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• W2003924215 abstract "In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage. Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator of genes subject to the integrated stress response (ISR). The ISR increases the expression of genes important for alleviating stress or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation is critical for stress-induced apoptosis. In this study, we show that eIF2 phosphorylation induces preferential translation of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5′-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF due to its poor start site context, and instead it allows scanning ribosomes to translate CHOP. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage. In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage. Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator of genes subject to the integrated stress response (ISR). The ISR increases the expression of genes important for alleviating stress or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation is critical for stress-induced apoptosis. In this study, we show that eIF2 phosphorylation induces preferential translation of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5′-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF due to its poor start site context, and instead it allows scanning ribosomes to translate CHOP. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage. CHOPing Away at Stress Damage♦: Phosphorylation of eIF2 Facilitates Ribosomal Bypass of an Inhibitory Upstream ORF to Enhance TranslationJournal of Biological ChemistryVol. 286Issue 13Preview♦ See referenced article, J. Biol. Chem. 2011, 286, 10939–10949 Full-Text PDF Open Access Rapid changes in global and gene-specific translation occur in response to many different environmental stresses. For example, translation is repressed in response to an accumulation of misfolded protein in the endoplasmic reticulum (ER), 2The abbreviations used are: ERendoplasmic reticulumISRintegrated stress responseuORFupstream ORFqRTquantitative RTRACErapid amplification of cDNA endsMEFmouse embryonic fibroblastTKthymidine kinase. which prevents further overload of the secretory pathway and provides time for the reconfiguration of gene expression with a focus on stress alleviation. A central mechanism for this translational control involves phosphorylation of eIF2 (eIF2∼P) by the protein kinase PERK/PEK. eIF2 is a translation initiation factor that combines with initiator Met-tRNAiMet and GTP and participates in the selection of the start codon (1Sonenberg N. Hinnebusch A.G. Cell. 2009; 136: 731-745Abstract Full Text Full Text PDF PubMed Scopus (2256) Google Scholar, 2Wek R.C. Jiang H.Y. Anthony T.G. Biochem. Soc. Trans. 2006; 34: 7-11Crossref PubMed Scopus (1021) Google Scholar, 3Ron D. Walter P. Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4879) Google Scholar). Phosphorylation of the α subunit of eIF2 at Ser-51 in response to ER stress blocks the exchange of eIF2-GDP to eIF2-GTP, thus reducing global translation initiation and subsequent protein synthesis. In addition to PERK, three other eIF2 kinases respond to other stress conditions, including GCN2 induced by nutritional deprivation, HRI activated by heme deficiency in erythroid cells, and PKR, which functions in an antiviral defense pathway. endoplasmic reticulum integrated stress response upstream ORF quantitative RT rapid amplification of cDNA ends mouse embryonic fibroblast thymidine kinase. Accompanying this repression of global translational initiation, eIF2∼P selectively enhances the translation of ATF4 mRNA, encoding a basic zipper transcriptional activator of stress-related genes involved in metabolism, protection against oxidative damage, and regulation of apoptosis (3Ron D. Walter P. Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4879) Google Scholar, 4Harding 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. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2381) Google Scholar, 5Schröder M. Kaufman R.J. Annu. Rev. Biochem. 2005; 74: 739-789Crossref PubMed Scopus (2444) Google Scholar). The idea that ATF4 is a common downstream target that integrates signaling from PERK and other eIF2 kinases has led to the eIF2∼P/ATF4 pathway being collectively referred to as the integrated stress response (ISR) (4Harding 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. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2381) Google Scholar). Preferential translation of ATF4 mRNA during eIF2∼P occurs by a mechanism involving two upstream ORFs (uORFs) (6Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2415) Google Scholar, 7Lu P.D. Harding H.P. Ron D. J. Cell Biol. 2004; 167: 27-33Crossref PubMed Scopus (662) Google Scholar, 8Vattem K.M. Wek R.C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 11269-11274Crossref PubMed Scopus (1135) Google Scholar). The 5′-proximal uORF1 is a positive-acting element that enables ribosomes to reinitiate translation at a downstream ORF in the ATF4 transcript (8Vattem K.M. Wek R.C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 11269-11274Crossref PubMed Scopus (1135) Google Scholar). When eIF2-GTP is readily available in nonstressed cells, ribosomes completing translation of uORF1 resume scanning downstream and reinitiate at the next coding region, uORF2, which is an inhibitory element that blocks ATF4 expression. During stress conditions, eIF2∼P and the lowered levels of eIF2-GTP increase the time required for the scanning ribosomes to reinitiate translation. Delayed reinitiation allows for ribosomes to scan through the inhibitory uORF2 and instead translate the ATF4 coding region (8Vattem K.M. Wek R.C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 11269-11274Crossref PubMed Scopus (1135) Google Scholar). Elevated ATF4 levels induce additional basic zipper transcriptional regulators, such as CHOP/GADD153 and ATF3, which together direct a program of gene expression important for cellular remediation or, alternatively, apoptosis (4Harding 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. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2381) Google Scholar, 5Schröder M. Kaufman R.J. Annu. Rev. Biochem. 2005; 74: 739-789Crossref PubMed Scopus (2444) Google Scholar, 9Jiang 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. Mol. Cell. Biol. 2004; 24: 1365-1377Crossref PubMed Scopus (390) Google Scholar, 10Marciniak S.J. Ron D. Physiol. Rev. 2006; 86: 1133-1149Crossref PubMed Scopus (779) Google Scholar). The mechanism of delayed translation reinitiation in response to eIF2∼P is also central for control of GCN4, a transcriptional activator of genes subject to the general amino acid control in the yeast Saccharomyces cerevisiae (11Hinnebusch A.G. Annu. Rev. Microbiol. 2005; 59: 407-450Crossref PubMed Scopus (903) Google Scholar, 12Abastado J.P. Miller P.F. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 486-496Crossref PubMed Scopus (168) Google Scholar, 13Dever T.E. Feng L. Wek R.C. Cigan A.M. Donahue T.F. Hinnebusch A.G. Cell. 1992; 68: 585-596Abstract Full Text PDF PubMed Scopus (565) Google Scholar). How are the ATF4-targeted ISR transcripts translated when there is repression of general protein synthesis as a consequence of eIF2∼P? One answer to this question is that ATF4 increases the expression of GADD34, which facilitates feedback control of the eIF2 kinase response by targeting the type 1 Ser/Thr protein phosphatase for dephosphorylation of eIF2∼P (10Marciniak S.J. Ron D. Physiol. Rev. 2006; 86: 1133-1149Crossref PubMed Scopus (779) Google Scholar, 14Novoa I. Zeng. H. Harding H.P. Ron D. J. Cell Biol. 2001; 153: 1011-1022Crossref PubMed Scopus (1040) Google Scholar, 15Connor J.H. Weiser D.C. Li S. Hallenbeck J.M. Shenolikar S. Mol. Cell. Biol. 2001; 21: 6841-6850Crossref PubMed Scopus (224) Google Scholar, 16Ma Y. Hendershot L.M. J. Biol. Chem. 2003; 278: 34864-34873Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 17Marciniak S.J. Yun C.Y. Oyadomari S. Novoa I. Zhang Y. Jungreis R. Nagata K. Harding H.P. Ron D. Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1498) Google Scholar). The resulting lowered eIF2∼P then allows for resumption of protein synthesis following the reprogramming of the stress-related transcriptome. However, many of the ISR gene products are highly expressed coincident with robust eIF2∼P, and their expression is central for implementation of the stress response pathway. For example, there are high levels of CHOP protein during ER or nutritional stress, whereas translation initiation is still repressed (9Jiang 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. Mol. Cell. Biol. 2004; 24: 1365-1377Crossref PubMed Scopus (390) Google Scholar, 18Zhou D. Palam L.R. Jiang L. Narasimhan J. Staschke K.A. Wek R.C. J. Biol. Chem. 2008; 283: 7064-7073Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Furthermore, CHOP expression has been suggested to be required for the transcriptional activation of GADD34 (17Marciniak S.J. Yun C.Y. Oyadomari S. Novoa I. Zhang Y. Jungreis R. Nagata K. Harding H.P. Ron D. Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1498) Google Scholar). This suggests that CHOP and other ISR target genes are subject to preferential translation when eIF2∼P levels are high (19Dang Do A.N. Kimball S.R. Cavener D.R. Jefferson L.S. Physiol. Genomics. 2009; 38: 328-341Crossref PubMed Scopus (56) Google Scholar, 20Chen Y.J. Tan B.C. Cheng Y.Y. Chen J.S. Lee S.C. Nucleic Acids Res. 2010; 38: 764-777Crossref PubMed Scopus (48) Google Scholar). Supporting this idea, CHOP mRNA is associated with polysomes during amino acid starvation (19Dang Do A.N. Kimball S.R. Cavener D.R. Jefferson L.S. Physiol. Genomics. 2009; 38: 328-341Crossref PubMed Scopus (56) Google Scholar). The extent and duration of CHOP protein synthesis are thought to be central for altering the ISR from an adaptive pathway that alleviates cellular injury to one that is maladaptive, thus triggering apoptosis (3Ron D. Walter P. Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4879) Google Scholar, 21Rutkowski 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. PLoS Biol. 2006; 4: e374Crossref PubMed Scopus (631) Google Scholar, 22Rutkowski D.T. Kaufman R.J. Trends Biochem. Sci. 2007; 32: 469-476Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). This study demonstrates that CHOP mRNA is subject to preferential translation during stress and describes a new mechanism for preferential translation in response to eIF2∼P. The CHOP mechanism of translational control involves a single uORF that blocks translation in the 5′-leader of the CHOP mRNA. However, with eIF2∼P induced by stress, scanning ribosomes bypass the inhibitory uORF by a process suggested to involve reduced efficiency of translation at initiation codons with a poor Kozak consensus sequence. This study indicates that eIF2∼P can regulate multiple mechanisms of preferential translation involving uORFs, and these mechanisms direct key ISR genes central to cell survival during stress conditions. A HindIII-NcoI DNA fragment encoding the 5′-leader of the CHOP mRNA, along with the initiation codon of the CHOP coding region, was inserted between the HindIII and NcoI restriction sites in a derivative of plasmid pGL3. The resulting TK-CHOP-Luc plasmid contains this 5′-leader sequence of CHOP fused to a luciferase reporter downstream of a constitutive TK promoter. Primer sequences used in this construct were as follows: sense 5′-GCTCAAGCTTGTTATCTTGAGCCTAACACGTCGATTAT-3′ and antisense 5′-TCATGAGTGCCATGACTGCACGTGG-3′. The ATG1 and ATG2 codons of the CHOP uORF were mutated individually or in combination to AGG in the PTK-CHOP-Luc plasmid, and this and subsequent mutations were generated using a site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions. To extend the uORF to overlap out-of-frame with the luciferase coding region, the encoded stop codon TGA in the uORF of TK-CHOP-Luc was mutated to GGA. Codon 24 (AGA) in the uORF of PTK-CHOP-Luc was mutated to TGA to generate the luciferase reporter with a shortened version of the uORF, which encoded amino acid residues 1–23. All plasmids were sequenced to ensure that there were only desired changes. Sequence changes upstream of the CHOP uORF involved the introduction of a PstI restriction site 10 nucleotides upstream of the uORF in PTK-CHOP-Luc plasmid. A previously described stem-loop structure 5′-CTGCAGCCACCACGGCCCCCAAGCTTGGGCCGTGGTGGCTGCAG-3′, with a ΔG value of −41 kcal/mol, was then inserted into the PstI site of the modified PTK-CHOP-Luc plasmid. Alternatively, an extension of the 5′-leader was achieved by inserting a 120-nucleotide sequence into the PstI site. This sequence was devoid of any start and stop codons or predicted strong secondary structures. The initiation codon context from the uORF1 of ATF4 that shares a Kozak consensus was substituted for the first four codons, including the ATG1 and ATG2, of the CHOP uORF in the in PTK-CHOP-Luc plasmid, generating PTK-ATGATF4-CHOP-Luc. This substitution involved replacing the TATATCATGTTGAAGATGA sequence in the CHOP uORF with GCCACCATGG. These substitutions were carried out by the sequence and ligation-independent cloning method (23Li M.Z. Elledge S.J. Nat. Methods. 2007; 4: 251-256Crossref PubMed Scopus (716) Google Scholar). The CHOP mRNA sequences from nucleotide 1 to 133 containing the entire coding region of the CHOP uORF were inserted in-frame with the firefly luciferase coding region in a modified version of pGL3. The resulting plasmid PTK-uORF-Luc expressed the uORF-Luc fusion protein from the TK promoter. Deletions in PTK-uORF-Luc were constructed with in-frame deletions of the CHOP uORF codons 14–34 (Δ14–34), 14–23 (Δ14–23), and 23–34 (Δ24–34). A version of the Δ24–34 version of the PTK-uORF-Luc was also constructed with the uORF1 of ATF4 substituted for the first four codons, including the ATG1 and ATG2, of the CHOP uORF portion of the fusion protein. MEF cells that were derived from S/S (wild-type eIF2α) and A/A (mutant eIF2α-S51A) mice were previously described (24Scheuner D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1091) Google Scholar, 25Jiang H.Y. Wek S.A. McGrath B.C. Scheuner D. Kaufman R.J. Cavener D.R. Wek R.C. Mol. Cell. Biol. 2003; 23: 5651-5663Crossref PubMed Scopus (355) Google Scholar). MEF cells were cultured in Dulbecco's modified Eagle's medium (Cellgro) supplemented with 10% FBS, 1 mm nonessential amino acids, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C. ER stress was induced in MEF cells by the addition of either 0.1 or 1 μm thapsigargin to the medium, followed by incubation for up to 12 h, as indicated. Plasmid transfections were performed using the S/S and A/A MEF cells grown to 40% confluency and the FuGENE 6 transfection reagent (Roche Applied Science). Co-transfections were carried out in triplicate using wild-type or mutant versions of the TK-CHOP-Luc or TK-uORF-Luc plasmids and a Renilla luciferase plasmid serving as an internal control (Promega). 24 h after transfection, MEF cells were treated with 0.1 μm thapsigargin for 12 h or with no ER stress. Shorter periods of time, from 4 to 6 h of stress, also showed significant elevation of CHOP-Luc expression in response to ER stress that supported the stated conclusions. Dual-Luciferase assays were carried out as described by the Promega instruction manual. Values are a measure of a ratio of firefly versus Renilla luciferase units (relative light units) and represent the mean values of three independent transfections. Renilla luciferase values did not change significantly in the dual reporter assays. Results are presented as means ± S.D. that were derived from three independent experiments. Parallel to the Dual-Luciferase assays, the amount of firefly luciferase mRNA in each transfected condition was measured by the qRT-PCR method. MEF cells cultured in the indicated stress conditions or no stress were washed twice with cold phosphate-buffered saline (pH 7.4), followed by lysis using a solution containing 50 mm Tris-HCl (pH 7.9), 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 100 mm NaF, 17.5 mm β-glycerol phosphate, 10% glycerol supplemented with protease inhibitors (100 μm of phenylmethylsulfonyl fluoride, 0.15 μm aprotinin, 1 μm leupeptin, and 1 μm of pepstatin). Lysates were subjected to sonication for 30 s and precleared by centrifugation. Protein content was determined by using a Bio-Rad protein quantitation kit according to the manufacturer's instructions. Equal amounts of proteins were separated by SDS-PAGE, and proteins were then transferred to nitrocellulose filters. Molecular weight markers were included for size determination of proteins in the immunoblot analyses. Transferred filters were then incubated in TBS-T solution containing 20 mm Tris-HCl (pH 7.9), 150 mm NaCl, and 0.2% Tween 20 supplemented with 4% nonfat milk, followed by incubation with TBS-T solution containing the primary antibody specific to the indicated protein. ATF4 antibody was prepared against recombinant protein (18Zhou D. Palam L.R. Jiang L. Narasimhan J. Staschke K.A. Wek R.C. J. Biol. Chem. 2008; 283: 7064-7073Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). CHOP (sc-7351) antibody was obtained from Santa Cruz Biotechnology, and β-actin monoclonal antibody (A5441) was purchased from Sigma. Polyclonal antibody that specifically recognizes phosphorylated eIF2α at Ser-51 was purchased from BioSource (44-728G). Monoclonal antibody that recognizes either phosphorylated or nonphosphorylated forms of eIF2α was provided by Dr. Scot Kimball (Pennsylvania State University, College of Medicine, Hershey). Cell lysates from MEF cells transfected with the indicated plasmids were blotted for firefly luciferase protein by using antibody obtained from Promega (G7451). Following incubation of the filters with the indicated antibodies, the filters were then washed three times in TBS-T followed by incubation with horseradish peroxidase-labeled secondary antibody and chemiluminescent substrate. Proteins in the immunoblot were visualized by exposing filters to x-ray film or by imaging using the LI-COR Odyssey system. Images shown in the figures are representative of three independent experiments. The cDNAs corresponding to the 5′-end of the CHOP mRNAs expressed in S/S MEF cells treated with 0.1 μm thapsigargin, or no stress, were amplified by using an RNA ligase-mediated RACE kit (RLM-RACE kit, Ambion) according to the manufacturer's instructions. Antisense primers corresponding endogenous CHOP mRNA that were used in the assays include the outer primer 5′-GGACGCAGGGTCAAGAGTAG-3′ and inner primer 5′-TCATGAGTGCCATGACTGCACGTGG-3′. The outer primer used for amplifying CHOP-Luc mRNA 5′-end was 5′-CGAATTCGAACACGCAGAT-3′, which was combined with the same inner primer listed above. The amplified product was then analyzed using electrophoresis on a 1.2% agarose gel. The prominent DNA bands were excised, gel-purified, and sequenced. The transcriptional start site was determined as the first nucleotide that is 3′ to the adapter sequence ligated to the 5′ of the mRNA transcripts. MEF cells were transfected with the indicated plasmids, treated with the designated stress conditions and harvested, and total cellular RNA was prepared using TRIzol reagent (Invitrogen) according to the instruction manual. Contaminating DNA was digested with RNase-free DNase (Promega). Single strand cDNA synthesis was carried out using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Quantitation of relative mRNA levels was performed using Light Cycler 480 PCR system (Roche Applied Science) and SYBR Green PCR mix from Applied Biosystems. The amount of firefly luciferase mRNA was measured using Renilla luciferase as an internal control. The oligonucleotide primers used were as follows: firefly luciferase 5′-CTCACTGAGACTACATCAGC-3′ and 5′-TCCAGATCCACAACCTTCGC-3′; Renilla luciferase 5′-GGAATTATAATGCTTATCTACGTGC-3′ and 5′-CTTGCGAAAAATGAAGACCTTTTAC-3′. The endogenous CHOP and ATF4 mRNA levels were measured using the following oligonucleotide primers: CHOP 5′-CCTAGCTTGGCTGACAGAGG-3′ and 5′-CTGCTCCTTCTCCTTCATGC-3′; ATF4 5′-GCCGGTTTAAGTTGTGTGCT-3′ and 5′-CTGGATTCGAGGAATGTGCT-3′. Quantitation of target genes was normalized using the reference β-actin. Primers used were 5′-GTATGGAATCCTGTGGCATC-3′ and 5′-AAGCACTTGCGGTGCACGAT-3′. Light Cycler 480 software (version 1.2.9.11) was used to perform quantification and to generate Cp values. Values are a representation of three independent experiments, with standard deviations as indicated. MEF cells were cultured as described above and treated with 1 μm thapsigargin for 6 h or no stress. 10 min prior to harvesting, cells were treated with 50 μg/ml cycloheximide. Cells were washed with cold phosphate-buffered saline (pH 7.4) solution containing 50 μg/ml cycloheximide, and cell lysates were prepared in a solution of 20 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 100 mm NaCl, and 0.4% Nonidet P-40 supplemented with 50 μg/ml cycloheximide. Cell lysates were passed though a 23-gauge needle and incubated on ice for 10 min. The cell lysate was precleared with brief centrifugation (10,000 rpm for 10 min at 4 °C) and then layered onto a 10–50% sucrose gradient solution containing 20 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 100 mm NaCl, and 50 μg/ml cycloheximide. The sucrose gradients were then subjected to centrifugation in a Beckman SW-41Ti rotor for 2 h at 40,000 rpm at 4 °C. A portion of unfractionated cell lysate was used for determining total mRNA levels of CHOP, ATF4, and β-actin. Gradients were fractionated using a Biocomp Gradient Station, and absorbance of RNA at 254 nm was recorded using an in-line UV monitor. Equivalent amounts of synthetic poly(A)+ luciferase RNA (10 ng/ml) purchased from Promega were added to each collected fraction. RNA was isolated from each fraction using the TRIzol LS reagent (Invitrogen), and synthesis of single-stranded cDNA was performed using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Prepared cDNA was used to measure the relative mRNA levels for CHOP, ATF4, and encoded β-actin. qRT-PCR data were normalized with the luciferase mRNA that was added prior to RNA isolation. Data represented are the result of three independent experiments with standard deviations as indicated. Alternatively, MEF cells cultured to 40% confluency were transfected with wild-type TK-CHOP-Luc plasmid or mutant variants with ΔATG1 and ΔATG2, individually or in combination, using the FuGENE 6 (Roche Applied Science) transfection reagent. After 24 h of transfection, cells were treated with 0.1 μm thapsigargin up to 6 h or no stress. Cell lysates prepared were then subjected to sucrose gradient analyses and fractionated as described above. Equivalent amounts of bacterial control RNA (Affymetrix) were added to each sucrose fraction to serve as internal control for the RNA isolation and in qRT-PCR analysis. RNA isolated from each fraction was used in preparation of single strand cDNA synthesis as described. The amount of firefly luciferase in each fraction was quantitated and normalized to the THR mRNA of bacterial control added in the RNA mixture. Oligonucleotide primers used for the THR mRNA measurement were as follows: forward 5′-AGGATGACGAGACCCAAATG-3′ and reverse 5′-TGATCGCAGCAATGAGGATA-3′. In response to ER stress, eIF2∼P triggers preferential translation of ATF4 mRNA concurrent with repressed global translation initiation. This is illustrated by treatment of MEF cells with thapsigargin, a potent ER stress agent (5Schröder M. Kaufman R.J. Annu. Rev. Biochem. 2005; 74: 739-789Crossref PubMed Scopus (2444) Google Scholar). Within 1 h of thapsigargin exposure, wild-type MEF cells displayed an enhanced eIF2∼P accompanied by increased expression of ATF4 protein (Fig. 1A). By contrast, MEF cells containing Ala for the eIF2α phosphorylation site Ser-51 (A/A) showed no eIF2∼P and minimal levels of ATF4 protein. In addition to translational control, ATF4 was reported to be subject to transcriptional regulation, with a 3-fold increase in ATF4 mRNA following 6 h of the ER stress (Fig. 1B) (26Dey S. Baird T.D. Zhou D. Palam L.R. Spandau D.F. Wek R.C. J. Biol. Chem. 2010; 285: 33165-33174Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). This increase in ATF4 mRNA levels in response to ER stress is substantially blocked in the A/A cells. ATF4 is a transcriptional activator of ISR genes, such as CHOP mRNA. Levels of CHOP protein and mRNA are sharply increased in response to ER stress by a mechanism requiring eIF2∼P (Fig. 1, A and B). Given that increased expression of CHOP protein occurs despite high levels of eIF2∼P, we reasoned that translation of CHOP mRNA may be favored even when global translation initiation is severely restricted. To test this idea, we carried out a polysome analysis using sucrose gradient centrifugation. Thapsigargin treatment of MEF cells significantly reduced polysome levels, concomitant with elevated levels of free ribosomes and monosomes, which is consistent with repressed translation initiation (Fig. 2). This reduction in translation initiation is dependent on eIF2∼P, as the polysome profile was largely unchanged when A/A cells were treated with thapsigargin (supplemental Fig. 1). During nonstressed conditions, the levels of ATF4 transcript, measured as the percentage of total ATF4 mRNA, were most abundant in the monosome and small polysome fractions of the sucrose gradient. In this condition, only 28% of the ATF4 mRNAs were associated with large polysomes consisting of transcripts associated with four or more ribosomes. By comparison, upon ER stress, there was a substantial shift of ATF4 transcripts to the large polysome fractions (67% associated with large polysomes), consistent with earlier reports that ATF4 mRNA is preferentially translated upon eIF2∼P (6Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2415) Google Scholar, 7Lu P.D. Harding H.P. Ron D. J. Cell Biol. 2004; 167: 27-33Crossref PubMed Scopus (662) Google Scholar, 8Vattem K.M. Wek R.C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 11269-11274Crossref PubMed Scopus (1135) Google Scholar). CHOP mRNA displayed a similar distribution pattern in the polysome profiles as the ATF4 transcripts (Fig. 2). In the absence of stress, CHOP mRNA was most abundant in the monosomes and small polysomes, whereas ER stress triggered increased association of this transcript with the large polysomes (25% associated with large polysomes in nonstressed conditions compared with 52% during ER stress). As a control, we also measured actin mRNA among the fractions in the sucrose gradient and found that this transcript was largely insensitive to ER stress. We next addressed whether the 5′-leader of the CHOP mRNA directs translational control in response to ER stress. The transcriptional start site of the CHOP transcript was determined in MEF cells in the presence or absence of stress by 5′-RACE and DNA sequencing (Fig. 3A). Transcription of CHOP occurs at the same site independent of stress conditions, leading to a 5′-leader sequence 162 nucleotides in length. The CHOP leader sequence encodes a single uORF representing a 34-residue polypeptide that is highly conserved among vertebrates (Fig. 3B) (27Jousse C. Bruhat A. Carraro V. Urano F. Ferrara M. Ron D. Fafournoux P. Nucleic Acids Res. 2001; 29: 4341-4351Crossref PubMed Scopus (107) Google Scholar). Notable among the conserved residues are Met residues at positions 1 and 4 (encoded by codons designated ATG1 and ATG2), providing for two possible initiation codon" @default.
• W2003924215 created "2016-06-24" @default.
• W2003924215 creator A5079999357 @default.
• W2003924215 creator A5082229689 @default.
• W2003924215 creator A5088156146 @default.
• W2003924215 date "2011-04-01" @default.
• W2003924215 modified "2023-10-05" @default.
• W2003924215 title "Phosphorylation of eIF2 Facilitates Ribosomal Bypass of an Inhibitory Upstream ORF to Enhance CHOP Translation" @default.
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