an interesting finding, because polyproline and Pro-Pro-Gly sequences have been shown to require the activity of an additional factor, eIF5A, for efficient translation (
      • Gutierrez E.
      • Shin B.S.
      • Woolstenhulme C.J.
      • Kim J.R.
      • Saini P.
      • Buskirk A.R.
      • Dever T.E.
      eIF5A promotes translation of polyproline motifs.
      ).
      Preferential translation of GADD34 in response to eIF2α-P and cellular stress occurs by scanning ribosome bypass of uORF2 by a process involving, at least in part, the poor start codon context of this inhibitory uORF (Fig. 3B) (
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ). Appropriate inhibition of GADD34 translation by uORF2 and its regulated bypass is critical for the magnitude and duration of translational control during the progression of a stress response. For example, deletion of the inhibitory GADD34 uORF2 results in an inappropriate increase in GADD34 expression even in the absence of stress that serves to sharply diminish levels of eIF2α-P even upon induction of the ISR (
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ). As a consequence, there are continued high levels of protein synthesis and increased cell sensitivity to cellular stress.
      Ribosomal bypass of an uORF is also central for preferential translation of CHOP in the ISR (
      • Palam L.R.
      • Baird T.D.
      • Wek R.C.
      Phosphorylation of eIF2 facilitates ribosomal bypass of an inhibitory upstream ORF to enhance CHOP translation.
      ,
      • Jousse C.
      • Bruhat A.
      • Carraro V.
      • Urano F.
      • Ferrara M.
      • Ron D.
      • Fafournoux P.
      Inhibition of CHOP translation by a peptide encoded by an open reading frame localized in the chop 5′UTR.
      ). Prolonged expression of CHOP during times of chronic stress and continued eIF2α-P can induce apoptosis (
      • Zinszner H.
      • Kuroda M.
      • Wang X.
      • Batchvarova N.
      • Lightfoot R.T.
      • Remotti H.
      • Stevens J.L.
      • Ron D.
      CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum.
      ,
      • Rutkowski D.T.
      • Arnold S.M.
      • Miller C.N.
      • Wu J.
      • Li J.
      • Gunnison K.M.
      • Mori K.
      • Sadighi Akha A.A.
      • Raden D.
      • Kaufman R.J.
      Adaptation to ER stress is mediated by differential stabilities of pro-survival and pro-apoptotic mRNAs and proteins.
      ,
      • Marciniak S.J.
      • Yun C.Y.
      • Oyadomari S.
      • Novoa I.
      • Zhang Y.
      • Jungreis R.
      • Nagata K.
      • Harding H.P.
      • Ron D.
      CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum.
      ,
      • Teske B.F.
      • Fusakio M.E.
      • Zhou D.
      • Shan J.
      • McClintick J.N.
      • Kilberg M.S.
      • Wek R.C.
      CHOP induces activating transcription factor 5 (ATF5) to trigger apoptosis in response to perturbations in protein homeostasis.
      ). Analogous to GADD34, bypass of the inhibitory CHOP uORF during eIF2α-P is also suggested to rely at least in part upon poor start codon context of the uORF (
      • Young S.K.
      • Palam L.R.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome elongation stall directs gene-specific translation in the integrated stress response.
      ,
      • Palam L.R.
      • Baird T.D.
      • Wek R.C.
      Phosphorylation of eIF2 facilitates ribosomal bypass of an inhibitory upstream ORF to enhance CHOP translation.
      ). The inhibitory nature of the CHOP uORF during basal conditions is centered upon an elongation stall at an encoded polypeptide segment (
      • Young S.K.
      • Palam L.R.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome elongation stall directs gene-specific translation in the integrated stress response.
      ). Emphasizing the importance of the uORF for appropriate CHOP expression in the ISR, mutations rendering the uORF nonfunctional lead to overexpression of CHOP and its downstream pro-apoptotic target genes, enhancing cell death during stress (
      • Young S.K.
      • Palam L.R.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome elongation stall directs gene-specific translation in the integrated stress response.
      ).
      We do not yet understand the mechanisms by which selection of translation start sites can be affected by eIF2α-P. It was suggested that eIF2α-P disrupts the stability of the interaction between the scanning ribosomal subunit and the mRNA at the −3 position of the start codon (
      • Hussain T.
      • Llácer J.L.
      • Fernández I.S.
      • Munoz A.
      • Martin-Marcos P.
      • Savva C.G.
      • Lorsch J.R.
      • Hinnebusch A.G.
      • Ramakrishnan V.
      Structural changes enable start codon recognition by the eukaryotic translation initiation complex.
      ). Another possibility is that eIF2α-P may modify the nature of the interaction between eIF2 and the Met-tRNAiMet that regulates start site selection (
      • Hussain T.
      • Llácer J.L.
      • Fernández I.S.
      • Munoz A.
      • Martin-Marcos P.
      • Savva C.G.
      • Lorsch J.R.
      • Hinnebusch A.G.
      • Ramakrishnan V.
      Structural changes enable start codon recognition by the eukaryotic translation initiation complex.
      ). Alternatively, the effects of eIF2α-P on start codon selection may be indirect. For example, eIF2α-P may alter the expression and stoichiometry of other initiation factors, such as eIF1, which play a role in translation start site selection (
      • Pestova T.V.
      • Kolupaeva V.G.
      The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection.
      ). The recently characterized small molecule ISRIB (integrated stress response inhibitor), which renders eIF2B largely insensitive to eIF2α-P (
      • Sidrauski C.
      • Acosta-Alvear D.
      • Khoutorsky A.
      • Vedantham P.
      • Hearn B.R.
      • Li H.
      • Gamache K.
      • Gallagher C.M.
      • Ang K.K.
      • Wilson C.
      • Okreglak V.
      • Ashkenazi A.
      • Hann B.
      • Nader K.
      • Arkin M.R.
      • et al.
      Pharmacological brake-release of mRNA translation enhances cognitive memory.
      ,
      • Sidrauski C.
      • McGeachy A.M.
      • Ingolia N.T.
      • Walter P.
      The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly.
      ,
      • Sekine Y.
      • Zyryanova A.
      • Crespillo-Casado A.
      • Fischer P.M.
      • Harding H.P.
      • Ron D.
      Mutations in a translation initiation factor identify the target of a memory-enhancing compound.
      ), may provide an important tool for discerning genome-wide changes in initiation codon selection afforded by eIF2α-P.

      Ribosome Bypass and Non-canonical Start Codons

      Recent ribosome profiling studies have illustrated that previously uncharacterized uORFs with non-canonical initiation codons are more commonly translated than previously thought (
      • Ingolia N.T.
      • Ghaemmaghami S.
      • Newman J.R.
      • Weissman J.S.
      Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.
      ,
      • Gao X.
      • Wan J.
      • Liu B.
      • Ma M.
      • Shen B.
      • Qian S.B.
      Quantitative profiling of initiating ribosomes in vivo.
      ,
      • Fritsch C.
      • Herrmann A.
      • Nothnagel M.
      • Szafranski K.
      • Huse K.
      • Schumann F.
      • Schreiber S.
      • Platzer M.
      • Krawczak M.
      • Hampe J.
      • Brosch M.
      Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting.
      ). This observation has been followed by the characterization of multiple mechanisms involving bypass of uORFs due to their non-canonical initiation codons that regulate gene-specific translation during cellular stress conditions (
      • Gao X.
      • Wan J.
      • Liu B.
      • Ma M.
      • Shen B.
      • Qian S.B.
      Quantitative profiling of initiating ribosomes in vivo.
      ,
      • Young S.K.
      • Baird T.D.
      • Wek R.C.
      Translation regulation of the glutamyl-prolyl-tRNA synthetase gene EPRS through bypass of upstream open reading frames with noncanonical initiation codons.
      ,
      • Starck S.R.
      • Tsai J.C.
      • Chen K.
      • Shodiya M.
      • Wang L.
      • Yahiro K.
      • Martins-Green M.
      • Shastri N.
      • Walter P.
      Translation from the 5′ untranslated region shapes the integrated stress response.
      ).
      One such example is the translation mechanism for glutamyl-prolyl tRNA synthetase, EPRS. Although EPRS expression is required for normal protein homeostasis, preferential translation of EPRS is suggested to increase the appropriately charged tRNA pool and prime the cell for resumption of protein synthesis once the cellular stress is remediated (
      • Young S.K.
      • Baird T.D.
      • Wek R.C.
      Translation regulation of the glutamyl-prolyl-tRNA synthetase gene EPRS through bypass of upstream open reading frames with noncanonical initiation codons.
      ,
      • Ibba M.
      • Soll D.
      Aminoacyl-tRNA synthesis.
      ). Of the five non-canonical initiation codons in the 5′-leader of EPRS only the 5′-proximal UUG and the subsequent CUG are considered to be the main regulators of EPRS preferential translation (
      • Young S.K.
      • Baird T.D.
      • Wek R.C.
      Translation regulation of the glutamyl-prolyl-tRNA synthetase gene EPRS through bypass of upstream open reading frames with noncanonical initiation codons.
      ). The uORF containing the CUG initiation codon overlaps out-of-frame with the EPRS CDS and was shown to be inhibitory to EPRS expression, presumably by ribosome dissociation from the mRNA past the initiation codon for the CDS (
      • Young S.K.
      • Baird T.D.
      • Wek R.C.
      Translation regulation of the glutamyl-prolyl-tRNA synthetase gene EPRS through bypass of upstream open reading frames with noncanonical initiation codons.
      ). The uORF encoded by the UUG, on the other hand, terminates 51 nucleotides 5′ of the CDS start codon, and was suggested to allow for a portion of the translating ribosomes to reinitiate at the downstream EPRS CDS (
      • Young S.K.
      • Baird T.D.
      • Wek R.C.
      Translation regulation of the glutamyl-prolyl-tRNA synthetase gene EPRS through bypass of upstream open reading frames with noncanonical initiation codons.
      ). Importantly, both inhibitory uORFs are bypassed due to their non-canonical initiation codons to a moderate extent during basal conditions and experience increased bypass efficiency during eIF2α-P and stress (
      • Young S.K.
      • Baird T.D.
      • Wek R.C.
      Translation regulation of the glutamyl-prolyl-tRNA synthetase gene EPRS through bypass of upstream open reading frames with noncanonical initiation codons.
      ).
      Another gene recently identified as containing a functional uORF with a non-canonical initiation codon is that of GADD45G, which regulates cell growth and apoptosis (
      • Gao X.
      • Wan J.
      • Liu B.
      • Ma M.
      • Shen B.
      • Qian S.B.
      Quantitative profiling of initiating ribosomes in vivo.
      ). The GADD45G mRNA contains an overlapping out-of-frame uORF with a CUG initiation codon. The uORF serves as a barrier to downstream translation during nonstressed conditions and is bypassed due to its noncanonical initiation codon during eIF2α-P (
      • Gao X.
      • Wan J.
      • Liu B.
      • Ma M.
      • Shen B.
      • Qian S.B.
      Quantitative profiling of initiating ribosomes in vivo.
      ). Also recently described is the model of translation control for BiP (GRP78/HSPA5) that participates in protein folding in the ER (
      • Starck S.R.
      • Tsai J.C.
      • Chen K.
      • Shodiya M.
      • Wang L.
      • Yahiro K.
      • Martins-Green M.
      • Shastri N.
      • Walter P.
      Translation from the 5′ untranslated region shapes the integrated stress response.
      ). The 5′-leader of the BiP mRNA features two uORFs that are both encoded by non-canonical initiation codons, as well as possible internal ribosome entry sequences (IRES), which may function in conjunction with uORFs to facilitate BiP translation during cellular stress (
      • Starck S.R.
      • Tsai J.C.
      • Chen K.
      • Shodiya M.
      • Wang L.
      • Yahiro K.
      • Martins-Green M.
      • Shastri N.
      • Walter P.
      Translation from the 5′ untranslated region shapes the integrated stress response.
      ,
      • Cho S.
      • Park S.M.
      • Kim T.D.
      • Kim J.H.
      • Kim K.T.
      • Jang S.K.
      BiP internal ribosomal entry site activity is controlled by heat-induced interaction of NSAP1.
      ,
      • Johannes G.
      • Sarnow P.
      Cap-independent polysomal association of natural mRNAs encoding c-myc, PiP, and eIF-4G conferred by internal ribosome entry sites.
      ,
      • Jackson R.J.
      The current status of vertebrate cellular mRNA IRESs.
      ).

      Other Complexities of Gene Expression Regulated by uORFs

      The observation that the 5′-leader of BiP mRNA contains both uORFs and an IRES that coordinate expression of BiP emphasizes that other mRNA structural features also contribute to translation regulation involving uORFs. The mRNA encoding the cationic amino acid transporter CAT1 contains both an uORF and an IRES that are required for induced expression of the CAT1 CDS during cellular stress (
      • Fernandez J.
      • Yaman I.
      • Huang C.
      • Liu H.
      • Lopez A.B.
      • Komar A.A.
      • Caprara M.G.
      • Merrick W.C.
      • Snider M.D.
      • Kaufman R.J.
      • Lamers W.H.
      • Hatzoglou M.
      Ribosome stalling regulates IRES-mediated translation in eukaryotes, a parallel to prokaryotic attenuation.
      ,
      • Fernandez J.
      • Yaman I.
      • Sarnow P.
      • Snider M.D.
      • Hatzoglou M.
      Regulation of internal ribosomal entry site-mediated translation by phosphorylation of the translation initiation factor eIF2α.
      ). The 5′-leader of CAT1 forms a stable secondary structure that prevents translation during nonstressed conditions. However, translation of the uORF during eIF2α-P is suggested to unfold the RNA structure, yielding an IRES that serves to increase translation of the CAT1 CDS (
      • Yaman I.
      • Fernandez J.
      • Liu H.
      • Caprara M.
      • Komar A.A.
      • Koromilas A.E.
      • Zhou L.
      • Snider M.D.
      • Scheuner D.
      • Kaufman R.J.
      • Hatzoglou M.
      The zipper model of translational control: a small upstream ORF is the switch that controls structural remodeling of an mRNA leader.
      ).
      The mRNA encoding the HER2 proto-oncogene contains an uORF that represses translation initiation at the primary HER2 initiation codon due to insufficient time for the scanning 40S ribosomal subunit to reacquire a new eIF2 ternary complex (
      • Mehta A.
      • Trotta C.R.
      • Peltz S.W.
      Derepression of the Her-2 uORF is mediated by a novel post-transcriptional control mechanism in cancer cells.
      ,
      • Spevak C.C.
      • Park E.H.
      • Geballe A.P.
      • Pelletier J.
      • Sachs M.S.
      her-2 upstream open reading frame effects on the use of downstream initiation codons.
      ). Instead translation reinitiation occurs at alternative downstream initiation codons that lead to the synthesis of N-terminally truncated HER2 protein isoforms (
      • Spevak C.C.
      • Park E.H.
      • Geballe A.P.
      • Pelletier J.
      • Sachs M.S.
      her-2 upstream open reading frame effects on the use of downstream initiation codons.
      ). However, full-length HER2 expression is enhanced in in both breast and ovarian cancers (
      • Mehta A.
      • Trotta C.R.
      • Peltz S.W.
      Derepression of the Her-2 uORF is mediated by a novel post-transcriptional control mechanism in cancer cells.
      ). This regulatory scheme is suggested to occur via interactions between RNA secondary structures in the 3′-UTR of the HER2 mRNA and the terminating ribosome at the uORF stop codon. This interaction is thought to retain the terminating ribosome and associated initiation factors on the HER2 mRNA until a new eIF2 ternary complex is acquired, thereby facilitating translation at the most immediate 5′-proximal AUG that leads to full-length HER2 protein synthesis (
      • Mehta A.
      • Trotta C.R.
      • Peltz S.W.
      Derepression of the Her-2 uORF is mediated by a novel post-transcriptional control mechanism in cancer cells.
      ).
      The three major regulators of mRNA abundance, transcription, mRNA processing, and mRNA degradation, also play major roles in uORF-mediated regulation of ISR-induced preferential translation (
      • Dey S.
      • Savant S.
      • Teske B.F.
      • Hatzoglou M.
      • Calkhoven C.F.
      • Wek R.C.
      Transcriptional repression of ATF4 gene by CCAAT/enhancer-binding protein β (C/EBPβ) differentially regulates integrated stress response.
      ,
      • Floor S.N.
      • Doudna J.A.
      Tunable protein synthesis by transcript isoforms in human cells.
      ,
      • Hansen M.B.
      • Mitchelmore C.
      • Kjaerulff K.M.
      • Rasmussen T.E.
      • Pedersen K.M.
      • Jensen N.A.
      Mouse ATF5: molecular cloning of two novel mRNAs, genomic organization, and odorant sensory neuron localization.
      ,
      • Watatani Y.
      • Ichikawa K.
      • Nakanishi N.
      • Fujimoto M.
      • Takeda H.
      • Kimura N.
      • Hirose H.
      • Takahashi S.
      • Takahashi Y.
      Stress-induced translation of ATF5 mRNA is regulated by the 5′-untranslated region.
      ). For example, ATF4 expression is potently induced during endoplasmic reticulum stress, but there are only low levels of ATF4 expression during UV irradiation (
      • Dey S.
      • Savant S.
      • Teske B.F.
      • Hatzoglou M.
      • Calkhoven C.F.
      • Wek R.C.
      Transcriptional repression of ATF4 gene by CCAAT/enhancer-binding protein β (C/EBPβ) differentially regulates integrated stress response.
      ,
      • Dey S.
      • Baird T.D.
      • Zhou D.
      • Palam L.R.
      • Spandau D.F.
      • Wek R.C.
      Both transcriptional regulation and translational control of ATF4 are central to the integrated stress response.
      ). Both conditions induce robust eIF2α-P, and preferential translation of ATF4 mRNA occurs with either stress (
      • Dey S.
      • Baird T.D.
      • Zhou D.
      • Palam L.R.
      • Spandau D.F.
      • Wek R.C.
      Both transcriptional regulation and translational control of ATF4 are central to the integrated stress response.
      ). However, increased LIP expression and binding to the ATF4 promoter during UV irradiation repress ATF4 transcription (
      • Dey S.
      • Savant S.
      • Teske B.F.
      • Hatzoglou M.
      • Calkhoven C.F.
      • Wek R.C.
      Transcriptional repression of ATF4 gene by CCAAT/enhancer-binding protein β (C/EBPβ) differentially regulates integrated stress response.
      ). Lowered levels of ATF4 mRNA available for translation during UV stress thus result in negligible ATF4 protein and transcriptional activity. These findings illustrate that ATF4 mRNAs with identical 5′-leaders and uORF configurations have sharply different induction capabilities in response to different stress conditions despite having comparable levels of eIF2α-P.
      Expression of the ISR transcriptional regulator ATF5 features two different mRNA isoforms (
      • Hansen M.B.
      • Mitchelmore C.
      • Kjaerulff K.M.
      • Rasmussen T.E.
      • Pedersen K.M.
      • Jensen N.A.
      Mouse ATF5: molecular cloning of two novel mRNAs, genomic organization, and odorant sensory neuron localization.
      ). The more abundant transcript, ATF5α, contains two uORFs that serve to promote preferential translation by delayed translation reinitiation (
      • Zhou D.
      • Palam L.R.
      • Jiang L.
      • Narasimhan J.
      • Staschke K.A.
      • Wek R.C.
      Phosphorylation of eIF2 directs ATF5 translational control in response to diverse stress conditions.
      ,
      • Watatani Y.
      • Ichikawa K.
      • Nakanishi N.
      • Fujimoto M.
      • Takeda H.
      • Kimura N.
      • Hirose H.
      • Takahashi S.
      • Takahashi Y.
      Stress-induced translation of ATF5 mRNA is regulated by the 5′-untranslated region.
      ). ATF5β encodes the same ATF5 CDS, but contains an alternative 5′-leader that does not contain any conserved uORFs and is not translationally regulated in a stress-dependent manner (
      • Watatani Y.
      • Ichikawa K.
      • Nakanishi N.
      • Fujimoto M.
      • Takeda H.
      • Kimura N.
      • Hirose H.
      • Takahashi S.
      • Takahashi Y.
      Stress-induced translation of ATF5 mRNA is regulated by the 5′-untranslated region.
      ). Thus, expression of ATF5 can also be modulated through the differential production of mRNA isoforms (
      • Hansen M.B.
      • Mitchelmore C.
      • Kjaerulff K.M.
      • Rasmussen T.E.
      • Pedersen K.M.
      • Jensen N.A.
      Mouse ATF5: molecular cloning of two novel mRNAs, genomic organization, and odorant sensory neuron localization.
      ). Recent genome-wide evidence has suggested that regulation of translation through mRNA splicing also plays a significant role in the presence of different 5′-leaders in mRNAs that can affect translational expression (
      • Floor S.N.
      • Doudna J.A.
      Tunable protein synthesis by transcript isoforms in human cells.
      ).
      uORF translation can also result in activation of the mRNA decay pathways, thus adding another layer to the mechanisms in which uORFs can negatively regulate downstream translation (
      • Gardner B.M.
      • Pincus D.
      • Gotthardt K.
      • Gallagher C.M.
      • Walter P.
      Endoplasmic reticulum stress sensing in the unfolded protein response.
      ). For example, CHOP was identified as a target of the nonsense-mediated mRNA decay pathway that recognizes the presence of a premature termination codon (
      • Mendell J.T.
      • Sharifi N.A.
      • Meyers J.L.
      • Martinez-Murillo F.
      • Dietz H.C.
      Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise.
      ). Depletion of the nonsense-mediated mRNA decay machinery from cells results in the stabilization of CHOP mRNA levels (
      • Gardner L.B.
      Hypoxic inhibition of nonsense-mediated RNA decay regulates gene expression and the integrated stress response.
      ). CHOP mRNA half-life was also increased more than 2-fold in cells in which the two initiating codons of the CHOP uORF were mutated (
      • Young S.K.
      • Palam L.R.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome elongation stall directs gene-specific translation in the integrated stress response.
      ). Combined, these studies suggest that the presence of an uORF can also serve to repress expression of the CDS through mechanisms involving mRNA decay.

      Evolutionary Conservation of uORF-mediated Translation Mechanisms

      Many uORF-mediated translation control schemes that rely on eIF2α-P are conserved in eukaryotes. For example, the uORF-mediated translational control mechanism for IBTKα is suggested to be largely conserved among mammals (Fig. 4A) (
      • Baird T.D.
      • Palam L.R.
      • Fusakio M.E.
      • Willy J.A.
      • Davis C.M.
      • McClintick J.N.
      • Anthony T.G.
      • Wek R.C.
      Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα.
      ). Interestingly, the Homo sapiens IBTKα mRNA has four uORFs, but only the two key uORFs that confer IBTKα translational control are consistently conserved among mammals (Fig. 4A) (
      • Baird T.D.
      • Palam L.R.
      • Fusakio M.E.
      • Willy J.A.
      • Davis C.M.
      • McClintick J.N.
      • Anthony T.G.
      • Wek R.C.
      Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα.
      ). This finding indicates that those uORFs that are retained throughout species likely maintain functional significance. This idea is emphasized in genome-wide analyses of uORF conservation in which the presence of an uORF and the regulatory nature of uORF(s) is suggested to be conserved throughout species (
      • Fritsch C.
      • Herrmann A.
      • Nothnagel M.
      • Szafranski K.
      • Huse K.
      • Schumann F.
      • Schreiber S.
      • Platzer M.
      • Krawczak M.
      • Hampe J.
      • Brosch M.
      Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting.
      ,
      • Iacono M.
      • Mignone F.
      • Pesole G.
      uAUG and uORFs in human and rodent 5′untranslated mRNAs.
      ,
      • Neafsey D.E.
      • Galagan J.E.
      Dual modes of natural selection on upstream open reading frames.
      ,
      • Bazzini A.A.
      • Johnstone T.G.
      • Christiano R.
      • Mackowiak S.D.
      • Obermayer B.
      • Fleming E.S.
      • Vejnar C.E.
      • Lee M.T.
      • Rajewsky N.
      • Walther T.C.
      • Giraldez A.J.
      Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation.
      ,
      • Johnstone T.G.
      • Bazzini A.A.
      • Giraldez A.J.
      Upstream ORFs are prevalent translational repressors in vertebrates.
      ).
      An example in which the regulatory function of an uORF is retained, but the underlying mechanisms vary among species is the inhibitory uORF located in the 5′-leader of GADD34 mRNA (
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ,
      • Lee Y.Y.
      • Cevallos R.C.
      • Jan E.
      An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2α phosphorylation.
      ,
      • Malzer E.
      • Szajewska-Skuta M.
      • Dalton L.E.
      • Thomas S.E.
      • Hu N.
      • Skaer H.
      • Lomas D.A.
      • Crowther D.C.
      • Marciniak S.J.
      Coordinate regulation of eIF2α phosphorylation by PPP1R15 and GCN2 is required during Drosophila development.
      ). The 5′-leaders of the Drosophila melanogaster and Mus musculus GADD34 transcripts each contain two uORFs, with the first uORF considered to be largely dispensable for GADD34 translation control (Fig. 4B) (
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ,
      • Lee Y.Y.
      • Cevallos R.C.
      • Jan E.
      An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2α phosphorylation.
      ,
      • Malzer E.
      • Szajewska-Skuta M.
      • Dalton L.E.
      • Thomas S.E.
      • Hu N.
      • Skaer H.
      • Lomas D.A.
      • Crowther D.C.
      • Marciniak S.J.
      Coordinate regulation of eIF2α phosphorylation by PPP1R15 and GCN2 is required during Drosophila development.
      ). uORF2 in D. melanogaster overlaps out-of-frame with the GADD34 CDS and is considered to be inhibitory by promoting uORF translation termination 3′ of the start codon for the GADD34 CDS (
      • Malzer E.
      • Szajewska-Skuta M.
      • Dalton L.E.
      • Thomas S.E.
      • Hu N.
      • Skaer H.
      • Lomas D.A.
      • Crowther D.C.
      • Marciniak S.J.
      Coordinate regulation of eIF2α phosphorylation by PPP1R15 and GCN2 is required during Drosophila development.
      ). By comparison, M. musculus uORF2, which terminates 23 nucleotides upstream of the GADD34 CDS, prohibits reinitiating downstream due to the inefficient translation termination (Fig. 4B) (
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ). Both inhibitory uORFs have been suggested to require ribosome bypass during cellular stress for preferential translation of GADD34 (
      • Young S.K.
      • Willy J.A.
      • Wu C.
      • Sachs M.S.
      • Wek R.C.
      Ribosome reinitiation directs gene-specific translation and regulates the integrated stress response.
      ,
      • Malzer E.
      • Szajewska-Skuta M.
      • Dalton L.E.
      • Thomas S.E.
      • Hu N.
      • Skaer H.
      • Lomas D.A.
      • Crowther D.C.
      • Marciniak S.J.
      Coordinate regulation of eIF2α phosphorylation by PPP1R15 and GCN2 is required during Drosophila development.
      ).
      Assessment of the GCN4 mRNAs in 12 fungal species revealed that uORFs range from three to six in number and are not positionally conserved (
      • Sundaram A.
      • Grant C.M.
      A single inhibitory upstream open reading frame (uORF) is sufficient to regulate Candida albicans GCN4 translation in response to amino acid starvation conditions.
      ,
      • Cvijović M.
      • Dalevi D.
      • Bilsland E.
      • Kemp G.J.
      • Sunnerhagen P.
      Identification of putative regulatory upstream ORFs in the yeast genome using heuristics and evolutionary conservation.
      ). Furthermore, the mechanism of GCN4 translation control for Candida albicans is reliant upon bypass of a single inhibitory uORF whereas GCN4 in S. cerevisiae relies on a mechanism involving delayed translation reinitiation that features multiple uORFs (Fig. 4C) (
      • Hinnebusch A.G.
      Translational regulation of GCN4 and the general amino acid control of yeast.
      ,
      • Sundaram A.
      • Grant C.M.
      A single inhibitory upstream open reading frame (uORF) is sufficient to regulate Candida albicans GCN4 translation in response to amino acid starvation conditions.
      ). These findings suggest that even among gene orthologs in different species, different uORF configurations and mechanisms can be implemented to achieve preferential translation in response to eIF2α-P.
      These examples of the evolutionary conservation of uORF-mediated translation control emphasize that there are multiple features of uORFs that can be combined in specific ways to generate uORFs of similar functions in regulation of translation. Furthermore, the proper composition and position of uORFs and their features are critical for uORF-mediated translation control mechanisms that direct regulated gene expression for optimal adaptation to environmental stress.
      A future challenge is the application of key tenets from uORF-mediated mechanisms described herein to genome-wide assessments of translation. The ubiquitous placement of uORFs throughout the genome indicates that the specific properties of uORFs themselves determine whether uORFs regulate gene expression in a positive or negative fashion. How can the specific features of uORFs be used to predict patterns of translation control? Furthermore, although uORFs regulate mRNA translation in a cis manner, can some uORF peptides function in trans to facilitate cellular homeostasis? Finally, base modifications in RNA, such as N6-methyladenosine, can occur differentially in the 5′-leaders of mRNAs in response to cellular stress (
      • Meyer K.D.
      • Patil D.P.
      • Zhou J.
      • Zinoviev A.
      • Skabkin M.A.
      • Elemento O.
      • Pestova T.V.
      • Qian S.B.
      • Jaffrey S.R.
      5′ UTR m6A promotes cap-independent translation.
      ,
      • Zhou J.
      • Wan J.
      • Gao X.
      • Zhang X.
      • Jaffrey S.R.
      • Qian S.B.
      Dynamic m6A mRNA methylation directs translational control of heat shock response.
      ). Additionally, N6-methyladenosine can promote translation of select mRNAs by binding to eIF3 and recruiting ribosomes independent of the 5′-cap. Can RNA base modifications in specific mRNAs alter ribosome scanning and uORF utilization affecting translational control in the ISR?

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