Global Translational Responses to Oxidative Stress Impact upon Multiple Levels of Protein Synthesis

Abstract

Global inhibition of protein synthesis is a common response to stress conditions. We have analyzed the regulation of protein synthesis in response to oxidative stress induced by exposure to H2O2 in the yeast Saccharomyces cerevisiae. Our data show that H2O2 causes an inhibition of translation initiation dependent on the Gcn2 protein kinase, which phosphorylates the α-subunit of eukaryotic initiation factor-2. Additionally, our data indicate that translation is regulated in a Gcn2-independent manner because protein synthesis was still inhibited in response to H2O2 in a gcn2 mutant. Polysome analysis indicated that H2O2 causes a slower rate of ribosomal runoff, consistent with an inhibitory effect on translation elongation or termination. Furthermore, analysis of ribosomal transit times indicated that oxidative stress increases the average mRNA transit time, confirming a post-initiation inhibition of translation. Using microarray analysis of polysome- and monosome-associated mRNA pools, we demonstrate that certain mRNAs, including mRNAs encoding stress protective molecules, increase in association with ribosomes following H2O2 stress. For some candidate mRNAs, we show that a low concentration of H2O2 results in increased protein production. In contrast, a high concentration of H2O2 promotes polyribosome association but does not necessarily lead to increased protein production. We suggest that these mRNAs may represent an mRNA store that could become rapidly activated following relief of the stress condition. In summary, oxidative stress elicits complex translational reprogramming that is fundamental for adaptation to the stress. Global inhibition of protein synthesis is a common response to stress conditions. We have analyzed the regulation of protein synthesis in response to oxidative stress induced by exposure to H2O2 in the yeast Saccharomyces cerevisiae. Our data show that H2O2 causes an inhibition of translation initiation dependent on the Gcn2 protein kinase, which phosphorylates the α-subunit of eukaryotic initiation factor-2. Additionally, our data indicate that translation is regulated in a Gcn2-independent manner because protein synthesis was still inhibited in response to H2O2 in a gcn2 mutant. Polysome analysis indicated that H2O2 causes a slower rate of ribosomal runoff, consistent with an inhibitory effect on translation elongation or termination. Furthermore, analysis of ribosomal transit times indicated that oxidative stress increases the average mRNA transit time, confirming a post-initiation inhibition of translation. Using microarray analysis of polysome- and monosome-associated mRNA pools, we demonstrate that certain mRNAs, including mRNAs encoding stress protective molecules, increase in association with ribosomes following H2O2 stress. For some candidate mRNAs, we show that a low concentration of H2O2 results in increased protein production. In contrast, a high concentration of H2O2 promotes polyribosome association but does not necessarily lead to increased protein production. We suggest that these mRNAs may represent an mRNA store that could become rapidly activated following relief of the stress condition. In summary, oxidative stress elicits complex translational reprogramming that is fundamental for adaptation to the stress. Cells must be able to maintain their intracellular homeostasis in the face of changing conditions. Typically, they respond by invoking complex regulatory mechanisms, including global inhibition of translation (1Clemens M.J. Prog. Mol. Subcell. Biol. 2001; 27: 57-89Crossref PubMed Scopus (171) Google Scholar, 2Proud C.G. Semin. Cell Dev. Biol. 2005; 16: 3-12Crossref PubMed Scopus (305) Google Scholar). This reduction in protein synthesis may prevent continued gene expression during potentially error-prone conditions as well as allow for the turnover of existing mRNAs and proteins, whilst gene expression is reprogrammed to deal with the stress. Four mammalian protein kinases that inhibit translation initiation by phosphorylating eukaryotic initiation factor-2 (eIF2) 2The abbreviations used are: eIF2, eukaryotic initiation factor-2; ER, endoplasmic reticulum; ROS, reactive oxygen species; TAP, tandem affinity purification; RT, reverse transcription. have been identified. GCN2 (the amino acid control kinase), PKR (the double-stranded protein kinase activated by RNA), HRI (the heme-regulated inhibitor), and PERK/PEK (the PKR-like endoplasmic reticulum eIF2α kinase) are regulated independently in response to various different cellular stresses (2Proud C.G. Semin. Cell Dev. Biol. 2005; 16: 3-12Crossref PubMed Scopus (305) Google Scholar, 3Dever T.E. Cell. 2002; 108: 545-556Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar). For example, PERK has been found in all multicellular eukaryotes and is a component of the unfolded protein response. Consistent with its central role in the endoplasmic reticulum (ER) stress response, cells lacking PERK fail to phosphorylate eIF2α and do not down-regulate protein synthesis during ER stress conditions (4Bertolotti A. Zhang Y. Hendershot L.M. Harding H.P. Ron D. Nat. Cell Biol. 2000; 2: 326-332Crossref PubMed Scopus (2133) Google Scholar, 5Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1944) Google Scholar). Attenuating protein synthesis may act to reduce the burden of newly synthesized ER client proteins on the ER folding machinery. Additionally, eIF2 phosphorylation induces translation of specific mRNAs, such as that encoding the metazoan activating transcription factor-4 (6Vattem K.M. Wek R.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11269-11274Crossref PubMed Scopus (1148) Google Scholar, 7Lu P.D. Harding H.P. Ron D. J. Cell Biol. 2004; 11: 27-33Crossref Scopus (667) Google Scholar). Activating transcription factor-4 mediates the integrated stress response, the targets of which include genes encoding proteins involved in amino acid metabolism and resistance to oxidative stress, ultimately protecting against the deleterious consequences of ER oxidation (8Harding 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 (2410) Google Scholar). In yeast, Gcn2 is the sole eIF2 kinase and phosphorylates eIF2α in response to nutrient starvation and sodium or rapamycin exposure. eIF2 is a guanine nucleotide-binding factor and, in the GTP-bound form, interacts with the initiator methionyl-tRNA (Met-tRNAiMet) to form a ternary complex (eIF2·GTP·Met-tRNAiMet) that is competent for translation initiation. Following each round of initiation, eIF2 is released from the ribosome as a binary complex with GDP. GDP is replaced by GTP in a guanine nucleotide exchange reaction promoted by eIF2B. Phosphorylation of eIF2α by Gcn2 converts eIF2 from a substrate to an inhibitor of the guanine nucleotide exchange factor eIF2B (9Pavitt G.D. Ramaiah K.V. Kimball S.R. Hinnebusch A.G. Genes Dev. 1998; 12: 514-526Crossref PubMed Scopus (213) Google Scholar). The resulting decrease in eIF2B activity leads to reduced ternary complex levels. Paradoxically, translation of the GCN4 mRNA is activated in response to low ternary complex levels in a mechanism involving short upstream open reading frames (10Hinnebusch A.G. Annu. Rev. Microbiol. 2005; 59: 407-450Crossref PubMed Scopus (910) Google Scholar). Gcn4 is a transcription factor that activates gene expression of many targets, including amino acid biosynthetic genes (11Natarajan K. Meyer M.R. Jackson B.M. Slade D. Roberts C. Hinnebusch A.G. Marton M.J. Mol. Cell. Biol. 2001; 21: 4347-4368Crossref PubMed Scopus (569) Google Scholar). Thus, analogous to the mammalian integrated stress response, activation of Gcn4 serves to overcome the imposed starvation, which initially led to the translational control. More recently, we used microarray analysis combined with polysome analysis to demonstrate that lowering ternary complex levels results in widespread translational reprogramming, identifying a fundamental role for translational control in the adaptation to nutrient limitation (12Smirnova K. G.D. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar). are to reactive oxygen such as the and the during the of metabolism or following exposure to to to PubMed Scopus Google Scholar, B. in and Scholar). against cells mechanisms, including and Cell Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). cells to oxidative stress by global including genes encoding and D. Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar, B. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar). we have that oxidative stress by exposure to H2O2 results in a and inhibition of protein synthesis D. J. 2003; PubMed Scopus Google Scholar). Thus, is in the gene expression are the cellular In we have analyzed the regulation of protein synthesis in response to oxidative stress induced by exposure to Our data show that H2O2 causes a inhibition of protein synthesis in by phosphorylation of In we that protein synthesis is by a Gcn2-independent inhibition of ribosomal We used microarray analysis to demonstrate that certain mRNAs are following oxidative stress, that translational control is a component of the cellular response to oxidative stress. and Saccharomyces used in are of PubMed Scopus Google Scholar). affinity from to by with a gene to with in yeast and or yeast amino and with amino and in and by the of of for the activity for used Hinnebusch A.G. C. Mol. Cell. Biol. PubMed Scopus Google Scholar). levels are as of of conditions on and and as Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). proteins by analysis complex of rate of protein synthesis was in cells with H2O2 for and for the with D. J. 2003; PubMed Scopus Google Scholar). proteins as B. M. B. 2001; PubMed Scopus Google Scholar). For the analysis of ribosome on yeast to and with H2O2 as and analyzed as Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar). and polysome For transit cells with and in and was in the and and protein by J. Cell. PubMed Scopus Google Scholar). times by the of amino proteins and proteins released from Polysome and as (12Smirnova K. G.D. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar). and for in a and was a For of and from the for and Thus, we on the and mRNAs, some mRNAs that are in the of the analysis by reverse transcription was the and the yeast to the of or was to the of targets was and to the for The the and with to and For with (12Smirnova K. G.D. Mol. Cell. Biol. 2005; PubMed Scopus Google we used analysis to the data as well as analysis the of in analysis was to of as from used to the not and that the we have is and all of the to microarray data A. J. C. J. T. J.C. H. A. U. S. J. R. J. M. Nat. 2001; PubMed Scopus Google Scholar). The data are with H2O2 for and the rate of protein synthesis was during the by the of H2O2 a inhibition of protein synthesis with inhibition The inhibition of protein synthesis by H2O2 to translational activity by the of are ribosomes that are be on and by from the including to and ribosomal and was a of ribosomes from the the or following with The of ribosomes in the of a is of translation initiation. Polysome from cells with during the analyzed to that effect not because of activating global mRNA in with was found to during effect on mRNA eIF2α of the translational regulatory in yeast phosphorylation of the eIF2α by the Gcn2 Gcn2 is activated in response to amino acid starvation and ultimately an inhibition of translation initiation (10Hinnebusch A.G. Annu. Rev. Microbiol. 2005; 59: 407-450Crossref PubMed Scopus (910) Google Scholar). the translation inhibition we eIF2α phosphorylation by analysis eIF2α Phosphorylation of eIF2α was in response to all and was H2O2 with polysome decrease of GCN2 phosphorylation of eIF2α and results in translational resistance to amino acid starvation (10Hinnebusch A.G. Annu. Rev. Microbiol. 2005; 59: 407-450Crossref PubMed Scopus (910) Google Scholar). inhibition of translation initiation was in a gcn2 in response to oxidative stress because of GCN2 the of in response to H2O2 in the gcn2 following H2O2 with the an effect the post-initiation of translation of translation initiation was in a a form of eIF2α lacking that is the of phosphorylation not The complex to Gcn2 and is to the activation of Gcn2 by The inhibition of translation initiation promoted by H2O2 stress was reduced by of or that the activation of Gcn2 by H2O2 is to a mechanism to amino acid starvation Phosphorylation of eIF2α causes a global inhibition of protein synthesis as well as translational activation of which a protein A.G. Sci. Full Text PDF PubMed Scopus Google Scholar). GCN4 expression has been a the GCN4 and expression of the This a to inhibition of eIF2B activity by eIF2α expression from was following H2O2 for a was in response to H2O2 This was dependent on Gcn2 because was in a gcn2 mutant. GCN4 expression was inhibited of H2O2 the that phosphorylation of eIF2α was H2O2 the for we the rate of protein synthesis in a gcn2 that could not inhibit translation initiation in response to H2O2 and found that protein synthesis was still inhibited in the gcn2 to a these data indicate that H2O2 exposure causes a global inhibition of protein synthesis inhibition of translation initiation as well as an mechanism that does not initiation. analysis that H2O2 an inhibition of translation initiation The for these in the of the translation elongation inhibitor to prevent continued elongation and ribosomal inhibition of ribosomal transit of be as a of are in the of a rate of ribosomal is and to I. R.J. in Scholar). We the of translation initiation by cells to lacking following the with from the results in a inhibition of translation initiation Mol. Biol. Cell. 2000; 11: PubMed Scopus Google and we that allow to effect on ribosomal transit the of translation initiation. Cells or with H2O2 to the to lacking in a inhibition of translation initiation as and ribosomal in control resulting in a of of the In contrast, following the H2O2 and of the for transit times be by the of protein and released from ribosomes J. Cell. PubMed Scopus Google Scholar). Using we that the average mRNA transit in a gcn2 is H2O2 increased the transit by to a gcn2 could not inhibit translation initiation these data that is an inhibition of translation elongation or in response to Global mRNAs following of expression such as microarray be used to protein synthesis T. 2004; PubMed Scopus Google Scholar). We used to mRNAs that are regulated in response to oxidative stress conditions. Cell from yeast cells with or H2O2 for because they a effect on translation initiation but inhibited protein synthesis to different The with H2O2 reduced by with of with The the of cells with the from the stress conditions and in or from and control yeast cells to The resulting and to The analysis was in and the data and the the of mRNAs in and and control yeast For example, translation is inhibited the of initiation, the association of mRNAs with the be used as a that the translational activity of specific mRNAs T. 2004; PubMed Scopus Google Scholar). We the as the of an mRNA and used to mRNAs that are in response to the amino acid starvation and (12Smirnova K. G.D. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar). translation is inhibited by as is the for increased polysome association be used as a of the translational of was to a to candidate mRNAs that are regulated following H2O2 stress. we the mRNAs in and during stress conditions with the mRNAs in and during control conditions We that mRNAs that overcome the initiation have ribosomes during stress resulting in an increase in with on a for and cells and the the in translational activity for the mRNAs This analysis the of mRNAs that overcome the in translation initiation because they ribosomes and following the stress of mRNAs, we that is to mRNAs that are to the ribosomal transit because they during stress conditions with control resulting in an increase in the and Thus, we able to mRNAs that overcome the inhibition of translation initiation the as well as the mRNAs that overcome the inhibition of ribosomal transit to an increase in the the stress conditions different translational Using a for the in translational activity mRNAs and and mRNAs and following with or and The of these mRNAs to the ribosomal transit because they during stress conditions with control conditions and and a of mRNAs are following each stress we mRNAs that following the on the and we the regulated mRNAs on the show that the mRNAs or different for each stress condition. the response to these stress conditions is in of and translational we the in translational activity against the in for each stress mRNAs that and the and mRNAs that and the are with regulated mRNAs and This analysis not in and the translational response to This may be because of the short exposure times used in these the genes that of and of induced the by the and H2O2 and of the and translation has been following rapamycin and amino acid starvation of yeast and has been (12Smirnova K. G.D. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar, T. J. Nat. Biol. 2003; PubMed Scopus Google Scholar). In contrast, was or for stress, that of the and translational activity is a (12Smirnova K. G.D. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar). We the microarray results analysis for a of mRNAs in of the of the mRNA and the in The used for the microarray analysis that are used for each This in or and for mRNAs global translation has been inhibited by the stress. This is by mRNAs, which by the or to the microarray the analysis that mRNA was with and following stress conditions consistent with the global inhibition of translation analysis could be used to the that from the microarray For example, analysis that the mRNA was increased in in and following stress that and mRNAs increased in and following the H2O2 and that the mRNA was increased in the following the H2O2 Our data show that mRNAs with in response to oxidative stress conditions. to results in increased protein we analyzed protein synthesis and levels for a of proteins that are with a protein cells with or H2O2 for and proteins with during the of analysis a global inhibition of protein synthesis protein was following but was for the H2O2 with proteins The microarray analysis indicated that the mRNA increased in in and following stress conditions. was by analysis during conditions In contrast, was increased following the microarray analysis indicated an increase in the translational activity of and in response to In with of proteins increased synthesis of proteins In the of increased protein levels by in the high levels of short the microarray and to the of mRNAs that to the in ribosomal transit the mRNA not show increase in following the H2O2 Thus, of mRNA in increased protein production. analysis indicated that the translational activity of and mRNAs was increased in response to increase in protein was for these proteins as analyzed by of proteins these data indicate that low results in increased protein high increases polyribosome association with certain mRNAs but does not necessarily in increased protein production. of mRNAs by data analyzed in of the of the regulated genes and the that in translational activity have on cellular to the and the Saccharomyces Genes that regulated the translational to the global are in these and The stress conditions of The low induced as be a and an which reduce H2O2 S. 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The of proteins in cells the is fundamental to their and because proteins of the in cells and and regulatory of protein levels translational regulation a to the because of the of the regulatory of protein levels is of the in the of cells during stress conditions. translation initiation is a complex regulated involving with ribosomal and mRNAs Annu. Rev. 2004; PubMed Scopus Google Scholar). The initiation of protein synthesis is and is a of In mammalian eIF2α is by PERK in response to oxidative stress as of the integrated stress response (8Harding 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 (2410) Google Scholar). does not and H2O2 stress has not been to inhibit protein Gcn2 phosphorylates eIF2α in response to nutrient starvation and sodium or rapamycin exposure. of amino leads to an of which activates the Gcn2 protein kinase its The activating Gcn2 in response to rapamycin or are not well to by phosphorylation of Gcn2 Hinnebusch A.G. Genes Dev. 2003; PubMed Scopus Google Scholar). activation of Gcn2 by rapamycin and still the of Gcn2 as well as and which are to the activation of Gcn2 by J. Wek R.C. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the inhibition of translation initiation in response to H2O2 and stress may an of a of amino and amino in proteins are to oxidation by 2003; PubMed Scopus Google Scholar). amino be in yeast and for example, and is following exposure to of H2O2 A. 2003; PubMed Scopus Google to used in the levels of amino are the oxidative on the amino acid may an amino acid starvation response. the proteins and that are for may be to resulting in an of and activation of For example, oxidative to including has been in the of J. 2005; PubMed Scopus Google Scholar, K. D. T. H. A. S. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google and the and activity of R. S. PubMed Scopus Google Scholar). We have the and of mRNAs that are regulated in response to stresses that eIF2B activity (12Smirnova K. G.D. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar). acid starvation leads to an of and activation of exposure to the eIF2B the stresses the translation initiation factor they have different in of the specific mRNAs that are This a specific stress response that adaptation to the stress (12Smirnova K. G.D. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar). We have these stresses act must be on the translational that ultimately which mRNAs are following stress. The of oxidative stress on ribosomal initiation and transit is because that control of the translational a to initiation be used to a In to amino acid starvation, protein synthesis is still inhibited in response to H2O2 in the of ribosomal is following H2O2 stress, consistent with an inhibition of translation elongation or termination. the average mRNA transit is increased by in a gcn2 confirming that H2O2 causes a post-initiation inhibition of protein of mRNA expression levels by translation elongation or is is that cells the rate of protein synthesis in response to different conditions or by changing the of elongation in C.G. N. of Scholar). oxidative stress in mammalian cells elicits a increase in eIF2 phosphorylation and oxidative which is to to an inhibition of translation J. A. C.G. J. 2002; PubMed Scopus Google Scholar, A. J. M. A. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Attenuating ribosomes in response to stress as to ribosomal initiation, the that ribosomes to mRNAs and rapidly protein synthesis the stress is or For an oxidative stress prevent continued protein synthesis during potentially error-prone conditions. are in which the expression of mRNAs is regulated of ribosomal transit times in N. N. of Scholar). is by the in which the rate of ribosomal transit is increased in response to Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Our data indicate that the of mRNAs that are following H2O2 stress ribosomes in following the stress condition. for be an of these mRNAs overcome translation The stress response genes that are activated or by a of stress including such as H2O2 D. Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar). The genes that are induced as of the stress response that are to against the stress as well as the resulting cellular our data indicate that of the mRNAs that are in response to H2O2 show increases in levels. levels in the of translation may a of mRNAs that become rapidly the stress is In the mRNAs that are during exposure to H2O2 may the that are to and H2O2 and its is that the low results in increased of stress protective proteins, the high increases the of ribosomes with certain mRNAs but does not in increased protein production. The low used in has been to an response cells become to a and with H2O2 PubMed Scopus Google Scholar). Our data indicate that may be by increased of stress protective proteins, including the low H2O2 In contrast, the high H2O2 increases ribosome association on mRNAs, which become rapidly the oxidative stress is In summary, our data indicate that the response to oxidative stress is translational and We the for the of S. A. and the of for and with