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.     

Hypoxia-inducible Factor-1α (HIF-1α) Promotes Cap-dependent Translation of Selective mRNAs through Up-regulating Initiation Factor eIF4E1 in Breast Cancer Cells under Hypoxia Conditions

Hypoxia promotes tumor evolution and metastasis, and hypoxia-inducible factor-1α (HIF-1α) is a key regulator of hypoxia-related cellular processes in cancer. The eIF4E translation initiation factors, eIF4E1, eIF4E2, and eIF4E3, are essential for translation initiation. However, whether and how HIF-1α affects cap-dependent translation through eIF4Es in hypoxic cancer cells has been unknown. Here, we report that HIF-1α promoted cap-dependent translation of selective mRNAs through up-regulation of eIF4E1 in hypoxic breast cancer cells. Hypoxia-promoted breast cancer tumorsphere growth was HIF-1α-dependent. We found that eIF4E1, not eIF4E2 or eIF4E3, is the dominant eIF4E family member in breast cancer cells under both normoxia and hypoxia conditions. eIF4E3 expression was largely sequestered in breast cancer cells at normoxia and hypoxia. Hypoxia up-regulated the expression of eIF4E1 and eIF4E2, but only eIF4E1 expression was HIF-1α-dependent. In hypoxic cancer cells, HIF-1α-up-regulated eIF4E1 enhanced cap-dependent translation of a subset of mRNAs encoding proteins important for breast cancer cell mammosphere growth. In searching for correlations, we discovered that human eIF4E1 promoter harbors multiple potential hypoxia response elements. Furthermore, using chromatin immunoprecipitation (ChIP) and luciferase and point mutation assays, we found that HIF-1α utilized hypoxia response elements in the human eIF4E1 proximal promoter region to activate eIF4E1 expression. Our study suggests that HIF-1α promotes cap-dependent translation of selective mRNAs through up-regulating eIF4E1, which contributes to tumorsphere growth of breast cancer cells at hypoxia. The data shown provide new insights into protein synthesis mechanisms in cancer cells at low oxygen levels.     

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.     

Measles virus N protein inhibits host translation by binding to eIF3-p40

The nonsegmented, negative-sense RNA genome of measles virus (MV) is encapsidated by the virus-encoded nucleocapsid protein (N). In this study, we searched for N-binding cellular proteins by using MV-N as bait and screening the human T-cell cDNA library by yeast two-hybrid assay and isolated the p40 subunit of eukaryotic initiation factor 3 (eIF3-p40) as a binding partner. The interaction between MV-N and eIF3-p40 in mammalian cells was confirmed by coimmunoprecipitation. Since eIF3-p40 is a translation initiation factor, we analyzed the potential inhibitory effect of MV-N on protein synthesis. Glutathione S-transferase (GST)-fused MV-N (GST-N) inhibited translation of reporter mRNAs in rabbit reticulocyte lysate translation system in a dose-dependent manner. Encephalomyocarditis virus internal ribosomal entry site-mediated translation, which requires canonical initiation factors to initiate translation, was also inhibited by GST-N. In contrast, a unique form of translation mediated by the intergenic region of Plautia stali intestine virus, which can assemble 80S ribosomes in the absence of canonical initiation factors, was scarcely affected by GST-N. In vivo expression of MV-N induced by the Cre/loxP switching system inhibited the synthesis of a transfected reporter protein, as well as overall protein synthesis. These results suggest that MV-N targets eIF3-p40 and may be involved in inhibiting MV-induced host translation.     

Viral stress-inducible protein p56 inhibits translation by blocking the interaction of eIF3 with the ternary complex eIF2.GTP.Met-tRNAi

Viral stress-inducible protein p56 is produced in response to viral stress-inducing agents such as double-stranded RNA and interferon, as well as other poorly understood mechanisms of viral infection. It has been shown previously that p56 is able to bind the eukaryotic initiation factor 3e(eIF3e) (p48/Int-6) subunit of the eukaryotic translation initiation factor eIF3 and function as an inhibitor of translation in vitro and in vivo. The exact mechanism by which p56 is able to interfere with protein synthesis is not understood. Based on the known roles of eIF3 in the initiation pathway, we employed assays designed to individually look at specific functions of eIF3 and the effect of p56 on these eIF3-mediated functions. These assays examined the effect of p56 on ribosome dissociation, the eIF3.eIF4F interaction, and enhancement of the ternary complex eIF2.GTP.Met-tRNAi formation. Here we report that p56 is able to inhibit translation initiation specifically at the level of eIF3.ternary complex formation. The effect of p56-mediated inhibition was also examined in two different contexts, cap-mediated and encephalomyocarditis virus internal ribosomal entry site-mediated translation. Whereas cap-dependent initiation was severely inhibited by p56, internal ribosomal entry site-mediated translation appeared to be insensitive to p56.     

Cloning and characterization of the p42 subunit of mammalian translation initiation factor 3 (eIF3): demonstration that eIF3 interacts with eIF5 in mammalian cells.

Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit protein complex that plays an essential role in the binding of the initiator methionyl-tRNA and mRNA to the 40S ribosomal subunit to form the 40S initiation complex. cDNAs encoding all the subunits of mammalian eIF3 except the p42 subunit have been cloned in several laboratories. Here we report the cloning and characterization of a human cDNA encoding the p42 subunit of mammalian eIF3. The open reading frame of the cDNA, which encodes a protein of 320 amino acids (calculated Mr35 614) has been expressed in Escherichia coli and the recombinant protein has been purified to homogeneity. The purified protein binds RNA in agreement with the presence of a putative RNA binding motif in the deduced amino acid sequence. The protein shows 33% identity and 53% similarity with the Tif35p subunit (YDR 429C) of yeast eIF3. Transfection experiments demonstrated that polyhistidine-tagged p42 protein, transiently expressed in human U20S cells, was incorporated into endogenous eIF3. Furthermore, eIF3 isolated from transfected cell lysates contains bound eIF5 indicating that a specific physical interaction between eIF5 and eIF3 may play an important role in the function of eIF5 during translation initiation in eukaryotic cells.     

Moe1 and spInt6, the fission yeast homologues of mammalian translation initiation factor 3 subunits p66 (eIF3d) and p48 (eIF3e), respectively, are required for stable association of eIF3 subunits

The protein encoded by the fission yeast gene, moe1(+) is the homologue of the p66/eIF3d subunit of mammalian translation initiation factor eIF3. In this study, we show that in fission yeast, Moe1 physically associates with eIF3 core subunits as well as with 40 S ribosomal particles as a constituent of the eIF3 protein complex that is similar in size to multisubunit mammalian eIF3. However, strains lacking moe1(+) (Deltamoe1) are viable and show no gross defects in translation initiation, although the rate of translation in the Deltamoe1 cells is about 30-40% slower than wild-type cells. Mutant Deltamoe1 cells are hypersensitive to caffeine and defective in spore formation. These phenotypes of Deltamoe1 cells are similar to those reported previously for deletion of the fission yeast int6(+) gene that encodes the fission yeast homologue of the p48/Int6/eIF3e subunit of mammalian eIF3. Further analysis of eIF3 subunits in Deltamoe1 or Deltaint6 cells shows that in these deletion strains, while all the eIF3 subunits are bound to 40 S particles, dissociation of ribosome-bound eIF3 results in the loss of stable association between the eIF3 subunits. In contrast, eIF3 isolated from ribosomes of wild-type cells are associated with one another in a protein complex. These observations suggest that Moe1 and spInt6 are each required for stable association of eIF3 subunits in fission yeast.     

EIF3 p170, a mediator of mimosine effect on protein synthesis and cell cycle progression

l-Mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been suggested to affect translation of mRNAs such as p27, the CDK inhibitor. However, the mechanism of this effect is not known. Regulation of translation generally occurs at the initiation step that, in mammalian cells, is a complex process that requires multiple eukaryotic initiation factors (eIFs) and ribosome. The effects of mimosine on initiation factors or regulators consequently will influence translation initiation. P170, a putative subunit of eIF3, has been suggested to be nonessential for eIF3 function to form preinitiation complexes and it may function as a regulator for translation of a subset of mRNAs. In this article, we tested this hypothesis and investigated whether eIF3 p170 mediates mimosine effect on mRNA translation. We found that p170 translation was dramatically reduced by mimosine due to its iron-chelating function. The decreased expression of p170 by mimosine caused diminished de novo synthesis of tyrosinated alpha-tubulin and elevated translation of p27 before cell cycle arrest. These observations suggest that p170 is likely an early response gene to mimosine treatment and a mediator for mimosine effect on mRNA translation. The effect of p170 on the synthesis of tyrosinated alpha-tubulin and p27 in a reciprocal manner also suggests that p170 functions as a regulator for mRNA translation.     

Dual function of eIF3j/Hcr1p in processing 20 S pre-rRNA and translation initiation

eIF3j/Hcr1p, a protein associated with eIF3, was shown to bind to, and stabilize, the multifactor complex containing eIFs 1, 2, 3, and 5 and Met-tRNA(i)(Met), whose formation is required for an optimal rate of translation initiation. Here we present evidence that eIF3j/Hcr1p is an RNA binding protein that enhances a late step in 40 S ribosome maturation involving cleavage of the 20 S precursor of 18 S rRNA in the cytoplasm. Immunofluorescence staining shows that eIF3j/Hcr1p is localized predominantly in the cytoplasm. The hcr1Delta mutant exhibits a decreased amount of 40 S subunits, hypersensitivity to paromomycin, and increased levels of 20 S pre-rRNA. Combining the hcr1Delta mutation with drs2Delta or rps0aDelta, deletions of two other genes involved in the same step of 40 S subunit biogenesis, produced a synthetic growth defect. p35, the human ortholog of eIF3j/Hcr1p, partially complemented the slow growth phenotype conferred by hcr1Delta when overexpressed in yeast. heIF3j/p35 was found physically associated with yeast eIF3 and 43 S initiation complexes in vitro and in vivo. Because it did not complement the 40 S biogenesis defect of hcr1Delta, it appears that heIF3j can substitute for eIF3j/Hcr1p only in translation initiation. We conclude that eIF3j/Hcr1p is required for rapid processing of 20 S to 18 S rRNA besides its role in translation initiation, providing an intriguing link between ribosome biogenesis and translation.     

Characterization of the p33 subunit of eukaryotic translation initiation factor-3 from Saccharomyces cerevisiae

Eukaryotic translation initiation factor-3 (eIF3) is a large multisubunit complex that binds to the 40 S ribosomal subunit and promotes the binding of methionyl-tRNAi and mRNA. The molecular mechanism by which eIF3 exerts these functions is incompletely understood. We report here the cloning and characterization of TIF35, the Saccharomyces cerevisiae gene encoding the p33 subunit of eIF3. p33 is an essential protein of 30,501 Da that is required in vivo for initiation of protein synthesis. Glucose repression of TIF35 expressed from a GAL1 promoter results in depletion of both the p33 and p39 subunits. Expression of histidine-tagged p33 in yeast in combination with Ni2+ affinity chromatography allows the isolation of a complex containing the p135, p110, p90, p39, and p33 subunits of eIF3. The p33 subunit binds both mRNA and rRNA fragments due to an RNA recognition motif near its C terminus. Deletion of the C-terminal 71 amino acid residues causes loss of RNA binding, but expression of the truncated form as the sole source of p33 nevertheless supports the slow growth of yeast. These results indicate that the p33 subunit of eIF3 plays an important role in the initiation phase of protein synthesis and that its RNA-binding domain is required for optimal activity.     

Autocrine Semaphorin3A stimulates eukaryotic initiation factor 4E-dependent RhoA translation in breast tumor cells

Translation of the small G protein RhoA in neurons is regulated by the eukaryotic translation initiation factor eIF4E. Here we show that this translation factor also regulates RhoA expression and activity in breast cancer cells. The introduction of eIF4E into breast tumor cells increased RhoA protein levels, while expression of an eIF4E siRNA reduced RhoA expression. Previous studies indicate that the axon repulsion factor Semaphorin3A (Sema3A) stimulates the eIF4E-dependent translation of RhoA in neurons, and breast tumor cells support autocrine Sema3A signaling. Accordingly, we next examined if autocrine Sema3A signaling drives eIF4E-dependent RhoA translation in breast cancer cells. The incubation of breast tumor cells with recombinant Sema3A rapidly increased eIF4E activity, RhoA protein levels, and RhoA activity. This Sema3A activity was blocked in tumor cells expressing an shRNA-specific for the Sema3A receptor, Neuropilin-1 (NP-1), as well as in cells incubated with an eIF4E inhibitor. Importantly, RhoA protein levels were reduced in Sema3A shRNA-expressing compared to control shRNA-expressing breast tumor cells, demonstrating that autocrine Sema3A increases RhoA expression in breast cancer. Considering that Sema3A suppresses axon extension by stimulating RhoA translation, we next examined if the Sema3A/RhoA axis impacts breast tumor cell migration. The incubation of control breast tumor cells, but not RhoA shRNA-expressing cells, with rSema3A significantly reduced their migration. Collectively, these studies indicate that Sema3A impedes breast tumor cell migration in part by stimulating RhoA. These findings identify common signaling pathways that regulate the navigation of neurons and breast cancer cells, thus suggesting novel targets for suppressing breast tumor cell migration.     

Expression of constitutively active 4EBP-1 enhances p27<Superscript>Kip1</Superscript> expression and inhibits proliferation of MCF7 breast cancer cells

BACKGROUND: Eukaryotic initiation factor 4E (eIF4E) is essential for cap-dependent initiation of translation. Cell proliferation is associated with increased activity of eIF4E and elevated expression of eIF4E leads to tumorigenic transformation. Many tumors express very high levels of eIF4E and this may be a critical factor in progression of the disease. In contrast, overexpression of 4EBP, an inhibitor of eIF4E, leads to cell cycle arrest and phenotypic reversion of some transformed cells. RESULTS: A constitutively active form of 4EBP-1 was inducibly expressed in the human breast cancer cell line MCF7. Induction of constitutively active 4EBP-1 led to cell cycle arrest. This was not associated with a general inhibition of protein synthesis but rather with changes in specific cell cycle regulatory proteins. Cyclin D1 was downregulated while levels of the CDK inhibitor p27Kip1 were increased. The levels of cyclin E and CDK2 were unaffected but the activity of CDK2 was significantly reduced due to increased association with p27Kip1. The increase in p27Kip1 did not reflect changes in p27Kip1 mRNA or degradation rates. Rather, it was associated with enhanced synthesis of the protein, even though 4EBP-1 is expected to inhibit translation. This could be explained, at least in part, by the ability of the p27Kip1 5'-UTR to mediate cap-independent translation, which was also enhanced by expression of constitutively active 4EBP-1. CONCLUSIONS: Expression of active 4EBP-1 in MCF7 leads to cell cycle arrest which is associated with downregulation of cyclin D1 and upregulation of p27Kip1. Upregulation of p27Kip1reflects increased synthesis which corresponds to enhanced cap-independent translation through the 5'-UTR of the p27Kip1 mRNA.     

Translation initiation factor modifications and the regulation of protein synthesis in apoptotic cells

The rate of protein synthesis is rapidly down-regulated in mammalian cells following the induction of apoptosis. Inhibition occurs at the level of polypeptide chain initiation and is accompanied by the phosphorylation of the alpha subunit of initiation factor eIF2 and the caspase-dependent cleavage of initiation factors eIF4G, eIF4B, eIF2alpha and the p35 subunit of eIF3. Proteolytic cleavage of these proteins yields characteristic products which may exert regulatory effects on the translational machinery. Inhibition of caspase activity protects protein synthesis from long-term inhibition in cells treated with some, but not all, inducers of apoptosis. This review describes the initiation factor modifications and the possible signalling pathways by which translation may be regulated during apoptosis. We discuss the significance of the initiation factor cleavages and other changes for protein synthesis, and the implications of these events for our understanding of the cellular changes associated with apoptosis.     

Association of the mammalian proto-oncoprotein Int-6 with the three protein complexes eIF3, COP9 signalosome and 26S proteasome

The mammalian Int-6 protein has been characterized as a subunit of the eIF3 translation initiation factor and also as a transforming protein when its C-terminal part is deleted. It includes a protein domain, which also exists in various subunits of eIF3, of the 26S proteasome and of the COP9 signalosome (CSN). By performing a two-hybrid screen with Int-6 as bait, we have isolated subunits belonging to all three complexes, namely eIF3-p110, Rpt4, CSN3 and CSN6. The results of transient expression experiments in COS7 cells confirmed the interaction of Int-6 with Rpt4, CSN3 and CSN6, but also showed that Int-6 is able to bind another subunit of the CSN: CSN7a. Immunoprecipitation experiments performed with the endogenous proteins showed that Int-6 binds the entire CSN, but in low amount, and also that Int-6 is associated with the 26S proteasome. Taken together these results show that the Int-6 protein can bind the three complexes with various efficiencies, possibly exerting a regulatory activity in both protein translation and degradation.     

Identification of partners of TIF34, a component of the yeast eIF3 complex, required for cell proliferation and translation initiation.

Eukaryotic initiation factor-3 (eIF3) in the yeast Saccharomyces cerevisiae plays a central role in initiation of translation. The eIF3 complex contains at least eight different proteins, but, as yet, little is known about the function of the individual proteins. In this study we have characterized the role of TIF34 (eIF3-p39), a recently identified WD-40 domain-containing protein of 39 kDa, in the eIF3 complex. Using temperature-sensitive mutants of TIF34 we show that this protein is required for cell cycle progression and for mating and plays an essential role in initiation of protein synthesis. By two-hybrid screening we have identified two partners that directly associate with TIF34: PRT1, a previously characterized eIF3 subunit, and a novel protein of 33 kDa (eIF3-p33) which is part of the eIF3 complex and has an RNA binding domain. TIF34 and p33 interact with each other and overexpression of p33 complements the growth defect of a tif34-ts mutant. Our results provide support for both physical and functional interactions between three subunits, TIF34, PRT1 and p33, in the eIF3 complex.     

Fission yeast homolog of murine Int-6 protein, encoded by mouse mammary tumor virus integration site, is associated with the conserved core subunits of eukaryotic translation initiation factor 3

The murine int-6 locus, identified as a frequent integration site of mouse mammary tumor viruses, encodes the 48-kDa eIF3e subunit of translation initiation factor eIF3. Previous studies indicated that the catalytically active core of budding yeast eIF3 consists of five subunits, all conserved in eukaryotes, but does not contain a protein closely related to eIF3e/Int-6. Whereas the budding yeast genome does not encode a protein closely related to murine Int-6, fission yeast does encode an Int-6 ortholog, designated here Int6. We found that fission yeast Int6/eIF3e is a cytoplasmic protein associated with 40 S ribosomes. FLAG epitope-tagged Tif35, a putative core eIF3g subunit, copurified with Int6 and all five orthologs of core eIF3 subunits. An int6 deletion (int6Delta) mutant was viable but grew slowly in minimal medium. This slow growth phenotype was accompanied by a reduction in the amount of polyribosomes engaged in translation and was complemented by expression of human Int-6 protein. These findings support the idea that human and Schizosaccharomyces pombe Int-6 homologs are involved in translation. Interestingly, haploid int6Delta cells showed unequal nuclear partitioning, possibly because of a defect in tubulin function, and diploid int6Delta cells formed abnormal spores. We propose that Int6 is not an essential subunit of eIF3 but might be involved in regulating the activity of eIF3 for translation of specific mRNAs in S. pombe.