Cellular energy status modulates translational control mechanisms in ischemic-reperfused rat hearts

Mechanisms regulating ischemia and reperfusion (I/R)-induced changes in mRNA translation in the heart are poorly defined, as are the factors that initiate these changes. Because cellular energy status affects mRNA translation under physiological conditions, it is plausible that I/R-induced changes in translation may in part be a result of altered cellular energy status. Therefore, the purpose of the studies described herein was to compare the effects of I/R with those of altered energy substrate availability on biomarkers of mRNA translation in the heart. Isolated adult rat hearts were perfused with glucose or a combination of glucose plus palmitate, and effects of I/R on various biomarkers of translation were subsequently analyzed. When compared with hearts perfused with glucose plus palmitate, hearts perfused with glucose alone exhibited increased phosphorylation of eukaryotic elongation factor (eEF)2, the alpha-subunit of eukaryotic initiation factor (eIF)2, and AMP-activated protein kinase (AMPK), and these hearts also exhibited enhanced association of eIF4E with eIF4E binding protein (4E-BP)1. Regardless of the energy substrate composition of the buffer, phosphorylation of eEF2 and AMPK was greater than control values after ischemia. Phosphorylation of eIF2alpha and eIF4E and the association of eIF4E with 4E-BP1 were also greater than control values after ischemia but only in hearts perfused with glucose plus palmitate. Reperfusion reversed the ischemia-induced increase in eEF2 phosphorylation in hearts perfused with glucose and reversed ischemia-induced changes in eIF4E, eEF2, and AMPK phosphorylation in hearts perfused with glucose plus palmitate. Because many ischemia-induced changes in mRNA translation are mimicked by the removal of a metabolic substrate under normal perfusion conditions, the results suggest that cellular energy status represents an important modulator of I/R-induced changes in mRNA translation.     

Evidence for a Negative Cooperativity between eIF5A and eEF2 on Binding to the Ribosome

eIF5A is the only protein known to contain the essential and unique amino acid residue hypusine. eIF5A functions in both translation initiation due to its stimulation of methionyl-puromycin synthesis and translation elongation, being highly required for peptide-bound formation of specific ribosome stalling sequences such as poly-proline. The functional interaction between eIF5A, tRNA, and eEF2 on the surface of the ribosome is further clarified herein. Fluorescence anisotropy assays were performed to determine the affinity of eIF5A to different ribosomal complexes and reveal its interaction exclusively and directly with the 60S ribosomal subunit in a hypusine-dependent manner (K_i^{60S-eIF5A-Hyp} = 16 nM, K_i^{60S-eIF5A-Lys} = 385 nM). A 3-fold increase in eIF5A affinity to the 80S is observed upon charged-tRNA_i^Met binding, indicating positive cooperativity between P-site tRNA binding and eIF5A binding to the ribosome. Previously identified conditional mutants of yeast eIF5A, eIF5A^Q22H/L93F and eIF5A^K56A, display a significant decrease in ribosome binding affinity. Binding affinity between ribosome and eIF5A-wild type or mutants eIF5A^K56A, but not eIF5A^Q22H/L93F, is impaired in the presence of eEF2 by 4-fold, consistent with negative cooperativity between eEF2 and eIF5A binding to the ribosome. Interestingly, high-copy eEF2 is toxic only to eIF5A^Q22H/L93F and causes translation elongation defects in this mutant. These results suggest that binding of eEF2 to the ribosome alters its conformation, resulting in a weakened affinity of eIF5A and impairment of this interplay compromises cell growth due to translation elongation defects.     

Sumoylation of eIF4E activates mRNA translation

Eukaryotic translation initiation factor 4E (eIF4E) is the cap‐binding protein that binds the 5′ cap structure of cellular messenger RNAs (mRNAs). Despite the obligatory role of eIF4E in cap‐dependent mRNA translation, how the translation activity of eIF4E is controlled remains largely undefined. Here, we report that mammalian eIF4E is regulated by SUMO1 (small ubiquitin‐related modifier 1) conjugation. eIF4E sumoylation promotes the formation of the active eIF4F translation initiation complex and induces the translation of a subset of proteins that are essential for cell proliferation and preventing apoptosis. Furthermore, disruption of eIF4E sumoylation inhibits eIF4E‐dependent protein translation and abrogates the oncogenic and antiapoptotic functions associated with eIF4E. These data indicate that sumoylation is a new fundamental regulatory mechanism of protein synthesis. Our findings suggest further that eIF4E sumoylation might be important in promoting human cancers.     

Thermal injury impairs cardiac protein synthesis and is associated with alterations in translation initiation

The purpose of the present study was to determine whether burn injury decreases myocardial protein synthesis and potential contributing mechanisms for this impairment. To address this aim, thermal injury was produced by a 40% total body surface area full-thickness scald burn in anesthetized rats, and the animals were studied 24 h late. Burn decreased the in vivo-determined rate of myocardial protein synthesis and translation efficiency by 25% but did not alter the protein synthetic rate in skeletal muscle. To identify potential mechanisms responsible for regulating mRNA translation in cardiac muscle, we examined several eukaryotic initiation factors (eIFs) and elongation factors (eEFs). Burn failed to alter eIF2B activity or the total amount or phosphorylation status of either eIF2 alpha or eIF2B epsilon in heart. In contrast, hearts from burned rats demonstrated 1) an increased binding of the translational repressor 4E-BP1 with eIF4E, 2) a decreased amount of eIF4E associated with eIF4G, and 3) a decreased amount of the hyperphosphorylated gamma-form of 4E-BP1. These changes in eIF4E availability were not seen in gastrocnemius muscle where burn injury did not decrease protein synthesis. Furthermore, constitutive phosphorylation of mTOR, S6K1, the ribosomal protein S6, and eIF4G were also decreased in hearts from burned rats. Burn did not appear to adversely affect elongation because there was no significant difference in the myocardial content of eEF1 alpha or eEF2 or the phosphorylation state of eEF2. The above-mentioned burn-induced changes in mRNA translation were associated with an impairment of in vitro myocardial performance. Finally, 24 h postburn, the cardiac mRNA content of IL-1 beta, IL-6, and high-mobility group protein B1 (but not TNF-alpha) was increased. In summary, these data suggest that thermal injury specifically decreases cardiac protein synthesis in part by decreasing mRNA translation efficiency resulting from an impairment in translation initiation associated with alterations in eIF4E availability and S6K1 activity.     

eIF5A interacts functionally with eEF2

eIF5A is highly conserved from archaea to mammals, essential for cell viability and the only protein known to contain the essential amino acid residue hypusine, generated by a unique posttranslational modification. eIF5A was originally identified as a translation initiation factor due to its ability to stimulate the formation of the first peptide bond. However, recent studies have shown that depletion of eIF5A causes a significant decrease in polysome run-off and an increase in the ribosome transit time, suggesting that eIF5A is actually involved in the elongation step of protein synthesis. We have previously shown that the depletion mutant tif51A-3 (eIF5A(C39Y/G118D)) shows a sicker phenotype when combined with the dominant negative mutant eft2 ( H699K ) of the elongation factor eEF2. In this study, we used the eIF5A(K56A) mutant to further investigate the relationship between eIF5A and eEF2. The eIF5A(K56A) mutant is temperature sensitive and has a defect in protein synthesis, but instead of causing depletion of the eIF5A protein, this mutant has a defect in hypusine modification. Like the mutant tif51A-3, the eIF5A(K56A) mutant is synthetic sick with the mutant eft2 ( H699K ) of eEF2. High-copy eEF2 not only improves cell growth of the eIF5A(K56A) mutant, but also corrects its increased cell size defect. Moreover, eEF2 suppression of the eIF5A(K56A) mutant is correlated with the improvement of total protein synthesis and with the increased resistance to the protein synthesis inhibitor hygromycin B. Finally, the polysome profile defect of the eIF5A(K56A) mutant is largely corrected by high-copy eEF2. Therefore, these results demonstrate that eIF5A is closely related to eEF2 function during translation elongation.     

Resveratrol ameliorates high glucose-induced protein synthesis in glomerular epithelial cells

High glucose-induced protein synthesis in the glomerular epithelial cell (GEC) is partly dependent on reduction in phosphorylation of AMP-activated protein kinase (AMPK). We evaluated the effect of resveratrol, a phytophenol known to stimulate AMPK, on protein synthesis. Resveratrol completely inhibited high glucose stimulation of protein synthesis and synthesis of fibronectin, an important matrix protein, at 3 days. Resveratrol dose-dependently increased AMPK phosphorylation and abolished high glucose-induced reduction in its phosphorylation. We examined the effect of resveratrol on critical steps in mRNA translation, a critical event in protein synthesis. Resveratrol inhibited high glucose-induced changes in association of eIF4E with eIF4G, phosphorylation of eIF4E, eEF2, eEF2 kinase and, p70S6 kinase, indicating that it affects important events in both initiation and elongation phases of mRNA translation. Upstream regulators of AMPK in high glucose-treated GEC were explored. High glucose augmented acetylation of LKB1, the upstream kinase for AMPK, and inhibited its activity. Resveratrol prevented acetylation of LKB1 and restored its activity in high glucose-treated cells; this action did not appear to depend on SIRT1, a class III histone deacetylase. Our data show that resveratrol ameliorates protein synthesis by regulating the LKB1–AMPK axis.     

Solution structure of human initiation factor eIF2alpha reveals homology to the elongation factor eEF1B

The GTP-bound form of the trimeric eukaryotic translation initiation factor 2 (eIF2) transfers aminoacylated initiator methionyl tRNA onto the 40S ribosome. We have solved with solution NMR the structure of the alpha subunit of human eIF2 (heIF2alpha). The protein consists of two domains that are mobile relative to each other. The N-terminal domain has an S1-type oligonucleotide/oligosaccharide binding-fold subdomain and an alpha-helical subdomain. The C-terminal domain adopts an alphabeta-fold very similar to the C-terminal domain of elongation factor (eEF) 1Balpha, the guanine-nucleotide exchange factor for eEF1A. The structural and functional similarities found between eIF2alpha/eIF2gamma and eEF1Balpha/eEF1A suggest a model for the interaction of eIF2alpha with eIF2gamma, and eIF2 with Met-tRNAiMet. It further indicates a previously unrecognized evolutionary lineage of eIF2alpha/gamma from the functionally related elongation factor eEF1Balpha/eEF1A complex.     

The elongation, termination, and recycling phases of translation in eukaryotes

This work summarizes our current understanding of the elongation and termination/recycling phases of eukaryotic protein synthesis. We focus here on recent advances in the field. In addition to an overview of translation elongation, we discuss unique aspects of eukaryotic translation elongation including eEF1 recycling, eEF2 modification, and eEF3 and eIF5A function. Likewise, we highlight the function of the eukaryotic release factors eRF1 and eRF3 in translation termination, and the functions of ABCE1/Rli1, the Dom34:Hbs1 complex, and Ligatin (eIF2D) in ribosome recycling. Finally, we present some of the key questions in translation elongation, termination, and recycling that remain to be answered.     

Exp5 exports eEF1A via tRNA from nuclei and synergizes with other transport pathways to confine translation to the cytoplasm

Importin beta-type transport receptors mediate the vast majority of transport pathways between cell nucleus and cytoplasm. We identify here the translation elongation factor 1A (eEF1A) as the predominant nuclear export substrate of RanBP21/exportin 5 (Exp5). This cargo-exportin interaction is rather un usual in that eEF1A binds the exportin not directly, but instead via aminoacylated tRNAs. Exp5 thus represents the second directly RNA-binding exportin and mediates tRNA export in parallel with exportin-t. It was suggested recently that 10-15% of the cellular translation would occur in the nucleus. Our data rule out such a scenario and instead suggest that nuclear translation is actively suppressed by the nuclear export machinery. We found that the vast majority of translation initiation factors (eIF2, eIF2B, eIF3, eIF4A1, eIF5 and eIF5B), all three elongation factors (eEF1A, eEF1B and eEF2) and the termination factor eRF1 are strictly excluded from nuclei. Besides Exp5 and importin 13, CRM1 and as yet unidentified exportins also contribute to the depletion of translation factors from nuclei.     

Analysis of the interacting partners eIF4F and 3′‐CITE required for Melon necrotic spot virus cap‐independent translation

We have shown previously that the translation of Melon necrotic spot virus (MNSV, family Tombusviridae, genus Carmovirus) RNAs is controlled by a 3′‐cap‐independent translation enhancer (CITE), which is genetically and functionally dependent on the eukaryotic translation initiation factor (eIF) 4E. Here, we describe structural and functional analyses of the MNSV‐Mα5 3′‐CITE and its translation initiation factor partner. We first mapped the minimal 3′‐CITE (Ma5TE) to a 45‐nucleotide sequence, which consists of a stem‐loop structure with two internal loops, similar to other I‐shaped 3′‐CITEs. UV crosslinking, followed by gel retardation assays, indicated that Ma5TE interacts in vitro with the complex formed by eIF4E + eIF4G_980–1159 (eIF4F_p20), but not with each subunit alone or with eIF4E + eIF4G_1003–1092, suggesting binding either through interaction with eIF4E following a conformational change induced by its binding to eIF4G_980–1159, or through a double interaction with eIF4E and eIF4G_980–1159. Critical residues for this interaction reside in an internal bulge of Ma5TE, so that their mutation abolished binding to eIF4E + eIF4G_1003–1092 and cap‐independent translation. We also developed an in vivo system to test the effect of mutations in eIF4E in Ma5TE‐driven cap‐independent translation, showing that conserved amino acids in a positively charged RNA‐binding motif around amino acid position 228, implicated in eIF4E–eIF4G binding or belonging to the cap‐recognition pocket, are essential for cap‐independent translation controlled by Ma5TE, and thus for the multiplication of MNSV.     

Pumpkin eIF5A isoforms interact with components of the translational machinery in the cucurbit sieve tube system

In yeast, eIF5A, in combination with eEF2, functions at the translation step, during the protein elongation cycle. This result is of significance with respect to functioning of the enucleate sieve tube system, as eIF5A was recently detected in Cucurbita maxima (pumpkin) phloem sap. In the present study, we further characterized four CmeIF5A isoforms, encoding three proteins, all of which were present in the phloem sap. Although hypusination of CmeIF5A was not necessary for entry into the sieve elements, this unique post‐translational modification was necessary for RNA binding. The two enzymes required for hypusination were detected in pumpkin phloem sap, where presumably this modification takes place. A combination of gel‐filtration chromatography and protein overlay assays demonstrated that, as in yeast, CmeIF5A interacts with phloem proteins, like eEF2, known to be involved in protein synthesis. These findings are discussed in terms of a potential role for eIF5A in regulating protein synthesis within the enucleate sieve tube system of the angiosperms.     

Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps

Insulin acutely activates protein synthesis in ventricular cardiomyocytes from adult rats. In this study, we have established the methodology for studying the regulation of the signaling pathways and translation factors that may be involved in this response and have examined the effects of acute insulin treatment on them. Insulin rapidly activated the 70-kDa ribosomal S6 kinase (p70 S6k), and this effect was inhibited both by rapamycin and by inhibitors of phosphatidylinositol 3-kinase. The activation of p70 S6k is mediated by a signaling pathway involving the mammalian target of rapamycin (mTOR), which also modulates other translation factors. These include the eukaryotic initiation factor (eIF) 4E binding proteins (4E-BPs) and eukaryotic elongation factor 2 (eEF2). Insulin caused phosphorylation of 4E-BP1 and induced its dissociation from eIF4E, and these effects were also blocked by rapamycin. Concomitant with this, insulin increased the binding of eIF4E to eIF4G. Insulin also activated protein kinase B (PKB), which may lie upstream of p70 S6k and 4E-BP1, with the activation of the different isoforms being in the order alpha>beta>gamma. Insulin also caused inhibition of glycogen synthase kinase 3, which lies downstream of PKB, and of eEF2 kinase. The phosphorylation of eEF2 itself was also decreased by insulin, and this effect and the inactivation of eEF2 kinase were attenuated by rapamycin. The activation of overall protein synthesis by insulin in cardiomyocytes was substantially inhibited by rapamycin (but not by inhibitors of other specific signaling pathways, e.g., mitogen-activated protein kinase), showing that signaling events linked to mTOR play a major role in the control of translation by insulin in this cell type.     

All translation elongation factors and the e, f, and h subunits of translation initiation factor 3 are encoded by 5'-terminal oligopyrimidine (TOP) mRNAs

Terminal oligopyrimidine (TOP) mRNAs (encoded by the TOP genes) are identified by a sequence of 6–12 pyrimidines at the 5′ end and by a growth-associated translational regulation. All vertebrate genes for the 80 ribosomal proteins and some other genes involved, directly or indirectly, in translation, are TOP genes. Among the numerous translation factors, only eEF1A and eEF2 are known to be encoded by TOP genes, most of the others having not been analyzed. Here, we report a systematic analysis of the human genes for translation factors. Our results show that: (1) all five elongation factors are encoded by TOP genes; and (2) among the initiation and termination factors analyzed, only eIF3e, eIF3f, and eIF3h exhibit the characteristics of TOP genes. Interestingly, these three polypeptides have been recently shown to constitute a specific subgroup among eIF3 subunits. In fact, eIF3e, eIF3f, and eIF3h are the part of the functional core of eIF3 that is not conserved in Saccharomyces cerevisiae. It has been hypothesized that they are regulatory subunits, and the fact that they are encoded by TOP genes may be relevant for their function.     

Leucine regulates translation of specific mRNAs in L6 myoblasts through mTOR-mediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6

Regulation of translation of mRNAs coding for specific proteins plays an important role in controlling cell growth, differentiation, and transformation. Two proteins have been implicated in the regulation of specific mRNA translation: eukaryotic initiation factor eIF4E and ribosomal protein S6. Increased phosphorylation of eIF4E as well as its overexpression are associated with stimulation of translation of mRNAs with highly structured 5'-untranslated regions. Similarly, phosphorylation of S6 results in preferential translation of mRNAs containing an oligopyrimidine tract at the 5'-end of the message. In the present study, leucine stimulated phosphorylation of the eIF4E-binding protein, 4E-BP1, in L6 myoblasts, resulting in dissociation of eIF4E from the inactive eIF4E.4E-BP1 complex. The increased availability of eIF4E was associated with a 1.6-fold elevation in ornithine decarboxylase relative to global protein synthesis. Leucine also stimulated phosphorylation of the ribosomal protein S6 kinase, p70(S6k), resulting in increased phosphorylation of S6. Hyperphosphorylation of S6 was associated with a 4-fold increase in synthesis of elongation factor eEF1A. Rapamycin, an inhibitor of the protein kinase mTOR, prevented all of the leucine-induced effects. Thus, leucine acting through an mTOR-dependent pathway stimulates the translation of specific mRNAs both by increasing the availability of eIF4E and by stimulating phosphorylation of S6.     

Dual regulation of translation initiation and peptide chain elongation during BDNF-induced LTP in vivo: evidence for compartment-specific translation control

Protein synthesis underlying activity-dependent synaptic plasticity is controlled at the level of mRNA translation. We examined the dynamics and spatial regulation of two key translation factors, eukaryotic initiation factor 4E (eIF4E) and elongation factor-2 (eEF2), during long-term potentiation (LTP) induced by local infusion of brain-derived neurotrophic factor (BDNF) into the dentate gyrus of anesthetized rats. BDNF-induced LTP led to rapid, transient phosphorylation of eIF4E and eEF2, and enhanced expression of eIF4E protein in dentate gyrus homogenates. Infusion of the extracellular signal-regulated kinase (ERK) inhibitor U0126 blocked BDNF-LTP and modulation of the translation factor activity and expression. Quantitative immunohistochemical analysis revealed enhanced staining of phospho-eIF4E and total eIF4E in dentate granule cells. The in vitro synaptodendrosome preparation was used to isolate the synaptic effects of BDNF in the dentate gyrus. BDNF treatment of synaptodendrosomes elicited rapid, transient phosphorylation of eIF4E paralleled by enhanced expression of alpha-calcium/calmodulin-dependent protein kinase II. In contrast, BDNF had no effect on eEF2 phosphorylation state in synaptodendrosomes. The results demonstrate rapid ERK-dependent regulation of the initiation and elongation steps of protein synthesis during BDNF-LTP in vivo. Furthermore, the results suggest a compartment-specific regulation in which initiation is selectively enhanced by BDNF at synapses, while both initiation and elongation are modulated at non-synaptic sites.     

Genetic removal of eIF2α kinase PERK in mice enables hippocampal L‐LTP independent of mTORC1 activity

Characterization of the molecular signaling pathways underlying protein synthesis‐dependent forms of synaptic plasticity, such as late long‐term potentiation (L‐LTP ), can provide insights not only into memory expression/maintenance under physiological conditions but also potential mechanisms associated with the pathogenesis of memory disorders. Here, we report in mice that L‐LTP failure induced by the mammalian (mechanistic) target of rapamycin complex 1 (mTORC 1) inhibitor rapamycin is reversed by brain‐specific genetic deletion of PKR ‐like ER kinase, PERK (PERK KO ), a kinase for eukaryotic initiation factor 2α (eIF 2α). In contrast, genetic removal of general control non‐derepressible‐2, GCN 2 (GCN 2 KO ), another eIF 2α kinase, or treatment of hippocampal slices with the PERK inhibitor GSK 2606414, does not rescue rapamycin‐induced L‐LTP failure, suggesting mechanisms independent of eIF 2α phosphorylation. Moreover, we demonstrate that phosphorylation of eukaryotic elongation factor 2 (eEF 2) is significantly decreased in PERK KO mice but unaltered in GCN 2 KO mice or slices treated with the PERK inhibitor. Reduction in eEF 2 phosphorylation results in increased general protein synthesis, and thus could contribute to the mTORC 1‐independent L‐LTP in PERK KO mice. We further performed experiments on mutant mice with genetic removal of eEF 2K (eEF 2K KO ), the only known kinase for eEF 2, and found that L‐LTP in eEF 2K KO mice is insensitive to rapamycin. These data, for the first time, connect reduction in PERK activity with the regulation of translation elongation in enabling L‐LTP independent of mTORC 1. Thus, our findings indicate previously unrecognized levels of complexity in the regulation of protein synthesis‐dependent synaptic plasticity.

Read the Editorial Highlight for this article on page 119 . Cover Image for this issue: doi: 10.1111/jnc.14185 .

     

Structure and functions of the translation initiation factor eIF4E and its role in cancer development and treatment

In eukaryotic cells, protein synthesis is a complex and multi-step process that has several mechanisms to start the translation including cap-dependent and cap-independent initiation. The translation control of eukaryotic gene expression occurs principally at the initiation step. In this context, it is critical that the eukaryotic translation initiation factor eIF4E bind to the 7-methylguanosine (m7G) cap present at the 5′-UTRs of most eukaryotic mRNAs. Combined with other initiation factors, eIF4E mediates the mRNA recruitment on ribosomes to start the translation. Moreover, the eIF4E nuclear bodies are involved in the export of specific mRNAs from the nucleus to the cytoplasm. In this review, we focus on the eIF4E structure and its physiological functions, and describe the role of eIF4E in cancer development and progression and the current therapeutic strategies to target eIF4E.     

eIF5A has a function in the elongation step of translation in yeast

The putative translation factor eIF5A is essential for cell viability and is highly conserved throughout evolution. Here, we describe genetic interactions between an eIF5A mutant and a translation initiation mutant (eIF4E) or a translation elongation mutant (eEF2). Polysome profile analysis of single and double mutants revealed that mutation in eIF5A reduces polysome run-off, contrarily to translation initiation mutants. Moreover, the polysome profile of an eIF5A mutant alone is very similar to that of a translation elongation mutant. Furthermore, depletion of eIF5A causes a significant decrease in total protein synthesis and an increase of the average ribosome transit time. Finally, we demonstrate that the formation of P bodies is inhibited in an eIF5A mutant, similarly to the effect of the translation elongation inhibitor cycloheximide. Taken together, these results not only reinforce a role for eIF5A in translation but also strongly support a function for eIF5A in the elongation step of protein synthesis.     

Hypoxic reactive oxygen species regulate the integrated stress response and cell survival

Under hypoxic conditions, cells suppress energy-intensive mRNA translation by modulating the mammalian target of rapamycin (mTOR) and pancreatic eIF2alpha kinase (PERK) pathways. Much is known about hypoxic inhibition of mTOR activity; however, the cellular processes activating PERK remain unclear. Since hypoxia is known to increase intracellular reactive oxygen species (ROS), we hypothesized that hypoxic ROS regulate mTOR and PERK to control mRNA translation and cell survival. Our data indicate that although exogenous ROS inhibit mTOR, eIF2alpha, and eEF2, mTOR and eEF2 were largely refractory to ROS generated under moderate hypoxia (0.5% O(2)). In direct contrast, the PERK/eIF2alpha/ATF4 integrated stress response (ISR) was activated by hypoxic ROS and contributed to global protein synthesis inhibition and adaptive ATF4-mediated gene expression. The ISR as well as exogenous growth factors were critical for cell viability during extended hypoxia, since ISR inhibition decreased the viability of cells deprived of O(2) and growth factors. Collectively, our data support an important role for ROS in hypoxic cell survival. Under conditions of moderate hypoxia, ROS induce the ISR, thereby promoting energy and redox homeostasis and enhancing cellular survival.     

A tale of two termini:: A functional interaction between the termini of an mRNA is a prerequisite for efficient translation initiation

A quarter of century following the prediction that mRNAs are translated in a circular form, recent biochemical and genetic evidence has accumulated to support the idea that communication between the termini of an mRNA is necessary to promote translation initiation. The poly(A)-binding protein (PABP) interacts with the cap-associated eukaryotic initiation factor (eIF) 4G (in yeast and plants) and eIF4B (in plants), a functional consequence of which is to increase the affinity of PABP for poly(A) and to increase the affinity of the cap-binding complex, eIF4F (of which eIF4G is a subunit) for the 5′ cap structure. In mammals, PABP interacts with a novel PABP-interacting protein that also binds eIF4A. The interaction between PABP and those initiation factors associated with the 5′ terminus of an mRNA may also explain the role of PABP during mRNA turnover, as it protects the 5′ cap from attack by Dcp1p, the decapping enzyme. Several of those mRNAs that have evolved functional equivalents to a cap or a poly(A) tail nevertheless require a functional interaction between terminal regulatory elements similar to that observed between the 5′ cap and poly(A) tail, suggesting that efficient translation is predicated on communication between largely-separated regulatory elements within an mRNA.