Conducting the initiation of protein synthesis: the role of eIF4G

The eukaryotic initiation factor eIF4G is a large modular protein which serves as a docking site for initiation factors and proteins involved in RNA translation. Together with eIF4E and eIF4A, eIF4G constitutes the eIF4F complex which is a key component in promoting ribosome binding to the mRNA. Thus, the central role of eIF4G in initiation makes it a valid target for events aimed at modulating translation. Such events occur during viral infection by picornaviruses and lentiviruses and result in the hijack of the translational machinery through cleavage of eIF4G. Proteolysis of eIF4G is also mediated by caspases during the onset of apoptosis causing inhibition of protein synthesis. We will review the role of eIF4G and protein partners as well as the cellular and viral events that modulate eIF4G activity in the initiation of translation.     

Interaction of translation initiation factor eIF4G with eIF4A in the yeast Saccharomyces cerevisiae.

Eukaryotic initiation factor (eIF) 4A is an essential protein that, in conjunction with eIF4B, catalyzes the ATP-dependent melting of RNA secondary structure in the 5'-untranslated region of mRNA during translation initiation. In higher eukaryotes, eIF4A is assumed to be recruited to the mRNA through its interaction with eIF4G. However, the failure to detect this interaction in yeast brought into question the generality of this model. The work presented here demonstrates that yeast eIF4G interacts with eIF4A both in vivo and in vitro. The eIF4A-binding site was mapped to amino acids 542-883 of yeast eIF4G1. Expression in yeast cells of the eIF4G1 domain that binds eIF4A results in cell growth inhibition, and addition of this domain to an eIF4A-dependent in vitro system inhibits translation in a dose-dependent manner. Both in vitro translation and cell growth can be specifically restored by increasing the eIF4A concentration. These data demonstrate that yeast eIF4A and eIF4G interact and suggest that this interaction is required for translation and cell growth.     

Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo

Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA 5' cap and brings the mRNA into a complex with other protein synthesis initiation factors and ribosomes. The activity of mammalian eIF4E is important for the translation of capped mRNAs and is thought to be regulated by two mechanisms. First, eIF4E is sequestered by binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce the release of eIF4E by stimulating the phosphorylation of 4EBP1. Second, mitogens and stresses induce the phosphorylation of eIF4E at Ser 209, increasing the affinity of eIF4E for capped mRNA and for an associated scaffolding protein, eIF4G. We previously showed that a mitogen- and stress-activated kinase, Mnk1, phosphorylates eIF4E in vitro at the physiological site. Here we show that Mnk1 regulates eIF4E phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies with eIF4G and eIF4E. We identified activating phosphorylation sites in Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogen-induced eIF4E phosphorylation, while expression of activated Mnk1 increases basal eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar kinase in cells and is independent of other mitogenic signals that release eIF4E from 4EBP1.     

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis

Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2alpha is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive. NMDA (N-methyl-D-aspartate) excitotoxicity is also involved in ischaemic damage, as exposure to NMDA impairs translation and promotes the synthesis of NO (nitric oxide), which can also inhibit translation. In the present study, we investigated whether NO was involved in NMDA-induced protein synthesis inhibition in neurons and studied the underlying molecular mechanisms. NMDA and the NO donor DEA/NO (diethylamine-nitric oxide sodium complex) both inhibited protein synthesis and this effect persisted after a 30 min exposure. Treatments with NMDA or NO promoted calpain-dependent eIF4G cleavage and 4E-BP1 (eIF4E-binding protein 1) dephosphorylation and also abolished the formation of eIF4E-eIF4G complexes; however, they did not induce eIF2alpha phosphorylation. Although NOS (NO synthase) inhibitors did not prevent protein synthesis inhibition during 30 min of NMDA exposure, they did abrogate the persistent inhibition of translation observed after NMDA removal. NOS inhibitors also prevented NMDA-induced eIF4G degradation, 4E-BP1 dephosphorylation, decreased eIF4E-eIF4G-binding and cell death. Although the calpain inhibitor calpeptin blocked NMDA-induced eIF4G degradation, it did not prevent 4E-BP1 dephosphorylation, which precludes eIF4E availability, and thus translation inhibition was maintained. The present study suggests that eIF4G integrity and hyperphosphorylated 4E-BP1 are needed to ensure appropriate translation in neurons. In conclusion, our data show that NO mediates NMDA-induced persistent translation inhibition and suggest that deficient eIF4F activity contributes to this process.     

Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.     

Translational control by the poly(A) binding protein: a check for mRNA integrity

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the capbinding protein eIF4E and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this "closed loop" mRNP among other effects enhance the affinity of eIF4E for the 5' cap by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picomavirus' internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to initiation complex formation. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP.     

RNA aptamers to mammalian initiation factor 4G inhibit cap-dependent translation by blocking the formation of initiation factor complexes

Eukaryotic translation initiation factor 4G (eIF4G) plays a crucial multimodulatory role in mRNA translation and decay by interacting with other translation factors and mRNA-associated proteins. In this study, we isolated eight different RNA aptamers with high affinity to mammalian eIF4G by in vitro RNA selection amplification. Of these, three aptamers (apt3, apt4, and apt5) inhibited the cap-dependent translation of two independent mRNAs in a rabbit reticulocyte lysate system. The cap-independent translation directed by an HCV internal ribosome entry site was not affected. Addition of exogenous eIF4G reversed the aptamer-mediated inhibition of translation. Even though apt3 and apt4 were selected independently, they differ only by two nucleotides. The use of truncated eIF4G variants in binding experiments indicated that apt4 (and probably apt3) bind to both the middle and C-terminal domains of eIF4G, while apt5 binds only to the middle domain of eIF4G. Corresponding to the difference in the binding sites in eIF4G, apt4, but not apt5, hindered eIF4G from binding to eIF4A and eIF3, in a purified protein solution system as well as in a crude lysate system. Therefore, the inhibition of translation by apt4 (and apt3) is due to the inhibition of formation of initiation factor complexes involving eIF4A and eIF3. On the other hand, apt5 had a much weaker affinity to eIF4G than apt4, but inhibited translation much more efficiently by an unknown mechanism. The five additional aptamers have sequences and predicted secondary structures that are largely different from each other and from apt3 through apt5. Therefore, we speculate that these seven sets of aptamers may bind to different regions in eIF4G in different fashions.     

Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1

Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5'-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2alpha is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.     

Eap1p, a novel eukaryotic translation initiation factor 4E-associated protein in Saccharomyces cerevisiae

Ribosome binding to eukaryotic mRNA is a multistep process which is mediated by the cap structure [m(7)G(5')ppp(5')N, where N is any nucleotide] present at the 5' termini of all cellular (with the exception of organellar) mRNAs. The heterotrimeric complex, eukaryotic initiation factor 4F (eIF4F), interacts directly with the cap structure via the eIF4E subunit and functions to assemble a ribosomal initiation complex on the mRNA. In mammalian cells, eIF4E activity is regulated in part by three related translational repressors (4E-BPs), which bind to eIF4E directly and preclude the assembly of eIF4F. No structural counterpart to 4E-BPs exists in the budding yeast, Saccharomyces cerevisiae. However, a functional homolog (named p20) has been described which blocks cap-dependent translation by a mechanism analogous to that of 4E-BPs. We report here on the characterization of a novel yeast eIF4E-associated protein (Eap1p) which can also regulate translation through binding to eIF4E. Eap1p shares limited homology to p20 in a region which contains the canonical eIF4E-binding motif. Deletion of this domain or point mutation abolishes the interaction of Eap1p with eIF4E. Eap1p competes with eIF4G (the large subunit of the cap-binding complex, eIF4F) and p20 for binding to eIF4E in vivo and inhibits cap-dependent translation in vitro. Targeted disruption of the EAP1 gene results in a temperature-sensitive phenotype and also confers partial resistance to growth inhibition by rapamycin. These data indicate that Eap1p plays a role in cell growth and implicates this protein in the TOR signaling cascade of S. cerevisiae.     

Translational control by the poly(A) binding protein: A check for mRNA integrity

The eukaryotic mRNA 3′ poly(A) tail and the 5′ cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the cap-binding protein eIF4E, and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this “closed loop” mRNA among other effects enhance the affinity of eIF4E for the 5′ cap, by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picornavirus’ internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to the formation of the initiation complex. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 684–693. The article is published in the original.     

Eukaryotic translation initiation: there are (at least) two sides to every story

The eukaryotic cap and poly(A) tail binding proteins, eIF4E and Pab1p, play important roles in the initiation of protein synthesis. The recent structures of the complex of eIF4E bound to the methylated guanosine (cap) found at the 5'end of messenger RNA (mRNA), the complex of eIF4E bound to peptide fragments of two related translation factors (eIF4G and 4E-BP1), and the complex of the N-terminal fragment of Pab1p bound to polyadenylate RNA have revealed that eIF4E and Pab1p contain at least two distinct functional surfaces. One surface is used for binding mRNA, and the other for binding proteins involved in translation initiation.     

High affinity RNA for mammalian initiation factor 4E interferes with mRNA-cap binding and inhibits translation

The eukaryotic translation initiation factor 4F (eIF4F) consists of three polypeptides (eIF4A, eIF4G, and eIF4E) and is responsible for recruiting ribosomes to mRNA. eIF4E recognizes the mRNA 5'-cap structure (m7GpppN) and plays a pivotal role in control of translation initiation, which is the rate-limiting step in translation. Overexpression of eIF4E has a dramatic effect on cell growth and leads to oncogenic transformation. Therefore, an inhibitory agent to eIF4E, if any, might serve as a novel therapeutic against malignancies that are caused by aberrant translational control. Along these lines, we developed two RNA aptamers, aptamer 1 and aptamer 2, with high affinity for mammalian eIF4E by in vitro RNA selection-amplification. Aptamer 1 inhibits the cap binding to eIF4E more efficiently than the cap analog m7GpppN or aptamer 2. Consistently, aptamer 1 inhibits specifically cap-dependent in vitro translation while it does not inhibit cap-independent HCV IRES-directed translation initiation. The interaction between eIF4E and eIF4E-binding protein 1 (4E-BP1), however, was not inhibited by aptamer 1. Aptamer 1 is composed of 86 nucleotides, and the high affinity to eIF4E is affected by deletions at both termini. Moreover, relatively large areas in the aptamer 1 fold are protected by eIF4E as determined by ribonuclease footprinting. These findings indicate that aptamers can achieve high affinity to a specific target protein via global conformational recognition. The genetic mutation and affinity study of variant eIF4E proteins suggests that aptamer 1 binds to eIF4E adjacent to the entrance of the cap-binding slot and blocks the cap-binding pocket, thereby inhibiting translation initiation.     

Cleavage of eukaryotic translation initiation factor 4GII correlates with translation inhibition during apoptosis

Eukaryotic translation initiation factor 4G (eIF4G), which has two homologs known as eIF4GI and eIF4GII, functions in a complex (eIF4F) which binds to the 5' cap structure of cellular mRNAs and facilitates binding of capped mRNA to 40S ribosomal subunits. Disruption of this complex in enterovirus-infected cells through eIF4G cleavage is known to block this step of translation initiation, thus leading to a drastic inhibition of cap-dependent translation. Here, we show that like eIF4GI, the newly identified homolog eIF4GII is cleaved during apoptosis in HeLa cells and can serve as a substrate for caspase 3. Proteolysis of both eIF4GI and eIF4GII occurs with similar kinetics and coincides with the profound translation inhibition observed in cisplatin-treated HeLa cells. Both eIF4GI and eIF4GII can be cleaved by caspase 3 with similar efficiency in vitro, however, eIF4GII is processed into additional fragments which destroy its core central domain and likely contributes to the shutoff of translation observed in apoptosis. Cell Death and Differentiation (2000) 7, 1234 - 1243.     

The Cap-binding protein eIF4E promotes folding of a functional domain of yeast translation initiation factor eIF4G1

The association of eucaryotic translation initiation factor eIF4G with the cap-binding protein eIF4E establishes a critical link between the mRNA and the ribosome during translation initiation. This association requires a conserved seven amino acid peptide within eIF4G that binds to eIF4E. Here we report that a 98-amino acid fragment of S. cerevisiae eIF4G1 that contains this eIF4E binding peptide undergoes an unfolded to folded transition upon binding to eIF4E. The folding of the eIF4G1 domain was evidenced by the eIF4E-dependent changes in its protease sensitivity and (1)H-(15)N HSQC NMR spectrum. Analysis of a series of charge-to-alanine mutations throughout the essential 55.4-kDa core of yeast eIF4G1 also revealed substitutions within this 98-amino acid region that led to reduced eIF4E binding in vivo and in vitro. These data suggest that the association of yeast eIF4E with eIF4G1 leads to the formation of a structured domain within eIF4G1 that could serve as a specific site for interactions with other components of the translational apparatus. They also suggest that the stability of the native eIF4E-eIF4G complex is determined by amino acid residues outside of the conserved seven-residue consensus sequence.     

Eukaryotic translation initiation factor 5 (eIF5) acts as a classical GTPase-activator protein

GTP hydrolysis occurs at several specific stages during the initiation, elongation, and termination stages of mRNA translation. However, it is unclear how GTP hydrolysis occurs; it has previously been suggested to involve a GTPase active center in the ribosome, although proof for this is lacking. Alternatively, it could involve the translation factors themselves, e.g., be similar to the situation for small G in which the GTPase active site involves arginine residues contributed by a further protein termed a GTPase-activator protein (GAP). During translation initiation in eukaryotes, initiation factor eIF5 is required for hydrolysis of GTP bound to eIF2 (the protein which brings the initiator Met-tRNA(i) to the 40S subunit). Here we show that eIF5 displays the hallmarks of a classical GAP (e.g., RasGAP). Firstly, its interaction with eIF2 is enhanced by AlF(4)(-). Secondly, eIF5 possesses a conserved arginine (Arg15) which, like the "arginine fingers" of classical GAPs, is flanked by hydrophobic residues. Mutation of Arg15 to methionine abolishes the ability of eIF5 either to stimulate GTP hydrolysis or to support mRNA translation in vitro. Mutation studies suggest that a second conserved arginine (Arg48) also contributes to the GTPase active site of the eIF2.eIF5 complex. Our data thus show that eIF5 behaves as a classical GAP and that GTP hydrolysis during translation involves proteins extrinsic to the ribosome. Indeed, inspection of their sequences suggests that other translation factors may also act as GAPs.     

General RNA‐binding proteins have a function in poly(A)‐binding protein‐dependent translation

The interaction between the poly(A)‐binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA‐binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB‐1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB‐1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP‐dependent after the addition of YB‐1. In this system, eIF4E binding to the cap structure is inhibited by YB‐1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB‐1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB‐1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP.     

Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.     

Inhibition of mammalian translation initiation by volatile anesthetics

Volatile anesthetics are essential for modern medical practice, but sites and mechanisms of action for any of their numerous cellular effects remain largely unknown. Previous studies with yeast showed that volatile anesthetics induce nutrient-dependent inhibition of growth through mechanisms involving inhibition of mRNA translation. Studies herein show that the volatile anesthetic halothane inhibits protein synthesis in perfused rat liver at doses ranging from 2 to 6%. A marked disaggregation of polysomes occurs, indicating that inhibition of translation initiation plays a key role. Dose- and time-dependent alterations that decrease the function of a variety of translation initiation processes are observed. At 6% halothane, a rapid and persistent increase in phosphorylation of the alpha-subunit of eukaryotic translation initiation factor (eIF)2 occurs. This is accompanied by inhibition of activity of the guanine nucleotide exchange factor eIF2B that is responsible for GDP-GTP exchange on eIF2. At lower doses, neither eIF2alpha phosphorylation nor eIF2B activity is altered. After extended exposure to 6% halothane, alterations in two separate responses regulated by the target of rapamycin pathway occur: 1) redistribution of eIF4E from its translation-stimulatory association with eIF4G to its translation-inactive complex with eIF4E-binding protein-1; and 2) decreased phosphorylation of ribosomal protein S6 (rpS6) with a corresponding decrease in active forms of a kinase that phosphorylates rpS6 (p70(S6K1)). Changes in the association of eIF4E and eIF4G are observed only after extended exposure to low anesthetic doses. Thus dose- and time-dependent alterations in multiple processes permit liver cells to adapt translation to variable degrees and duration of stress imposed by anesthetic exposure.     

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.     

Y-box Binding Protein 1: Providing a New Angle on Translational Regulation

Current models of translational regulation are mostly focused on how translational factors engage a messenger mRNA to the ribosome to initiate translation. Since the majority of mRNAs in eukaryotes are translated in a cap-dependent manner, the mRNA 5’ cap-binding protein eIF4E was characterized as a key player responsible for the recruitment of mRNAs to the initiation complex. The availability of eIF4E is believed to be especially critical for translational activation of mRNAs with extensive secondary structures in their 5’UTRs, many of which code for labile regulatory proteins essential for cell growth or viability. Surprisingly, little attention is paid to the other side of translational control, e.g., to define mechanisms responsible for translational silencing and storage of the above messages. In this review, we discuss the possibility that eIF4E per se may not be sufficient to release mRNAs from translational block. We found that many growth- and stress-related mRNAs are associated with the translational repressor YB-1, which can compete with the eIF4E-driven translation initiation complex for binding to the capped 5’ mRNA terminus. Moreover, the cap-dependent repressor activity of YB-1 appears to be negatively regulated via Akt-mediated phosphorylation of the Ser-102 residue of YB-1. Taken together with recent evidence suggesting that translational activation of growth-related messages is a primary cellular response to activation of Ras-Erk and PI3K-Akt signaling pathways, our data suggest that differential expression of specific mRNA subsets is regulated by the PI3K-Akt pathway and achieved via coordinated activation of the components of translational machinery and inactivation of general translational repressors such as YB-1.