Distinct regulation of internal ribosome entry site-mediated translation following cellular stress is mediated by apoptotic fragments of eIF4G translation initiation factor family members eIF4GI and p97/DAP5/NAT1

Many cellular stresses lead to the inhibition of protein synthesis. Despite this, some cellular mRNAs are selectively translated under these conditions. It was suggested that the presence of internal ribosome entry site (IRES) sequences in the 5'-untranslated regions allow these mRNAs to be actively translated despite the overall cessation of protein synthesis. Here we tested the hypothesis that the IRES elements of genes that are involved in the control of cell survival are distinctly regulated by cellular stresses. We show that the transient conditions of cellular stress favor the translation of pro-survival IRES, while the severe apoptotic conditions support translation of pro-death IRES elements. Furthermore, activation of pro-death IRES during the etoposide-induced apoptosis is caspase-dependent and correlates with the expression of apoptotic fragments of two members of the eIF4G translation initiation factor family, p97/DAP5/NAT1 and eIF4GI. Our results suggest that the regulation of IRES translation during stress contributes to the fine-tuning of cell fate.     

Internal ribosome entry site-mediated translation of a mammalian mRNA is regulated by amino acid availability

The cationic amino acid transporter, Cat-1, facilitates the uptake of the essential amino acids arginine and lysine. Amino acid starvation causes accumulation and increased translation of cat-1 mRNA, resulting in a 58-fold increase in protein levels and increased arginine uptake. A bicistronic mRNA expression system was used to demonstrate the presence of an internal ribosomal entry sequence (IRES) within the 5'-untranslated region of the cat-1 mRNA. This study shows that IRES-mediated translation of the cat-1 mRNA is regulated by amino acid availability. This IRES causes an increase in translation under conditions of amino acid starvation. In contrast, cap-dependent protein synthesis is inhibited during amino acid starvation, which is well correlated with decreased phosphorylation of the cap-binding protein, eIF4E. These findings reveal a new aspect of mammalian gene expression and regulation that provides a cellular stress response; when the nutrient supply is limited, the activation of IRES-mediated translation of mammalian mRNAs results in the synthesis of proteins essential for cell survival.     

Regulation of internal ribosomal entry site-mediated translation by phosphorylation of the translation initiation factor eIF2alpha

Initiation of translation from most cellular mRNAs occurs via scanning; the 40 S ribosomal subunit binds to the m(7)G-cap and then moves along the mRNA until an initiation codon is encountered. Some cellular mRNAs contain internal ribosome entry sequences (IRESs) within their 5'-untranslated regions, which allow initiation independently of the 5'-cap. This study investigated the ability of cellular stress to regulate the activity of IRESs in cellular mRNAs. Three stresses were studied that cause the phosphorylation of the translation initiation factor, eIF2alpha, by activating specific kinases: (i) amino acid starvation, which activates GCN2; (ii) endoplasmic reticulum (ER) stress, which activates PKR-like ER kinase, PERK kinase; and (iii) double-stranded RNA, which activates double-stranded RNA-dependent protein kinase (PKR) by mimicking viral infection. Amino acid starvation and ER stress caused transient phosphorylation of eIF2alpha during the first hour of treatment, whereas double-stranded RNA caused a sustained phosphorylation of eIF2alpha after 2 h. The effects of these treatments on IRES-mediated initiation were investigated using bicistronic mRNA expression vectors. No effect was seen for the IRESs from the mRNAs for the chaperone BiP and the protein kinase Pim-1. In contrast, translation mediated by the IRESs from the cationic amino acid transporter, cat-1, and of the cricket paralysis virus intergenic region, were stimulated 3- to 10-fold by all three treatments. eIF2alpha phosphorylation was required for the response because inactivation of phosphorylation prevented the stimulation. It is concluded that cellular stress can stimulate translation from some cellular IRESs via a mechanism that requires the phosphorylation of eIF2alpha. Moreover, there are distinct regulatory patterns for different cellular mRNAs that contain IRESs within their 5'-untranslated regions.     

Translation of eukaryotic translation initiation factor 4GI (eIF4GI) proceeds from multiple mRNAs containing a novel cap-dependent internal ribosome entry site (IRES) that is active during poliovirus infection

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential scaffolding protein required to recruit the 43 S complex to the 5'-end of mRNA during translation initiation. We have previously demonstrated that eIF4GI protein expression is translationally regulated. This regulation is mediated by cis-acting RNA elements, including an upstream open reading frame and an IRES that directs synthesis of five eIF4GI protein isoforms via alternative AUG initiation codon selection. Here, we further characterize eIF4GI IRES function and show that eIF4GI is expressed from several distinct mRNAs that vary via alternate promoter use and alternate splicing. Several mRNA variants contain the IRES element. We found that IRES activity mapped to multiple regions within the eIF4GI RNA sequence, but not within the 5'-UTR per se. However, the 5'-UTR enhanced IRES activity in vivo and played a role in initiation codon selection. The eIF4GI IRES was active when transfected into cells in an RNA form, and thus, does not require nuclear processing events for its function. However, IRES activity was found to be dependent upon the presence, in cis, of a 5' m7guanosine-cap. Despite this requirement, the eIF4GI IRES was activated by 2A protease cleavage of eIF4GI, in vitro, and retained the ability to promote translation during poliovirus-mediated inhibition of cap-dependent translation. These data indicate that intact eIF4GI protein is not required for the de novo synthesis of eIF4GI, suggesting its expression can continue under stress or infection conditions where eIF4GI is cleaved.     

Cellular IRES-mediated translation: The war of ITAFs in pathophysiological states

Translation of cellular mRNAs via initiation at internal ribosome entry sites (IRESs) has received increased attention during recent years due to its emerging significance for many physiological and pathological stress conditions in eukaryotic cells. Expression of genes bearing IRES elements in their mRNAs is controlled by multiple molecular mechanisms, with IRES-mediated translation favored under conditions when cap-dependent translation is compromised. In this review, we discuss recent advances in the field and future directions that may bring us closer to understanding the complex mechanisms that guide cellular IRES-mediated expression. We present examples in which the competitive action of IRES-transacting factors (ITAFs) plays a pivotal role in IRES-mediated translation and thereby controls cell-fate decisions leading to either pro-survival stress adaptation or cell death.Key words: translation initiation, IRES, canonical initiation factors, ITAFs, stress response, eIF2, angiogenesis, mitosis, nutrient-signaling, hyperosmolar stress     

Insights into the role of yeast eIF2A in IRES-mediated translation

Eukaryotic initiation factor 2A is a single polypeptide that acts to negatively regulate IRES-mediated translation during normal cellular conditions. We have found that eIF2A (encoded by YGR054w) abundance is reduced at both the mRNA and protein level during 6% ethanol stress (or 37°C heat shock) under conditions that mimic the diauxic shift in the yeast Saccharomyces cerevisiae. Furthermore, eIF2A protein is posttranslationally modified during ethanol stress. Unlike ethanol and heat shock stress, H(2)O(2) and sorbitol treatment induce the loss of eIF2A mRNA, but not protein and without protein modification. To investigate the mechanism of eIF2A function we employed immunoprecipitation-mass spectrometry and identified an interaction between eIF2A and eEF1A. The interaction between eIF2A and eEF1A increases during ethanol stress, which correlates with an increase in IRES-mediated translation from the URE2 IRES element. These data suggest that eIF2A acts as a switch to regulate IRES-mediated translation, and eEF1A may be an important mediator of translational activation during ethanol stress.     

Interaction of translation initiation factor eIF4B with the poliovirus internal ribosome entry site

Poliovirus translation is initiated at the internal ribosome entry site (IRES). Most likely involving the action of standard initiation factors, this highly structured cis element in the 5" noncoding region of the viral RNA guides the ribosome to an internal silent AUG. The actual start codon for viral protein synthesis further downstream is then reached by ribosomal scanning. In this study we show that two of the secondary structure elements of the poliovirus IRES, domain V and, to a minor extent, domain VI, are the determinants for binding of the eukaryotic initiation factor eIF4B. Several mutations in domain V which are known to greatly affect poliovirus growth also seriously impair the binding of eIF4B. The interaction of eIF4B with the IRES is not dependent on the presence of the polypyrimidine tract-binding protein, which also binds to the poliovirus IRES. In contrast to its weak interaction with cellular mRNAs, eIF4B remains tightly associated with the poliovirus IRES during the formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. These results indicate that the interaction of eIF4B with the 3" region of the poliovirus IRES may be directly involved in translation initiation.     

IRES-mediated translation of cellular messenger RNA operates in eIF2α- independent manner during stress

Physiological and pathophysiological stress attenuates global translation via phosphorylation of eIF2α. This in turn leads to the reprogramming of gene expression that is required for adaptive stress response. One class of cellular messenger RNAs whose translation was reported to be insensitive to eIF2α phosphorylation-mediated repression of translation is that harboring an Internal Ribosome Entry Site (IRES). IRES-mediated translation of several apoptosis-regulating genes increases in response to hypoxia, serum deprivation or gamma irradiation and promotes tumor cell survival and chemoresistance. However, the molecular mechanism that allows IRES-mediated translation to continue in an eIF2α-independent manner is not known. Here we have used the X-chromosome linked Inhibitor of Apoptosis, XIAP, IRES to address this question. Using toeprinting assay, western blot analysis and polysomal profiling we show that the XIAP IRES supports cap-independent translation when eIF2α is phosphorylated both in vitro and in vivo. During normal growth condition eIF2α-dependent translation on the IRES is preferred. However, IRES-mediated translation switches to eIF5B-dependent mode when eIF2α is phosphorylated as a consequence of cellular stress.     

Continuous heat shock enhances translational initiation directed by internal ribosomal entry site

Many cellular mRNAs contain internal ribosomal entry sites (IRES) that become functional under conditions of cellular stress, when the rate of protein synthesis for most cellular mRNA is reduced. Internal ribosomal entry increases in response to hypoxia, cell differentiation, apoptosis, gamma irradiation, and heat shock. Heat shock is the principal cellular stress in which general cap-dependent translation is inhibited. On the other hand, heat shock induces the preferential translation of a small class of mRNA, called heat shock protein (HSP) mRNAs, which probably occurs because little or no eIF4F activity is required for their translation. In this study, we found that continuous heat stress enhances expression of the heat shock protein BiP at the level of translation. Interestingly, heat stress also enhanced the viral IRES-dependent translation of encephalomyocarditis virus and hepatitis C virus but not poliovirus. Although several BiP inducers increased BiP protein expression, BiP IRES-dependent translation was enhanced only during heat shock, suggesting that heat shock is a specific inducer for BiP IRES-dependent translation. Taken together, these results indicate that the mechanism of IRES-dependent translation can be used during heat shock and suggest that this translational mechanism may be critical to the survival and proliferation of cells under stress.     

Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a nucleocytoplasmic shuttling protein that regulates gene expression through its action on mRNA metabolism and translation. The cytoplasmic redistribution of hnRNP A1 is a regulated process during viral infection and cellular stress. Here, we show that hnRNP A1 is an internal ribosome entry site (IRES) trans-acting factor that binds specifically to the 5' untranslated region of both the human rhinovirus-2 and the human apoptotic peptidase activating factor 1 (apaf-1) mRNAs, thereby regulating their translation. Furthermore, the cytoplasmic redistribution of hnRNP A1 after rhinovirus infection leads to enhanced rhinovirus IRES-mediated translation, whereas the cytoplasmic relocalization of hnRNP A1 after UVC irradiation limits the UVC-triggered translational activation of the apaf-1 IRES. Therefore, this study provides a direct demonstration that IRESs behave as translational enhancer elements regulated by specific trans-acting mRNA binding proteins in given physiological conditions. Our data highlight a new way to regulate protein synthesis in eukaryotes through the subcellular relocalization of a nuclear mRNA-binding protein.     

Internal ribosome entry site-mediated translation of Apaf-1, but not XIAP, is regulated during UV-induced cell death

Components of the cellular translation machinery are targets of caspase-mediated cleavage during apoptosis that correlates with the inhibition of protein synthesis, which accompanies apoptosis. Paradoxically, protein synthesis is required for apoptosis to occur in many experimental settings. Previous studies showed that two proteins that regulate apoptosis by controlling caspase activity, XIAP and Apaf-1, are translated by a unique, cap-independent mechanism mediated by an internal ribosome entry site (IRES) that is used preferentially under conditions in which normal cap-dependent translation is repressed. We investigated the regulation of XIAP and Apaf-1 following UVC irradiation. We show that UVC irradiation leads to the inhibition of translation and cell death. Furthermore, IRES-mediated translation of Apaf-1, but not XIAP, is enhanced by UVC irradiation, and this increase in Apaf-1 translation correlated with cell death. The enhanced Apaf-1 IRES-mediated translation is caspase-independent but is negatively modulated by the eIF2alpha kinase protein kinase RNA-like endoplasmic reticulum kinase. These data suggest that progression of UV-induced apoptosis requires IRES-mediated translation of Apaf-1 to ensure continuous levels of Apaf-1 despite an overall suppression of protein synthesis.     

Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry.

Translation of picornavirus RNA is initiated after ribosomal binding to an internal ribosomal entry site (IRES) within the 5' untranslated region. We have reconstituted IRES-mediated initiation on encephalomyocarditis virus RNA from purified components and used primer extension analysis to confirm the fidelity of 48S preinitiation complex formation. Eukaryotic initiation factor 2 (eIF2), eIF3, and eIF4F were required for initiation; eIF4B and to a lesser extent the pyrimidine tract-binding protein stimulated this process. We show that eIF4F binds to the IRES in a novel cap-independent manner and suggest that cap- and IRES-dependent initiation mechanisms utilize different modes of interaction with this factor to promote ribosomal attachment to mRNA.     

Cap-independent translation conferred by the 5' leader of tobacco etch virus is eukaryotic initiation factor 4G dependent

The 5' leader of tobacco etch virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.     

Translation initiation factor 4E inhibits differentiation of erythroid progenitors

Stem cell factor (SCF) delays differentiation and enhances the expansion of erythroid progenitors. Previously, we performed expression-profiling experiments to link signaling pathways to target genes using polysome-bound mRNA. SCF-induced phosphoinositide-3-kinase (PI3K) appeared to control polysome recruitment of specific mRNAs associated with neoplastic transformation. To evaluate the role of mRNA translation in the regulation of expansion versus differentiation of erythroid progenitors, we examined the function of the eukaryote initiation factor 4E (eIF4E) in these cells. SCF induced a rapid and complete phosphorylation of eIF4E-binding protein (4E-BP). Overexpression of eIF4E did not induce factor-independent growth but specifically impaired differentiation into mature erythrocytes. Overexpression of eIF4E rendered polysome recruitment of mRNAs with structured 5' untranslated regions largely independent of growth factor and resistant to the PI3K inhibitor LY294002. In addition, overexpression of eIF4E rendered progenitors insensitive to the differentiation-inducing effect of LY294002, indicating that control of mRNA translation is a major pathway downstream of PI3K in the regulation of progenitor expansion.     

Defects in translational regulation mediated by the alpha subunit of eukaryotic initiation factor 2 inhibit antiviral activity and facilitate the malignant transformation of human fibroblasts

Suppression of protein synthesis through phosphorylation of the translation initiation factor alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) is known to occur in response to many forms of cellular stress. To further study this, we have developed novel cell lines that inducibly express FLAG-tagged versions of either the phosphomimetic eIF2alpha variant, eIF2alpha-S51D, or the phosphorylation-insensitive eIF2alpha-S51A. These variants showed authentic subcellular localization, were incorporated into endogenous ternary complexes, and were able to modulate overall rates of protein synthesis as well as influence cell division. However, phosphorylation of eIF2alpha failed to induce cell death or sensitize cells to killing by proapoptotic stimuli, though it was able to inhibit viral replication, confirming the role of eIF2alpha in host defense. Further, although the eIF2alpha-S51A variant has been shown to transform NIH 3T3 cells, it was unable to transform the murine fibroblast 3T3 L1 cell line. To therefore clarify this issue, we explored the role of eIF2alpha in growth control and demonstrated that the eIF2alpha-S51A variant is capable of collaborating with hTERT and the simian virus 40 large T antigen in the transformation of primary human kidney cells. Thus, dysregulation of translation initiation is indeed sufficient to cooperate with defined oncogenic elements and participate in the tumorigenesis of human tissue.     

eIF2A mediates translation of hepatitis C viral mRNA under stress conditions

Translation of most mRNAs is suppressed under stress conditions. Phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF2), which delivers initiator tRNA (Met-tRNA(i)) to the P site of the 40S ribosomal subunit, is responsible for such translational suppression. However, translation of hepatitis C viral (HCV) mRNA is refractory to the inhibitory effects of eIF2α phosphorylation, which prevents translation by disrupting formation of the eIF2-GTP-Met-tRNA(i) ternary complex. Here, we report that eIF2A, an alternative initiator tRNA-binding protein, has a key role in the translation of HCV mRNA during HCV infection, in turn promoting eIF2α phosphorylation by activating the eIF2α kinase PKR. Direct interaction of eIF2A with the IIId domain of the HCV internal ribosome entry site (IRES) is required for eIF2A-dependent translation. These data indicate that stress-independent translation of HCV mRNA occurs by recruitment of eIF2A to the HCV IRES via direct interaction with the IIId domain and subsequent loading of Met-tRNA(i) to the P site of the 40S ribosomal subunit.     

Internal ribosome initiation of translation and the control of cell death

The majority of cellular stresses lead to the inhibition of cap-dependent translation. Some mRNAs, however, are translated by a cap-independent mechanism, mediated by ribosome binding to internal ribosome entry site (IRES) elements located in the 5' untranslated region. Interestingly, IRES elements are found in the mRNAs of several survival factors, oncogenes and proteins crucially involved in the control of apoptosis. These mRNAs are translated under a variety of stress conditions, including hypoxia, serum deprivation, irradiation and apoptosis. Thus, IRES-mediated translational control might have evolved to regulate cellular responses in acute but transient stress conditions that would otherwise lead to cell death.     

Deficiency of dietary EAA preferentially inhibits mRNA translation of ribosomal proteins in liver of meal-fed rats

The goal of these studies was to investigate the mechanisms by which amino acid supply regulates global rates of protein synthesis as well as the translation of ribosomal protein (rp) mRNAs in liver. In the experiments conducted, male weanling rats were trained over a 2-wk period to consume their daily food intake within 3 h. On day 14, rats were fed the control diet or an isocaloric, isonitrogenous diet lacking glycine, tryptophan, leucine, or the branched-chain amino acids (BCAA) for 1 h. Feeding Trp-, Leu-, or BCAA-deficient diets resulted in significant reductions in serum insulin, hepatic protein synthesis, eukaryotic initiation factor 2B (eIF2B) activity, and phosphorylation of eIF4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K1). Phosphorylation of eIF2alpha was inversely related to eIF2B activity under all conditions. Alterations in the hepatic synthesis of rp were assessed by changes in the distribution of rp (S4, S8, L26) mRNAs across sucrose density gradients and compared with non-rp (beta-actin, albumin) mRNAs. In all dietary treatments, non-rp mRNAs were mostly polysome associated. Conversely, the proportion of rp mRNAs residing in polysomes was two- to fivefold less in rats fed diets lacking tryptophan, leucine, or BCAA compared with rats fed the control diet. Total hepatic abundance of all mRNAs examined did not differ among treatment groups. For all parameters examined, there were no differences between rats fed the glycine-deficient diet and rats fed the control diet. The data suggest that essential amino acid (EAA) deficiency inhibits global rates of liver protein synthesis via a block in translation initiation. Additionally, the translation of rp mRNAs is preferentially repressed in association with decreased S6K1 phosphorylation.     

Characterization of the functional role of nucleotides within the URE2 IRES element and the requirements for eIF2A-mediated repression

Cap-independent initiation of translation is thought to promote protein synthesis on some mRNAs during times when cap-dependent initiation is down-regulated. However, the mechanism of cap-independent initiation is poorly understood. We have previously reported the secondary structure within the yeast minimal URE2 IRES element. In this study, we sought to investigate the mechanism of internal initiation in yeast by assessing the functional role of nucleotides within the minimal URE2 IRES element, and delineating the cis-sequences that modulate levels of internal initiation using a monocistronic reporter vector. Furthermore, we compared the eIF2A sensitivity of the URE2 IRES element with some of the invasive growth IRES elements using ΔeIF2A yeast. We found that the stability of the stem–loop structure within the minimal URE2 IRES element is not a critical determinant of optimal IRES activity, and the downstream sequences that modulate URE2 IRES-mediated translation can be defined to discrete regions within the URE2 coding region. Repression of internal initiation on the URE2 minimal IRES element by eIF2A is not dependent on the stability of the secondary structure within the URE2 IRES element. Our data also indicate that eIF2A-mediated repression is not specific to the URE2 IRES element, as both the GIC1 and PAB1 IRES elements are repressed by eIF2A. These data provide valuable insights into the mRNA requirements for internal initiation in yeast, and insights into the mechanism of eIF2A-mediated suppression.