Analysis of the isoform of Xenopus euakryotic translation initiation factor 4E

We have found two isoforms of the eukaryotic translation initiation factor 4E (eIF4E) in Xenopus laevis. These proteins differ in length by 18 amino acids. Overexpression of either of the two eIF4E proteins modestly increase translation in Xenopus oocytes. The results suggest that both of these two isoforms function in translation.     

Inhibition of translation and progesterone-induced maturation of Xenopus oocytes by expressing the amino-terminal portion of the eukaryotic translation initiation factor 4G

The eukaryotic translation initiation factor 4G (eIF4G) plays a pivotal role in translation. EIF4G interacts with several other factors including eIF4E, which is a cap-binding protein, and the poly(A)-binding protein (PABP). In this work, we demonstrate that the expression of the amino-terminal one-third of eIF4G, which interacts with eIF4E and PABP, in Xenopus oocyte inhibits translation and progesterone-induced maturation.     

Translational control by neuroguidin, a eukaryotic initiation factor 4E and CPEB binding protein

CPEB-mediated translation is important in early development and neuronal synaptic plasticity. Here, we describe a new eukaryotic initiation factor 4E (eIF4E) binding protein, Neuroguidin (Ngd), and its interaction with CPEB. In the mammalian nervous system, Ngd is detected as puncta in axons and dendrites and in growth cones and filopodia. Ngd contains three motifs that resemble those present in eIF4G, 4EBP, Cup, and Maskin, all of which are eIF4E binding proteins. Ngd binds eIF4E directly, and all three motifs must be deleted to abrogate the interaction between these two proteins. In injected Xenopus oocytes, Ngd binds CPEB and, most importantly, represses translation in a cytoplasmic polyadenylation element (CPE)-dependent manner. In Xenopus embryos, Ngd is found in both neural tube and neural crest cells. The injection of morpholino-containing antisense oligonucleotides directed against ngd mRNA disrupts neural tube closure and neural crest migration; however, the wild-type phenotype is restored by the injection of a rescuing ngd mRNA. These data suggest that Ngd guides neural development by regulating the translation of CPE-containing mRNAs.     

The stem-loop binding protein stimulates histone translation at an early step in the initiation pathway

Metazoan replication-dependent histone mRNAs do not have a poly(A) tail but end instead in a conserved stem-loop structure. Efficient translation of these mRNAs is dependent on the stem-loop binding protein (SLBP). Here we explore the mechanism by which SLBP stimulates translation in vertebrate cells, using the tethered function assay and analyzing protein-protein interactions. We show for the first time that translational stimulation by SLBP increases during oocyte maturation and that SLBP stimulates translation at the level of initiation. We demonstrate that SLBP can interact directly with subunit h of eIF3 and with Paip1; however, neither of these interactions is sufficient to mediate its effects on translation. We find that Xenopus SLBP1 functions primarily at an early stage in the cap-dependent initiation pathway, targeting small ribosomal subunit recruitment. Analysis of IRES-driven translation in Xenopus oocytes suggests that SLBP activity requires eIF4E. We propose a model in which a novel factor contacts eIF4E bound to the 5' cap and SLBP bound to the 3' end simultaneously, mediating formation of an alternative end-to-end complex.     

CDK1 and calcineurin regulate Maskin association with eIF4E and translational control of cell cycle progression

Maskin regulates assembly of the eIF4F translation initiation complex on messenger RNAs that contain cytoplasmic polyadenylation elements (CPEs) in their 3' untranslated regions. Because Maskin and eIF4G contain similar peptide motifs that bind eIF4E, they compete for occupancy of this factor and consequently control translation. One mRNA that is regulated by Maskin encodes cyclin B1, whose translation oscillates with the early cell cycles of Xenopus laevis embryos. Here we show that Maskin phosphorylation-dephosphorylation also oscillates with the cell cycle and is controlled by the kinase CDK1 and the phosphatase calcineurin. These phosphorylation events control the Maskin-eIF4E interaction and, as a result, translation of cyclin B1 mRNA. Cell cycle progression requires this Maskin-mediated translational regulation.     

Binding analysis of Xenopus laevis translation initiation factor 4E (eIF4E) in initiation complex formation.

A translation initiation factor, eIF4E, of Xenopus laevis was purified by affinity column chromatography after the gene expression as a full-length protein in a baculovirus-insect cell system. Interaction between X. laevis eIF4E and 4E-BP2 was analyzed by affinity column chromatography, gel permeation chromatography (GPC), and surface plasmon resonance (SPR). It was found that the interaction of eIF4E with an mRNA cap-analogue enhanced the binding activity of eIF4E with 4E-BP2. Furthermore, the SPR analysis showed that the eIF4E-cap-analogue interaction was very weak regardless of complex formation of 4E-BP2 with eIF4E; the dissociation constant of eIF4E for the cap-analogue was estimated to be 10(-2)-10(-4) M. These results suggest that the participation of another initiation factor is required for eIF4E to recognize the cap structure in vivo. The results reported in this paper support "the performed complex model" of Lee et al., in which eIF4E binds to the mRNA cap structure after the initiation factors have formed the initiation complex eIF4F.     

Differential phosphorylation controls Maskin association with eukaryotic translation initiation factor 4E and localization on the mitotic apparatus

Several cytoplasmic polyadenylation element (CPE)-containing mRNAs that are repressed in Xenopus oocytes become active during meiotic maturation. A group of factors that are anchored to the CPE are responsible for this repression and activation. Two of the most important are CPEB, which binds directly to the CPE, and Maskin, which associates with CPEB. In oocytes, Maskin also binds eukaryotic translation initiation factor 4E (eIF4E), an interaction that excludes eIF4G and prevents formation of the eIF4F initiation complex. When the oocytes are stimulated to reenter the meiotic divisions (maturation), CPEB promotes cytoplasmic polyadenylation. The newly elongated poly(A) tail becomes bound by poly(A) binding protein (PABP), which in turn binds eIF4G and helps it displace Maskin from eIF4E, thereby inducing translation. Here we show that Maskin undergoes several phosphorylation events during oocyte maturation, some of which are important for its dissociation from eIF4E and translational activation of CPE-containing mRNA. These sites are T58, S152, S311, S343, S453, and S638 and are phosphorylated by cdk1. Mutation of these sites to alanine alleviates the cdk1-induced dissociation of Maskin from eIF4E. Prior to maturation, Maskin is phosphorylated on S626 by protein kinase A. While this modification has no detectable effect on translation during oocyte maturation, it is critical for this protein to localize on the mitotic apparatus in somatic cells. These results show that Maskin activity and localization is controlled by differential phosphorylation.     

Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus oocyte maturation

The poly(A)-binding protein Pab1p interacts directly with the eukaryotic translation initiation factor 4G (eIF4G) to facilitate translation initiation of polyadenylated mRNAs in yeast [1,2]. Although the eIF4G-PABP interaction has also been demonstrated in a mammalian system [3,4], its biological significance in vertebrates is unknown. In Xenopus oocytes, cytoplasmic polyadenylation of several mRNAs coincides with their translational activation and is critical for maturation [5-7]. Because the amount of PABP is very low in oocytes [8], it has been argued that the eIF4G-PABP interaction does not play a major role in translational activation during oocyte maturation. Also, overexpression of PABP in Xenopus oocytes has only a modest stimulatory effect on translation of polyadenylated mRNA and does not alter either the efficiency or the kinetics of progesterone-induced maturation [9]. Here, we report that the expression of an eIF4GI mutant defective in PABP binding in Xenopus oocytes reduces translation of polyadenylated mRNA and dramatically inhibits progesterone-induced maturation. Our results show that the eIF4G-PABP interaction is critical for translational control of maternal mRNAs during Xenopus development.     

CPEB interacts with an ovary-specific eIF4E and 4E-T in early Xenopus oocytes

CPEB (cytoplasmic polyadenylation element-binding protein) is an important regulator of translation in oocytes and neurons. Although previous studies of CPEB in late Xenopus oocytes involve the eIF4E-binding protein maskin as the key factor for the repression of maternal mRNA, a second mechanism must exist, since maskin is absent earlier in oogenesis. Using co-immunoprecipitation and gel filtration assays, we show that CPEB specifically interacts, via protein/protein interactions, with the RNA helicase Xp54, the RNA-binding proteins P100(Pat1) and RAP55, the eIF4E-binding protein 4E-T, and an eIF4E protein. Remarkably, these CPEB complex proteins have been characterized, in one or more organism, as P-body, maternal, or neuronal granule components. We do not detect interactions with eIF4E1a, the canonical cap-binding factor, eIF4G, or eIF4A or with proteins expressed late in oogenesis, including maskin, PARN, and 4E-BP1. The eIF4E protein was identified as eIF4E1b, a close homolog of eIF4E1a, whose expression is restricted to oocytes and early embryos. Although eIF4E1b possesses all residues required for cap and eIF4G binding, it binds m(7)GTP weakly, and in pull-down assays, rather than binding eIF4G, it binds 4E-T, in a manner independent of the consensus eIF4E-binding site, YSKEELL. Wild type and Y-A mutant 4E-T (which binds eIF4E1b but not eIF4E1a), when tethered to a reporter mRNA, represses its translation in a cap-dependent manner, and injection of eIF4E1b antibody accelerates meiotic maturation. Altogether, our data suggest that CPEB, partnered with several highly conserved RNA-binding partners, inhibits protein synthesis in oocytes using a novel pairing of 4E-T and eIF4E1b.     

Eukaryotic Translation Initiation Factor 4AIII (eIF4AIII) Is Functionally Distinct from eIF4AI and eIF4AII

Eukaryotic initiation factor 4A (eIF4A) is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' proximal secondary structure of eukaryotic mRNAs to facilitate attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors (eIF4E and eIF4G). Two isoforms of eIF4A, eIF4AI and eIF4AII, which are encoded by two different genes, are functionally indistinguishable. A third member of the eIF4A family, eIF4AIII, whose human homolog exhibits 65% amino acid identity to human eIF4AI, has also been cloned from Xenopus and tobacco, but its function in translation has not been characterized. In this study, human eIF4AIII was characterized biochemically. While eIF4AIII, like eIF4AI, exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, it fails to substitute for eIF4AI in an in vitro-reconstituted 40S ribosome binding assay. Instead, eIF4AIII inhibits translation in a reticulocyte lysate system. In addition, whereas eIF4AI binds independently to the middle and carboxy-terminal fragments of eIF4G, eIF4AIII binds to the middle fragment only. These functional differences between eIF4AI and eIF4AIII suggest that eIF4AIII might play an inhibitory role in translation under physiological conditions.     

Interference with interaction between eukaryotic translation initiation factor 4G and poly(A)-binding protein in Xenopus oocytes leads to inhibition of polyadenylated mRNA translation and oocyte maturation.

The interaction between eukaryotic translation initiation factor 4G (eIF4G) and the poly(A)-binding protein (PABP) facilitates translational initiation of polyadenylated mRNAs. It was shown recently that the expression of an eIF4GI mutant defective in PABP binding in Xenopus oocytes reduces polyadenylated mRNA translation and dramatically inhibits progesterone-induced oocyte maturation. These results strongly suggest that the eIF4G-PABP interaction plays a critical role in the translational control of maternal mRNAs during oocyte maturation. In the present work, we employed another strategy to interfere eIF4G-PABP interaction in Xenopus oocytes. The amino-terminal part of eIF4GI containing the PABP-binding site (4GNt-M1) was expressed in Xenopus oocytes. 4GNt-M1 could bind to PABP in oocytes, which suggests that 4GNt-M1 may evict PABP from the endogenous eIF4G. The expression of 4GNt-M1 resulted in reduction of polyadenylated mRNA translation. Furthermore, 4GNt-M1 inhibited progesterone-induced oocyte maturation. In contrast, 4GNt-M2, in which the PABP-binding sequences were mutated to abolish the PABP-binding activity, could not inhibit polyadenylated mRNA translation or oocyte maturation. These results further support the idea that the eIF4G-PABP interaction is critical for translational regulation of maternal mRNAs in oocytes.     

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.     

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.     

Translational homeostasis via the mRNA cap-binding protein, eIF4E

Translational control of gene expression plays a key role in many biological processes. Consequently, the activity of the translation apparatus is under tight homeostatic control. eIF4E, the mRNA 5' cap-binding protein, facilitates cap-dependent translation and is a major target for translational control. eIF4E activity is controlled by a family of repressor proteins, termed 4E-binding proteins (4E-BPs). Here, we describe the surprising finding that despite the importance of eIF4E for translation, a drastic knockdown of eIF4E caused only minor reduction in translation. This conundrum can be explained by the finding that 4E-BP1 is degraded in eIF4E-knockdown cells. Hypophosphorylated 4E-BP1, which binds to eIF4E, is degraded, whereas hyperphosphorylated 4E-BP1 is refractory to degradation. We identified the KLHL25-CUL3 complex as the E3 ubiquitin ligase, which targets hypophosphorylated 4E-BP1. Thus, the activity of eIF4E is under homeostatic control via the regulation of the levels of its repressor protein 4E-BP1 through ubiquitination.     

Targeting the eIF4F translation initiation complex for cancer therapy

Abstract In multiple human cancers, the function of the eukaryotic translation initiation factor 4E (eIF4E) is elevated and directly related to disease progression. Overexpression or hyperactivation of eIF4E in experimental models can drive cellular transformation and malignant progression. Elevated eIF4E function triggers enhanced assembly of the eIF4F translation initiation complex and thereby drives cap-dependent translation. Though all capped mRNAs require eIF4F for translation, a pool of mRNAs are exceptionally dependent on elevated eIF4F activity for translation and are thereby selectively and disproportionately affected by altered eIF4F activity. These mRNAs encode proteins that play significant roles in all aspects of malignancy including angiogenesis factors (VEGF, FGF-2), onco-proteins (c-myc, cyclin D1, ODC), pro-survival proteins (survivin, BCL-2) and proteins involved in tumor invasion and metastasis (MMP-9, heparanase). Recent advances in targeting the eIF4F complex have highlighted the role for this complex in tumor cell survival and angiogenesis and have illuminated the enhanced susceptibility of the tumor cells to inhibition of the eIF4F complex. These studies have demonstrated the attractiveness and plausibility of targeting eIF4E and the eIF4F translation initiation complex for cancer therapy and have prompted the advance of the first eIF4E-specific therapy to the clinic.     

Regulation of eukaryotic initiation factor 4E phosphorylation in the nervous system of Aplysia californica

We have used an antibody that specifically recognizes eukaryotic initiation factor 4E (eIF4E) when it is phosphorylated at Ser(207) to characterize eIF4E phosphorylation in the nervous system of APLYSIA: The level of phosphorylated eIF4E, but not the level of total eIF4E, was significantly correlated with the basal rate of translation measured from different animals. Serotonin (5-HT), a transmitter that regulates the rate of translation in APLYSIA: neurons, had mixed effects on eIF4E phosphorylation. 5-HT decreased eIF4E phosphorylation in sensory cell clusters through activation of protein kinase C. 5-HT increased eIF4E phosphorylation in the whole pleural ganglia. In the APLYSIA: nervous system, eIF4E phosphorylation correlated with phosphorylation of the p38 MAP kinase, but not the p42 MAP kinase (ERK). Furthermore, an inhibitor of the p38 MAP kinase significantly decreased basal eIF4E phosphorylation, but an inhibitor of the MAP or ERK kinase (MEK) did not. Despite the correlation of eIF4E phosphorylation with the basal rate of translation, inhibition of eIF4E phosphorylation by an inhibitor of the p38 MAP kinase did not significantly decrease the rate of translation.     

eIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo

The mRNA's cap-binding protein eukaryotic translation initiation factor (eIF)4E is a major target for the regulation of translation initiation. eIF4E activity is controlled by a family of translation inhibitors, the eIF4E-binding proteins (4E-BPs). We have previously shown that a rapid dissociation of 4E-BP from eIF4E is related with the dramatic rise in protein synthesis that occurs following sea urchin fertilization. Here, we demonstrate that 4E-BP is destroyed shortly following fertilization and that 4E-BP degradation is sensitive to rapamycin, suggesting that proteolysis could be a novel means of regulating 4E-BP function. We also show that eIF4E/4E-BP dissociation following fertilization is sensitive to rapamycin. Furthermore, while rapamycin modestly affects global translation rates, the drug strongly inhibits cyclin B de novo synthesis and, consequently, precludes the completion of the first mitotic cleavage. These results demonstrate that, following sea urchin fertilization, cyclin B translation, and thus the onset of mitosis, are regulated by a rapamycin-sensitive pathway. These processes are effected at least in part through eIF4E/4E-BP complex dissociation and 4E-BP degradation.     

A new translational regulator with homology to eukaryotic translation initiation factor 4G.

Translation initiation in eukaryotes is facilitated by the cap structure, m7GpppN (where N is any nucleotide). Eukaryotic translation initiation factor 4F (eIF4F) is a cap binding protein complex that consists of three subunits: eIF4A, eIF4E and eIF4G. eIF4G interacts directly with eIF4E and eIF4A. The binding site of eIF4E resides in the N-terminal third of eIF4G, while eIF4A and eIF3 binding sites are present in the C-terminal two-thirds. Here, we describe a new eukaryotic translational regulator (hereafter called p97) which exhibits 28% identity to the C-terminal two-thirds of eIF4G. p97 mRNA has no initiator AUG and translation starts exclusively at a GUG codon. The GUG-initiated open reading frame (907 amino acids) has no canonical eIF4E binding site. p97 binds to eIF4A and eIF3, but not to eIF4E. Transient transfection experiments show that p97 suppresses both cap-dependent and independent translation, while eIF4G supports both translation pathways. Furthermore, inducible expression of p97 reduces overall protein synthesis. These results suggest that p97 functions as a general repressor of translation by forming translationally inactive complexes that include eIF4A and eIF3, but exclude eIF4E.     

The 3' cap-independent translation element of Barley yellow dwarf virus binds eIF4F via the eIF4G subunit to initiate translation

The 3' cap-independent translation element (BTE) of Barley yellow dwarf virus RNA confers efficient translation initiation at the 5' end via long-distance base pairing with the 5'-untranslated region (UTR). Here we provide evidence that the BTE functions by recruiting translation initiation factor eIF4F. We show that the BTE interacts specifically with the cap-binding initiation factor complexes eIF4F and eIFiso4F in a wheat germ extract (wge). In wge depleted of cap-interacting factors, addition of eIF4F (and to a lesser extent, eIFiso4F) allowed efficient translation of an uncapped reporter construct (BLucB) containing the BTE in its 3' UTR. Translation of BLucB required much lower levels of eIF4F or eIFiso4F than did a capped, nonviral mRNA. Both full-length eIF4G and the carboxy-terminal half of eIF4G lacking the eIF4E binding site stimulated translation to 70% of the level obtained with eIF4F, indicating a minor role for the cap-binding protein, eIF4E. In wge inhibited by either BTE in trans or cap analog, eIF4G alone restored translation nearly as much as eIF4F, while addition of eIF4E alone had no effect. The BTE bound eIF4G (Kd = 177 nm) and eIF4F (Kd = 37 nm) with high affinity, but very weakly to eIF4E. These interactions correlate with the ability of the factors to facilitate BTE-mediated translation. These results and previous observations are consistent with a model in which eIF4F is delivered to the 5' UTR by the BTE, and they show that eIF4G, but not eIF4E, plays a major role in this novel mechanism of cap-independent translation.     

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.