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Gene expression during acute and prolonged hypoxia is regulated by distinct mechanisms of translational control

2006; Springer Nature; Volume: 25; Issue: 5 Linguagem: Inglês

10.1038/sj.emboj.7600998

1460-2075

Marianne Koritzinsky, Michaël G. Magagnin, Twan van den Beucken, Renaud Seigneuric, Kim G.M. Savelkouls, Josée Dostie, Stéphane Pyronnet, Randal J. Kaufman, Sherry A. Weppler, Jan Willem Voncken, Philippe Lambin, Constantinos Koumenis, Nahum Sonenberg, Bradly G. Wouters,

Mitochondrial Function and Pathology

Article9 February 2006free access Gene expression during acute and prolonged hypoxia is regulated by distinct mechanisms of translational control Marianne Koritzinsky Marianne Koritzinsky Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Michaël G Magagnin Michaël G Magagnin Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Twan van den Beucken Twan van den Beucken Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Renaud Seigneuric Renaud Seigneuric Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Kim Savelkouls Kim Savelkouls Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Josée Dostie Josée Dostie Department of Biochemistry, McGill Cancer Centre, McGill University, Canada Search for more papers by this author Stéphane Pyronnet Stéphane Pyronnet Department of Biochemistry, McGill Cancer Centre, McGill University, Canada Search for more papers by this author Randal J Kaufman Randal J Kaufman Howard Hughes Medical Institute, University of Michigan Medical Center, USA Search for more papers by this author Sherry A Weppler Sherry A Weppler Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Jan Willem Voncken Jan Willem Voncken Department of Molecular Genetics, University of Maastricht, The Netherlands Search for more papers by this author Philippe Lambin Philippe Lambin Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Constantinos Koumenis Constantinos Koumenis Departments of Radiation Oncology and Cancer Biology, Wake Forest University School of Medicine, USA Search for more papers by this author Nahum Sonenberg Nahum Sonenberg Department of Biochemistry, McGill Cancer Centre, McGill University, Canada Search for more papers by this author Bradly G Wouters Corresponding Author Bradly G Wouters Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Marianne Koritzinsky Marianne Koritzinsky Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Michaël G Magagnin Michaël G Magagnin Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Twan van den Beucken Twan van den Beucken Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Renaud Seigneuric Renaud Seigneuric Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Kim Savelkouls Kim Savelkouls Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Josée Dostie Josée Dostie Department of Biochemistry, McGill Cancer Centre, McGill University, Canada Search for more papers by this author Stéphane Pyronnet Stéphane Pyronnet Department of Biochemistry, McGill Cancer Centre, McGill University, Canada Search for more papers by this author Randal J Kaufman Randal J Kaufman Howard Hughes Medical Institute, University of Michigan Medical Center, USA Search for more papers by this author Sherry A Weppler Sherry A Weppler Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Jan Willem Voncken Jan Willem Voncken Department of Molecular Genetics, University of Maastricht, The Netherlands Search for more papers by this author Philippe Lambin Philippe Lambin Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Constantinos Koumenis Constantinos Koumenis Departments of Radiation Oncology and Cancer Biology, Wake Forest University School of Medicine, USA Search for more papers by this author Nahum Sonenberg Nahum Sonenberg Department of Biochemistry, McGill Cancer Centre, McGill University, Canada Search for more papers by this author Bradly G Wouters Corresponding Author Bradly G Wouters Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands Search for more papers by this author Author Information Marianne Koritzinsky1, Michaël G Magagnin1,‡, Twan van den Beucken1,‡, Renaud Seigneuric1, Kim Savelkouls1, Josée Dostie2, Stéphane Pyronnet2, Randal J Kaufman3, Sherry A Weppler1, Jan Willem Voncken4, Philippe Lambin1, Constantinos Koumenis5, Nahum Sonenberg2 and Bradly G Wouters 1 1Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, The Netherlands 2Department of Biochemistry, McGill Cancer Centre, McGill University, Canada 3Howard Hughes Medical Institute, University of Michigan Medical Center, USA 4Department of Molecular Genetics, University of Maastricht, The Netherlands 5Departments of Radiation Oncology and Cancer Biology, Wake Forest University School of Medicine, USA ‡These authors contributed equally to this work *Corresponding author. Department of Radiation Oncology (Maastro), GROW Research Institute, University of Maastricht, UNS50/23 Postbus 616, 6200 MD Maastricht, The Netherlands. Tel.: +31 43 388 2912; Fax: +31 43 388 4540; E-mail: [email protected] The EMBO Journal (2006)25:1114-1125https://doi.org/10.1038/sj.emboj.7600998 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Hypoxia has recently been shown to activate the endoplasmic reticulum kinase PERK, leading to phosphorylation of eIF2α and inhibition of mRNA translation initiation. Using a quantitative assay, we show that this inhibition exhibits a biphasic response mediated through two distinct pathways. The first occurs rapidly, reaching a maximum at 1–2 h and is due to phosphorylation of eIF2α. Continued hypoxic exposure activates a second, eIF2α-independent pathway that maintains repression of translation. This phase is characterized by disruption of eIF4F and sequestration of eIF4E by its inhibitor 4E-BP1 and transporter 4E-T. Quantitative RT–PCR analysis of polysomal RNA indicates that the translation efficiency of individual genes varies widely during hypoxia. Furthermore, the translation efficiency of individual genes is dynamic, changing dramatically during hypoxic exposure due to the initial phosphorylation and subsequent dephosphorylation of eIF2α. Together, our data indicate that acute and prolonged hypoxia regulates mRNA translation through distinct mechanisms, each with important contributions to hypoxic gene expression. Introduction The presence of hypoxic and anoxic areas in human tumors is well documented, and is prognostic for poor outcome (reviewed in Harris, 2002; Wouters et al, 2002). The clinical importance of tumor hypoxia results from its ability to protect cells against both radiation and chemotherapy and from the fact that it can provide a selection pressure for apoptotically resistant cells (Graeber et al, 1996). Furthermore, the cellular response to hypoxia causes important changes in gene expression that affect cell behavior and influence patient prognosis. There has been particular focus on changes mediated through the family of hypoxia-inducible transcription factors (HIFs). HIF-1 and HIF-2 promote transcription of more than 60 putative downstream genes (for a review see Semenza, 2003) that affect hypoxia tolerance, energy homeostasis, angiogenesis and tumor growth. Although the transcriptional response to hypoxia is clearly very important (Ryan et al, 1998; Tang et al, 2004; Leek et al, 2005), tumor cells also experience short, transient exposures to hypoxia and/or anoxia that occur over time frames too fast for an effective transcriptional response. Transient changes in oxygenation occur owing to the abnormal vasculature found in most tumors, characterized by immature, leaky and improperly formed vessels. Perfusion of these vessels can change dynamically in time, leading to rapid but transient episodes of severe hypoxia in the tumor cells dependent upon them (Bennewith and Durand, 2004; Cardenas-Navia et al, 2004). Consequently, post-transcriptional responses are presumably important for adaptation to cycling oxygenation in tumors. Control of mRNA translation during hypoxia is emerging as an important cellular response to hypoxia (Koumenis et al, 2002; Koritzinsky et al, 2005; Wouters et al, 2005). As protein synthesis is energy costly, inhibition of mRNA translation may represent an active response to prevent loss of energy homeostasis during hypoxia. Indeed, it has been shown that overall mRNA translation is severely but reversibly inhibited during hypoxia (Koumenis et al, 2002; Erler et al, 2004; Bi et al, 2005) with kinetics that precede ATP depletion (Lefebvre et al, 1993). Furthermore, regulation of mRNA translation can have a significant and rapid impact on individual gene expression. This is because the sensitivity of individual genes to changes in overall translation varies widely and in a manner that reflects the molecular mechanisms responsible for controlling translation (Johannes et al, 1999; Harding et al, 2000). Regulation of gene expression through control of mRNA translation is important during various pathologies including cancer (Holland et al, 2004). The mechanisms responsible for inhibiting translation during hypoxia are not yet fully understood. We have previously investigated the involvement of the endoplasmic reticulum (ER) kinase PERK in the hypoxia-induced downregulation of protein synthesis (Koumenis et al, 2002). PERK is activated as part of the evolutionarily conserved unfolded protein response (UPR) (reviewed in Schroder and Kaufman, 2005). It phosphorylates eIF2α, a subunit of eIF2, which in its GTP-bound form recruits the aminoacylated tRNA to the 40S ribosomal subunit. The exchange of GDP for GTP is mediated by the guanine nucleotide exchange factor eIF2B. Ser51-phosphorylated eIF2α inhibits eIF2B, resulting in inhibition of translation initiation. eIF2α phosphorylation results in a set of molecular events collectively termed the integrated stress response. These include the inhibition of global mRNA translation in conjunction with induced expression of the transcription factor ATF4 and its downstream target genes (Harding et al, 2003). We showed that hypoxia rapidly activated PERK, which led to reversible phosphorylation of eIF2α (Koumenis et al, 2002). Hypoxia-induced inhibition of protein synthesis was severely attenuated in cells without functional PERK. After prolonged periods of hypoxia, PERK-deficient cells did show partial inhibition, suggesting that protein synthesis is regulated through additional mechanisms. Another candidate mechanism for inhibiting translation during hypoxia is disruption of the cap-binding protein complex eIF4F, which consists of eIF4E, eIF4A and eIF4G (for recent reviews see Gebauer and Hentze, 2004; Holcik and Sonenberg, 2005). eIF4E participates in a protein bridge between the mRNA and the ribosome by its simultaneous interaction with the mRNA 5′ cap structure and the large scaffolding protein eIF4G, which in turn interacts with eIF3 that is bound to the 40S ribosomal subunit. eIF4E is regulated through a set of binding proteins (4E-BPs) that bind reversibly to eIF4E in their hypophosphorylated form, and this obstructs the interaction between eIF4E and eIF4G. The 4E-BP1 protein becomes hyperphosphorylated in response to a number of stimuli, such as insulin, hormones, growth factors, mitogens and cytokines, as a result of activation of the PI3-kinase/Akt/FRAPmTOR pathway (Hay and Sonenberg, 2004). It remains unclear to what degree the lack of eIF4F assembly contributes to inhibition of translation during tumor hypoxia. Several studies have investigated the combined consequences of ischemia/reperfusion on eIF4F-related proteins in rat brains (reviewed in DeGracia et al, 2002). Proteolysis of eIF4G was reported during ischemia and reperfusion in vivo (Neumar et al, 1998; Martin de la Vega et al, 2001), but not in neuronal cells cultured in vitro (NGF differentiated PC12 cells) (Martin et al, 2000). The reports addressing the expression and phosphorylation status of eIF4E during ischemia are conflicting, but 4E-BP1 dephosphorylation has been demonstrated both in vivo and in vitro (Martin et al, 2000; Martin de la Vega et al, 2001). The acuteness and complexity of ischemia/reperfusion stress and the high sensitivity of neurons to deprivation and reconstitution of both oxygen and nutrients are distinct properties of this model system and thus difficult to extrapolate to tumor hypoxia. In rat hepatocytes, 4E-BP1 becomes dephosphorylated and associates with eIF4E rapidly (15–60 min) upon mild hypoxia, but this could not explain the observed downregulation of protein synthesis (Tinton and Buc-Calderon, 1999). More recently, it was reported that hypoxia could influence 4E-BP1 phosphorylation by affecting the activity of mTOR (Arsham et al, 2003). Serum-starved and hypoxic human embryonic kidney cells failed to activate mTOR, phosphorylate 4E-BP1 and dissociate 4E-BP1 from eIF4E in response to insulin treatment. Nonetheless, it remains unknown whether hypoxia alone is sufficient to disrupt the eIF4F complex and to what extent this influences overall translation during hypoxia. Here we show that hypoxia induces a biphasic inhibition of mRNA translation characterized by transient phosphorylation of eIF2α and subsequent dissociation of eIF4F. These two mechanisms operate independently of each other and both have important consequences for gene expression during hypoxia. Results Kinetics of translation inhibition To determine the effects of hypoxia on mRNA translation initiation in HeLa cells, we examined the association of ribosomes with mRNA at various time points. In this assay, the number of ribosomes found within the ‘polysomal’ fraction of mRNA (mRNA containing two or more ribosomes) is a reflection of de novo protein synthesis. This technique is advantageous to other methods such as 35S incorporation, which requires prior amino-acid starvation, a procedure that can itself influence translation initiation (Kimball and Jefferson, 2000). Figure 1A shows that at all time points examined, hypoxia causes a large decrease in polysomal mRNA and a corresponding increase in free ribosomes and ribosomal subunits. The reduction in translation is not influenced by cell death, as cell viability remains above 90% following 16 h of hypoxia (data not shown). Furthermore, the inhibition of translation is completely reversible upon reoxygenation (data not shown). Figure 1.Hypoxia inhibits mRNA translation. HeLa cells were exposed to 0.0% O2 for 0–16 h and cell lysates were separated on a sucrose gradient. (A) The optical density (OD) at 254 nm is shown as a function of gradient depth for each time point. Actively translated mRNA is associated with high-molecular-weight polysomes deep in the gradient. (B) Translation efficiency in HeLa cells as a function of time in 0.0% O2. As a measure of overall translation efficiency, the relative amount of rRNA participating in polysomes was estimated. This fraction is proportional to the integrated area under the curve containing polysomes, as marked in (A). (C) The average number of ribosomes per mRNA in the polysomes as a function of time in 0.0% O2. This was calculated by differential integration of the profiles in (A). Download figure Download PowerPoint To assess quantitatively overall mRNA translation from the polysome profiles, we calculated the percentage of rRNA participating in polysomes and defined this as the overall translation efficiency. This value is reduced from 62 to 24% after 1 h of hypoxia, and then recovers somewhat stabilizing at ∼30% (Figure 1B). The drop in translation reproducibly exhibited this biphasic response with maximum inhibition after 1–2 h, followed by a small recovery. The magnitude of inhibition is comparable to that observed following complete disruption of the cellular redox environment with 1 mM dithiothreitol (DTT) (17%) (data not shown). Analysis of the polysome profiles in Figure 1A shows that hypoxia also causes a change in the distribution of the polysomal mRNA, with proportionally less signal in the higher molecular weight fractions. This indicates that the average number of ribosomes per mRNA transcript is also decreased during hypoxia, reflecting a reduction in translation initiation efficiency even for those transcripts that remain translated. From the polysome profiles, we calculated the average number of ribosomes per translated transcript (i.e. mRNAs containing two or more ribosomes) at different time points during hypoxia (Figure 1C). The kinetics of this parameter follow in large part that of the overall translation. eIF2α regulates translation during acute hypoxia The eIF2α kinase PERK is at least partly responsible for protein synthesis inhibition during acute hypoxia, as measured by radioactive labeling of newly synthesized proteins (Koumenis et al, 2002). Thus, we hypothesized that the rapid inhibition and subsequent partial recovery in translation is due to changes in eIF2α phosphorylation. Indeed, we found that the phosphorylation of eIF2α is greatest after 1–2 h and then decreases by 8 h of hypoxia in several cell lines (Figure 2A). ATF4 protein levels also increase and then decrease during hypoxia in a manner that mirrors eIF2α phosphorylation. The dynamics of eIF2α phosphorylation and ATF4 protein induction thus correlate with the initial inhibition of translation and its subsequent recovery. Figure 2.Inhibition of translation during acute hypoxia is dependent on eIF2α. HeLa cells, A549 cells, human normal fibroblasts (NF) and WT or S51A MEFs were exposed to 0.0% O2 for 0–16 h, 1 mM DTT or serum starvation (SS) for 1 h. Cell lysates were separated by SDS–PAGE. Immunoblots for (A) HeLa, A549 and NF or (B) MEFs were performed using antibodies against total or phosphorylated eIF2α, ATF4 and β-actin. In (A), optical densitometry for phosphorylated eIF2α or ATF4 normalized by total eIF2α is also shown. Total eIF2α expression has previously been shown to be constant during hypoxia (Koumenis et al, 2002). (C) Cell lysates were separated on a sucrose gradient, and OD at 254 nm was recorded. Translation efficiency as a function of time in 0.0% O2 in WT and S51A MEFs was estimated as in Figure 1. (D) Average number of ribosomes per mRNA in the polysomes in WT and S51A MEFs as a function of time in 0.0% O2 was calculated as in Figure 1. Download figure Download PowerPoint To assess the requirement of eIF2α phosphorylation for translation inhibition during hypoxia we examined the response of mouse embryo fibroblasts (MEFs) derived from eIF2α knock-in mice containing an S51A mutation (Scheuner et al, 2001). As expected, these cells were defective in phosphorylation of eIF2α during hypoxia (Figure 2B). The translation efficiency in wild-type (WT) MEFs is similar to that in HeLa cells, with a rapid drop during acute hypoxia followed by a partial recovery (Figure 2C). In contrast, S51A MEFs display a substantial defect in their ability to inhibit translation during the initial phase. Nonetheless, after 16 h of hypoxia, both cell lines show a similar loss in translation efficiency. These data indicate that eIF2α phosphorylation is indeed necessary for inhibition of translation during acute hypoxia, but not at later times. When the polysome profiles are analyzed in terms of the average number of ribosomes per translated transcript, S51A MEFs exhibit an even stronger defect in their response during acute hypoxia. Despite a small but detectable drop in translation efficiency during the first 4 h of hypoxia (Figure 2C), S51A MEFs show no decrease in the average number of ribosomes per translated transcript (Figure 2D). The same result was found in cells treated with DTT, a known activator of PERK that causes eIF2α phosphorylation. In contrast, WT MEFs show a strong reduction in average ribosomes per transcript during both acute hypoxia and DTT treatment. Interestingly, after 8 h of hypoxia, the average number of ribosomes per translated transcript increases again toward normal levels in WT cells and is equivalent to that in S51A MEFs by 16 h. These data provide further evidence that the inhibition of translation that occurs after acute and prolonged hypoxia is mechanistically distinct. Disruption of the eIF4F complex during hypoxia The assembly of the cap-binding complex eIF4F is a common control point for translation initiation and was thus a likely candidate for maintaining low rates of translation during prolonged hypoxia. We examined the levels of eIF4E and proteins that associate with it as an active complex (eIF4GI) or as an inactive complex (4E-BP1). Figure 3A shows that the levels of eIF4E do not change during hypoxia. In contrast, 4E-BP1 (Figure 3B) shows both a small induction at 8 h and a strong dephosphorylation after 16 h of hypoxia. This protein runs as different migrating bands representing different phosphorylation levels (Pause et al, 1994). The fastest migrating band is substantially increased after 16 h of hypoxia, and represents the hypophosphorylated 4E-BP1, which is known to have a higher affinity for eIF4E. A small decrease in the abundance of the scaffold protein eIF4GI (Figure 3C) was observed after 8 h, consistent with a decrease in its rate of synthesis measured in a microarray study using polysomal RNA (unpublished data). Overexposure of the blots indicated no reproducible changes in the cleavage of eIF4G. The influence of hypoxia on 4E-BP1 phosphorylation appears to be largely independent of eIF2α phosphorylation, as it is not differentially affected in the WT and S51A MEFs (unpublished data). However, until the relative contributions of various upstream signaling pathways to 4E-BP phosphorylation under hypoxia are better understood, it is premature to conclude that no connection between eIF2α and eIF4F exists. Figure 3.Expression of eIF4E, 4E-BP1 and eIF4GI during hypoxia. HeLa cells were exposed to 0.0% O2 for 0–16 h and cell lysates separated by SDS–PAGE. Immunoblots were performed using antibodies against actin, (A) eIF4E, (B) 4E-BP1 and (C) eIF4GI. The phosphorylation forms of 4E-BP1 have different electrophoretic mobilities and are represented by several bands on the immunoblot. Full-length eIF4GI run at about 220 kDa; the blot is overexposed to detect cleavage products. Download figure Download PowerPoint To more strictly assess the influence of hypoxia on eIF4F, we investigated the association of eIF4E with eIF4GI and eIF4GII as well as with its inhibitor 4E-BP1 in HeLa cells. During aerobic conditions where translation is efficient, eIF4E is associated with large amounts of both eIF4GI and eIF4GII, and only a small amount of 4E-BP1 (cap lanes in Figure 4A and B). Cap-associated eIF4G migrated somewhat slower than the overall pool of eIF4G, suggesting a possible modification of this phospho-protein when bound to the cap. In contrast, after 4 or 16 h of hypoxia, there is a dramatic loss in binding to both eIF4GI and eIF4GII, indicating dissociation of the eIF4F complex. At 16 h, this dissociation correlates with a large increase in binding between eIF4E and 4E-BP1, consistent with the increase in the hypophosphorylated levels of 4E-BP1 at this time. It also correlated with decreased phosphorylation of eIF4E (Supplementary Figure S1) at 16 h, but the physiological significance of this remains unclear. However, although dissociation of eIF4G and eIF4E is complete after 4 h of hypoxia, a corresponding change in eIF4E phosphorylation or eIF4E/4E-BP1 association is not seen at this time point. This suggests that a mechanism distinct from 4E-BP1 dephosphorylation may also inhibit eIF4F during hypoxia. Figure 4.eIF4F is disrupted during prolonged hypoxia. HeLa cells were exposed to 0.0% O2 for 0–16 h and cell lysates probed for the presence of various eIF4E complexes. Lysates were incubated with an m7-cap analogue (‘Cap’) or uncapped resin as a negative control. Immunoblots were performed with antibodies against actin, (A) eIF4GI, eIF4E, 4E-BP1 and (B) eIF4GII. ‘Cap’: proteins bound to the capped resin; ‘Resin’: proteins bound to the uncapped resin; ‘C-FT’: unbound fraction after incubation with capped resin; ‘R-FT’: unbound fraction after incubation with uncapped resin. Download figure Download PowerPoint Translocation of eIF4E by 4E-T A potential cause of eIF4F disruption that has not been well characterized is the translocation of eIF4E to the nucleus or to cytoplasmic bodies of mRNA processing (P-bodies). A 5–20% fraction of eIF4E is known to localize to the cell nucleus (Lejbkowicz et al, 1992). The shuttling protein 4E-T is the only known regulator of eIF4E localization and is capable of binding and transporting it to the cell nucleus (Dostie et al, 2000). eIF4E also colocalizes with 4E-T in P-bodies, where mRNA is degraded or stored (Andrei et al, 2005). Hypoxia caused a redistribution of both eIF4E and 4E-T from predominantly cytoplasmic staining under aerobic conditions to substantial nuclear staining during hypoxia (Figure 5A–C). This redistribution occurred progressively over time in hypoxic conditions, correlating with the gradual dephosphorylation of 4E-T (Figure 5D). In addition, hypoxic cells exhibit significant eIF4E and 4E-T staining in the perinuclear area, which may be associated with the nuclear envelope or the ER. Interestingly, hypoxia also increased the number of 4E-T speckles, which have been described as P-bodies (Ferraiuolo et al, 2005). Figure 5.4E-T and eIF4E relocalize during hypoxia. HeLa cells were treated with 0.0% O2 for 0–16 h. Cells were stained with DAPI and (A) a polyclonal antibody against eIF4E, (B) a polyclonal antibody against 4E-T or (C) a monoclonal antibody against eIF4E and a polyclonal antibody against 4E-T. Cells were visualized by confocal microscopy and individual pictures merged to determine colocalization. (D) Cell lysates were separated by SDS–PAGE and immunoblots performed using antibodies against 4E-T. The ratio of the individual bands was quantified with optical densitometry. A crossreacting band is indicated (*). Download figure Download PowerPoint Gene-specific regulation of translation As translation efficiency is highly gene specific, we anticipated that individual genes would show different patterns of translation efficiency during acute and prolonged hypoxia. To investigate this, we fractionated polysomal mRNA and subsequently measured the mRNA abundance of individual genes by quantitative RT–PCR (Figure 6A). We first confirmed that concomitant with an increase in polysome association, the non/subpolysomal abundance decreased (Supplementary Figure S2). Subsequently, we quantified both the transcript recruitment and distribution within the polysomes (expressed as the relative fraction of translated transcripts and the average number of ribosomes per translated transcript, respectively). Figure 6.Gene-specific regulation of translation during hypoxia. HeLa cells were exposed to 0.0% O2 for 0–16 h and cell lysates were separated on sucrose gradients. (A) Fractions were collected as indicated, RNA was isolated and reverse transcribed. Thereafter, the total mRNA abundance of (B) β-actin, (C) CAIX, (D) ATF4, (E) CHOP and (F) GADD34 was determined using real-time quantitative PCR. The left panel shows total mRNA levels from unfractionated samples, normalized by 18S rRNA signal. The following three panels use black, gray and white bars to represent the gene abundance in polysome fractions following 0, 1 or 16 h hypoxia, respectively. The last two graphs show components of translation efficiency. This includes the relative fraction of transcripts in polysomes (i.e. corrected for total mRNA abundance) and the average number of ribosomes per mRNA. Graphs show the average from two independent experiments, and the histograms show the results from one representative experiment. Download figure Download PowerPoint We first measured the translational profile of the housekeeping gene β-actin (Figure 6B). In aerobic cells, it is efficiently translated with a majority of the mRNA in polysome fractions 5 and 6. After 1 h of hypoxia, there is a marked reduction in translation, as evidenced by a shift toward the lower polysome fractions, which recovers considerably by 16 h. The drop in translation efficiency at 1 h is due to reductions in the relative fraction of translated mRNA and in the average number of ribosomes per translated transcript (Figure 6B). At later time points, only the average number of ribosomes per transcript remained low. The kinetic changes in translation efficiency for β-actin are similar to those observed for overall translation efficiency.