Journal articles on the topic 'Eukaryotic initiation factor eIF4GI'
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1
Gradi, Alessandra, Hiroaki Imataka, Yuri V. Svitkin, Eran Rom, Brian Raught, Shigenobu Morino, and Nahum Sonenberg. "A Novel Functional Human Eukaryotic Translation Initiation Factor 4G." Molecular and Cellular Biology 18, no. 1 (January 1, 1998): 334–42. http://dx.doi.org/10.1128/mcb.18.1.334.
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ABSTRACT Mammalian eukaryotic translation initiation factor 4F (eIF4F) is a cap-binding protein complex consisting of three subunits: eIF4E, eIF4A, and eIF4G. In yeast and plants, two related eIF4G species are encoded by two different genes. To date, however, only one functional eIF4G polypeptide, referred to here as eIF4GI, has been identified in mammals. Here we describe the discovery and functional characterization of a closely related homolog, referred to as eIF4GII. eIF4GI and eIF4GII share 46% identity at the amino acid level and possess an overall similarity of 56%. The homology is particularly high in certain regions of the central and carboxy portions, while the amino-terminal regions are more divergent. Far-Western analysis and coimmunoprecipitation experiments were used to demonstrate that eIF4GII directly interacts with eIF4E, eIF4A, and eIF3. eIF4GII, like eIF4GI, is also cleaved upon picornavirus infection. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A to cleave endogenous eIF4G. Finally, eIF4GII exists as a complex with eIF4E in HeLa cells, because eIF4GII and eIF4E can be purified together by cap affinity chromatography. Taken together, our findings indicate that eIF4GII is a functional homolog of eIF4GI. These results may have important implications for the understanding of the mechanism of shutoff of host protein synthesis following picornavirus infection.
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Robert, Francis, Regina Cencic, Renying Cai, T. Martin Schmeing, and Jerry Pelletier. "RNA-tethering assay and eIF4G:eIF4A obligate dimer design uncovers multiple eIF4F functional complexes." Nucleic Acids Research 48, no. 15 (August 4, 2020): 8562–75. http://dx.doi.org/10.1093/nar/gkaa646.
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Abstract Eukaryotic cellular mRNAs possess a 5′ cap structure (m7GpppN) which plays a critical role in translation initiation mediated by eukaryotic initiation factor (eIF) 4F. The heterotrimeric eIF4F complex possesses several activities imparted by its subunits that include cap recognition (by eIF4E), RNA unwinding (eIF4A), and factor/ribosome recruitment (eIF4G). Mammalian cells have paralogs of all three eIF4F subunits and it remains an open question as to whether these all can participate in the process of ribosome recruitment. To query the activities of the eIF4F subunits in translation initiation, we adopted an RNA-tethering assay in which select subunits are recruited to a specific address on a reporter mRNA template. We find that all eIF4F subunits can participate in the initiation process. Based on eIF4G:eIF4A structural information, we also designed obligate dimer pairs to probe the activity of all combinations of eIF4G and eIF4A paralogs. We demonstrate that both eIF4GI and eIF4GII can associate with either eIF4A1 or eIF4A2 to recruit ribosomes to mRNA templates. In combination with eIF4E and eIF4E3, our results indicate the presence of up to eight eIF4F complexes that can operate in translation initiation.
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Coldwell, Mark J., Ulrike Sack, Joanne L. Cowan, Rachel M. Barrett, Markete Vlasak, Keiley Sivakumaran, and Simon J. Morley. "Multiple isoforms of the translation initiation factor eIF4GII are generated via use of alternative promoters, splice sites and a non-canonical initiation codon." Biochemical Journal 448, no. 1 (October 18, 2012): 1–11. http://dx.doi.org/10.1042/bj20111765.
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During the initiation stage of eukaryotic mRNA translation, the eIF4G (eukaryotic initiation factor 4G) proteins act as an aggregation point for recruiting the small ribosomal subunit to an mRNA. We previously used RNAi (RNA interference) to reduce expression of endogenous eIF4GI proteins, resulting in reduced protein synthesis rates and alterations in the morphology of cells. Expression of EIF4G1 cDNAs, encoding different isoforms (f–a) which arise through selection of alternative initiation codons, rescued translation to different extents. Furthermore, overexpression of the eIF4GII paralogue in the eIF4GI-knockdown background was unable to restore translation to the same extent as eIF4GIf/e isoforms, suggesting that translation events governed by this protein are different. In the present study we show that multiple isoforms of eIF4GII exist in mammalian cells, arising from multiple promoters and alternative splicing events, and have identified a non-canonical CUG initiation codon which extends the eIF4GII N-terminus. We further show that the rescue of translation in eIF4GI/eIF4GII double-knockdown cells by our novel isoforms of eIF4GII is as robust as that observed with either eIF4GIf or eIF4GIe, and more than that observed with the original eIF4GII. As the novel eIF4GII sequence diverges from eIF4GI, these data suggest that the eIF4GII N-terminus plays an alternative role in initiation factor assembly.
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Yanagiya, Akiko, Yuri V. Svitkin, Shoichiro Shibata, Satoshi Mikami, Hiroaki Imataka, and Nahum Sonenberg. "Requirement of RNA Binding of Mammalian Eukaryotic Translation Initiation Factor 4GI (eIF4GI) for Efficient Interaction of eIF4E with the mRNA Cap." Molecular and Cellular Biology 29, no. 6 (December 29, 2008): 1661–69. http://dx.doi.org/10.1128/mcb.01187-08.
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ABSTRACT Eukaryotic mRNAs possess a 5′-terminal cap structure (cap), m7GpppN, which facilitates ribosome binding. The cap is bound by eukaryotic translation initiation factor 4F (eIF4F), which is composed of eIF4E, eIF4G, and eIF4A. eIF4E is the cap-binding subunit, eIF4A is an RNA helicase, and eIF4G is a scaffolding protein that bridges between the mRNA and ribosome. eIF4G contains an RNA-binding domain, which was suggested to stimulate eIF4E interaction with the cap in mammals. In Saccharomyces cerevisiae, however, such an effect was not observed. Here, we used recombinant proteins to reconstitute the cap binding of the mammalian eIF4E-eIF4GI complex to investigate the importance of the RNA-binding region of eIF4GI for cap interaction with eIF4E. We demonstrate that chemical cross-linking of eIF4E to the cap structure is dramatically enhanced by eIF4GI fragments possessing RNA-binding activity. Furthermore, the fusion of RNA recognition motif 1 (RRM1) of the La autoantigen to the N terminus of eIF4GI confers enhanced association between the cap structure and eIF4E. These results demonstrate that eIF4GI serves to anchor eIF4E to the mRNA and enhance its interaction with the cap structure.
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Caron, Sandrine, Martine Charon, Elisabeth Cramer, Nahum Sonenberg, and Isabelle Dusanter-Fourt. "Selective Modification of Eukaryotic Initiation Factor 4F (eIF4F) at the Onset of Cell Differentiation: Recruitment of eIF4GII and Long-Lasting Phosphorylation of eIF4E." Molecular and Cellular Biology 24, no. 11 (June 1, 2004): 4920–28. http://dx.doi.org/10.1128/mcb.24.11.4920-4928.2004.
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ABSTRACT mRNA translation is mainly regulated at the level of initiation, a process that involves the synergistic action of the 5′ cap structure and the 3′ poly(A) tail at the ends of eukaryotic mRNA. The eukaryote initiation factor 4G(eIF4G) is a pivotal scaffold protein that forms a critical link between mRNA cap structure, poly(A) tail, and the small ribosomal subunit. There are two functional homologs of eIF4G in mammals, the original eIF4G, renamed eIF4GI, and eIF4GII that functionally complements eIF4GI. To date, biochemical and functional analysis have not identified differential activities for eIF4GI and eIF4GII. In this report, we demonstrate that eIF4GII, but not eIF4GI, is selectively recruited to capped mRNA at the onset of cell differentiation. This recruitment is coincident with a strong and long-lasting phosphorylation of eIF4E and the release of 4E-BP1, a suppressor of eIF4E function, from the cap structure, without a concomitant change in 4E-BP1's phosphorylation. Our data further indicate that cytokines such as thrombopoietin can differentially regulate eIF4GI/II activities. These results provide the first evidence that eIF4GI/II does fulfill selective roles in mammalian cells.
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Álvarez, Enrique, Luis Menéndez-Arias, and Luis Carrasco. "The Eukaryotic Translation Initiation Factor 4GI Is Cleaved by Different Retroviral Proteases." Journal of Virology 77, no. 23 (December 1, 2003): 12392–400. http://dx.doi.org/10.1128/jvi.77.23.12392-12400.2003.
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ABSTRACT The initiation factor eIF4G plays a central role in the regulation of translation. In picornaviruses, as well as in human immunodeficiency virus type 1 (HIV-1), cleavage of eIF4G by the viral protease leads to inhibition of protein synthesis directed by capped cellular mRNAs. In the present work, cleavage of both eIF4GI and eIF4GII has been analyzed by employing the proteases encoded within the genomes of several members of the family Retroviridae, e.g., Moloney murine leukemia virus (MoMLV), mouse mammary tumor virus, human T-cell leukemia virus type 1, HIV-2, and simian immunodeficiency virus. All of the retroviral proteases examined were able to cleave the initiation factor eIF4GI both in intact cells and in cell-free systems, albeit with different efficiencies. The eIF4GI hydrolysis patterns obtained with HIV-1 and HIV-2 proteases were very similar to each other but rather different from those obtained with MoMLV protease. Both eIF4GI and eIF4GII were cleaved very efficiently by the MoMLV protease. However, eIF4GII was a poor substrate for HIV proteases. Proteolytic cleavage of eIF4G led to a profound inhibition of cap-dependent translation, while protein synthesis driven by mRNAs containing internal ribosome entry site elements remained unaffected or was even stimulated in transfected cells.
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Coldwell, Mark J., and Simon J. Morley. "Specific Isoforms of Translation Initiation Factor 4GI Show Differences in Translational Activity." Molecular and Cellular Biology 26, no. 22 (September 18, 2006): 8448–60. http://dx.doi.org/10.1128/mcb.01248-06.
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ABSTRACT The eukaryotic initiation factor (eIF) 4GI gene locus (eIF4GI) contains three identified promoters, generating alternately spliced mRNAs, yielding a total of five eIF4GI protein isoforms. Although eIF4GI plays a critical role in mRNA recruitment to the ribosomes, little is known about the functions of the different isoforms, their partner binding capacities, or the role of the homolog, eIF4GII, in translation initiation. To directly address this, we have used short interfering RNAs (siRNAs) expressed from DNA vectors to silence the expression of eIF4GI in HeLa cells. Here we show that reduced levels of specific mRNA and eIF4GI isoforms in HeLa cells promoted aberrant morphology and a partial inhibition of translation. The latter reflected dephosphorylation of 4E-BP1 and decreased eIF4F complex levels, with no change in eIF2α phosphorylation. Expression of siRNA-resistant Myc-tagged eIF4GI isoforms has allowed us to show that the different isoforms exhibit significant differences in their ability to restore translation rates. Here we quantify the efficiency of eIF4GI promoter usage in mammalian cells and demonstrate that even though the longest isoform of eIF4GI (eIF4GIf) was relatively poorly expressed when reintroduced, it was more efficient at promoting the translation of cellular mRNAs than the more highly expressed shorter isoforms used in previous functional studies.
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Patel, Krishnaben, Grishma K. Shah, Sai Shilpa Kommaraju, and Woon-Kai Low. "Investigation of the conserved glutamate immediately following the DEAD box in eukaryotic translation initiation factor 4AI." Biochemistry and Cell Biology 92, no. 1 (February 2014): 33–42. http://dx.doi.org/10.1139/bcb-2013-0076.
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The DExD-box family (DEAD-box) of proteins was surveyed for eukaryotic translation initiation factor 4A-specific sequences surrounding the DEAD box. An eIF4A-unique glutamate residue (E186 in eIF4AI) was identified immediately following the D-E-A-D sequence in eIF4AI, II, and III that was found to be conserved from yeast to Man. Mutation to a selection of alternative amino acids was performed within recombinant eIF4AI expressed in Escherichia coli and mutant proteins were surveyed for RNA-dependent ATPase activity. The mutants were also investigated for changes in activity in the presence of the two eIF4AI-binding domains of eIF4GI as well as for co-purification ability to these two domains. The E186 residue was found to be of significance for RNA-dependent ATPase activity for eIF4AI alone and in the presence of eIF4AI-binding domains of eIF4GI through point-mutation analysis. Furthermore, binding interactions between eIF4AI and eIF4GI domains were also significantly influenced by mutation of E186, as observed through co-purification assays. Thus, this residue appears to be of functional significance for eIF4A.
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Neff, Carrie L., and Alan B. Sachs. "Eukaryotic Translation Initiation Factors 4G and 4A from Saccharomyces cerevisiae Interact Physically and Functionally." Molecular and Cellular Biology 19, no. 8 (August 1, 1999): 5557–64. http://dx.doi.org/10.1128/mcb.19.8.5557.
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ABSTRACT The initiation of translation in eukaryotes requires several multisubunit complexes, including eukaryotic translation initiation factor 4F (eIF4F). In higher eukaryotes eIF4F is composed of the cap binding protein eIF4E, the adapter protein eIF4G, and the RNA-stimulated ATPase eIF4A. The association of eIF4A withSaccharomyces cerevisiae eIF4F has not yet been demonstrated, and therefore the degree to which eIF4A’s conserved function relies upon this association has remained unclear. Here we report an interaction between yeast eIF4G and eIF4A. Specifically, we found that the growth arrest phenotype associated with three temperature-sensitive alleles of yeast eIF4G2 was suppressed by excess eIF4A and that this suppression was allele specific. In addition, in vitro translation extracts derived from an eIF4G2 mutant strain could be heat inactivated, and this inactivation could be reversed upon the addition of recombinant eIF4A. Finally, in vitro binding between yeast eIF4G and eIF4A was demonstrated, as was diminished binding between mutant eIF4G2 proteins and eIF4A. In total, these data indicate that yeast eIF4G and eIF4A physically associate and that this association performs an essential function.
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Li, Qiyu, Hiroaki Imataka, Shigenobu Morino, George W. Rogers, Nancy J. Richter-Cook, William C. Merrick, and Nahum Sonenberg. "Eukaryotic Translation Initiation Factor 4AIII (eIF4AIII) Is Functionally Distinct from eIF4AI and eIF4AII." Molecular and Cellular Biology 19, no. 11 (November 1, 1999): 7336–46. http://dx.doi.org/10.1128/mcb.19.11.7336.
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ABSTRACT 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.
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Svitkin, Yuri V., Alessandra Gradi, Hiroaki Imataka, Shigenobu Morino, and Nahum Sonenberg. "Eukaryotic Initiation Factor 4GII (eIF4GII), but Not eIF4GI, Cleavage Correlates with Inhibition of Host Cell Protein Synthesis after Human Rhinovirus Infection." Journal of Virology 73, no. 4 (April 1, 1999): 3467–72. http://dx.doi.org/10.1128/jvi.73.4.3467-3472.1999.
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ABSTRACT For many members of the Picornaviridae family, infection of cells results in a shutoff of host protein synthesis. For rhinoviruses and enteroviruses, the shutoff has been explained in part by the cleavage of eukaryotic initiation factor 4GI (eIF4GI), a component of the cap-binding protein complex eIF4F. The cleavage of eIF4GI is mediated by the virus-specific proteinase 2Aproand results in inhibition of cap-dependent, but not cap-independent, translation. The inhibition of host protein synthesis after infection with human rhinovirus 14 (HRV-14) lags behind the cleavage of eIF4GI. Recently, we discovered a functional homolog of eIF4GI, termed eIF4GII, and showed that cleavage of eIF4GII coincides with the shutoff of host cell protein synthesis after poliovirus infection (Gradi et al., Proc. Natl. Acad. Sci. USA 95:11089–11094, 1998). We wished to determine whether eIF4GII cleavage kinetics could also explain the lack of correlation between the kinetics of eIF4GI cleavage and the shutoff of host protein synthesis after rhinovirus infection. In this study, we examined the correlation between human rhinovirus-induced shutoff of host protein synthesis and cleavage of eIF4GI and eIF4GII. In HRV-14-infected HeLa cells, almost no intact eIF4GI could be detected by 4 h postinfection, while only 4% of eIF4GII was cleaved at this time. By 6 h, however, 67% of eIF4GII was cleaved, and this cleavage coincided with a significant (60%) decline of host translation. These results suggest that cleavage of both eIF4GI and eIF4GII is required for HRV-mediated inhibition of host cell protein synthesis and that the cleavage of eIF4GII is the rate-limiting step in the shutoff of host cell protein synthesis after rhinovirus infection.
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Lomakin, Ivan B., Christopher U. T. Hellen, and Tatyana V. Pestova. "Physical Association of Eukaryotic Initiation Factor 4G (eIF4G) with eIF4A Strongly Enhances Binding of eIF4G to the Internal Ribosomal Entry Site of Encephalomyocarditis Virus and Is Required for Internal Initiation of Translation." Molecular and Cellular Biology 20, no. 16 (August 15, 2000): 6019–29. http://dx.doi.org/10.1128/mcb.20.16.6019-6029.2000.
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ABSTRACT Mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three similarly sized regions. The central region (amino acids [aa] 613 to 1090) binds eIF3, eIF4A, and the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and mediates initiation on this RNA. We identified the regions of eIF4GI that are responsible for its specific interaction with the IRES and that are required to mediate 48S complex formation on the IRES in vitro. Mutational analysis demarcated the IRES binding fragment of eIF4GI (aa 746 to 949) and indicated that it does not resemble an RNA recognition motif (RRM)-like domain. An additional amino-terminal sequence (aa 722 to 746) was required for binding eIF4A and for 48S complex formation. eIF4GI bound the EMCV IRES and β-globin mRNA with similar affinities, but association with eIF4A increased its affinity for the EMCV IRES (but not β-globin RNA) by 2 orders of magnitude. On the other hand, eIF4GI mutants with defects in binding eIF4A were defective in mediating 48S complex formation even if they bound the IRES normally. These data indicate that the eIF4G-eIF4A complex, rather than eIF4G alone, is required for specific high-affinity binding to the EMCV IRES and for internal ribosomal entry on this RNA.
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Aragón, Tomás, Susana de la Luna, Isabel Novoa, Luis Carrasco, Juan Ortín, and Amelia Nieto. "Eukaryotic Translation Initiation Factor 4GI Is a Cellular Target for NS1 Protein, a Translational Activator of Influenza Virus." Molecular and Cellular Biology 20, no. 17 (September 1, 2000): 6259–68. http://dx.doi.org/10.1128/mcb.20.17.6259-6268.2000.
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ABSTRACT Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix showed that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we showed that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5′ untranslated region (5′ UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.
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Walsh, Derek, Carolina Arias, Cesar Perez, David Halladin, Martin Escandon, Takeshi Ueda, Rie Watanabe-Fukunaga, Rikiro Fukunaga, and Ian Mohr. "Eukaryotic Translation Initiation Factor 4F Architectural Alterations Accompany Translation Initiation Factor Redistribution in Poxvirus-Infected Cells." Molecular and Cellular Biology 28, no. 8 (February 4, 2008): 2648–58. http://dx.doi.org/10.1128/mcb.01631-07.
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ABSTRACT Despite their self-sufficient ability to generate capped mRNAs from cytosolic DNA genomes, poxviruses must commandeer the critical eukaryotic translation initiation factor 4F (eIF4F) to recruit ribosomes. While eIF4F integrates signals to control translation, precisely how poxviruses manipulate the multisubunit eIF4F, composed of the cap-binding eIF4E and the RNA helicase eIF4A assembled onto an eIF4G platform, remains obscure. Here, we establish that the poxvirus infection of normal, primary human cells destroys the translational repressor eIF4E binding protein (4E-BP) and promotes eIF4E assembly into an active eIF4F complex bound to the cellular polyadenylate-binding protein (PABP). Stimulation of the eIF4G-associated kinase Mnk1 promotes eIF4E phosphorylation and enhances viral replication and protein synthesis. Remarkably, these eIF4F architectural alterations are accompanied by the concentration of eIF4E and eIF4G within cytosolic viral replication compartments surrounded by PABP. This demonstrates that poxvirus infection redistributes, assembles, and modifies core and associated components of eIF4F and concentrates them within discrete subcellular compartments. Furthermore, it suggests that the subcellular distribution of eIF4F components may potentiate the complex assembly.
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Pestova, T. V., I. N. Shatsky, and C. U. Hellen. "Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes." Molecular and Cellular Biology 16, no. 12 (December 1996): 6870–78. http://dx.doi.org/10.1128/mcb.16.12.6870.
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Eukaryotic translation is initiated following binding of ribosomes either to the capped 5' end of an mRNA or to an internal ribosomal entry site (IRES) within its 5' nontranslated region. These processes are both mediated by eukaryotic initiation factor 4F (eIF4F), which consists of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G subunits. Here we present a functional analysis of eIF4F which defines the subunits and subunit domains necessary for its function in initiation mediated by the prototypical IRES element of encephalomyocarditis virus. In an initiation reaction reconstituted in vitro from purified translation components and lacking eIF4A and -4F, IRES-mediated initiation did not require the cap-binding protein eIF4E but was absolutely dependent on eIF4A and the central third of eIF4G. This central domain of eIF4G bound strongly and specifically to a structural element within the encephalomyocarditis virus IRES upstream of the initiation codon in an ATP-independent manner and with the same specificity as eIF4F. The carboxy-terminal third of eIF4G did not bind to the IRES. The central domain of eIF4G was itself UV cross-linked to the IRES and strongly stimulated UV cross-linking of eIF4A to the IRES in conjunction with either eIF4B or with the carboxy-terminal third of eIF4G.
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Byrd, Marshall P., Miguel Zamora, and Richard E. Lloyd. "Generation of Multiple Isoforms of Eukaryotic Translation Initiation Factor 4GI by Use of Alternate Translation Initiation Codons." Molecular and Cellular Biology 22, no. 13 (July 1, 2002): 4499–511. http://dx.doi.org/10.1128/mcb.22.13.4499-4511.2002.
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ABSTRACT Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5′ end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5′ sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5′ untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.
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Sen, Neelam Dabas, Fujun Zhou, Michael S. Harris, Nicholas T. Ingolia, and Alan G. Hinnebusch. "eIF4B stimulates translation of long mRNAs with structured 5′ UTRs and low closed-loop potential but weak dependence on eIF4G." Proceedings of the National Academy of Sciences 113, no. 38 (September 6, 2016): 10464–72. http://dx.doi.org/10.1073/pnas.1612398113.
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DEAD-box RNA helicases eukaryotic translation initiation factor 4A (eIF4A) and Ded1 promote translation by resolving mRNA secondary structures that impede preinitiation complex (PIC) attachment to mRNA or scanning. Eukaryotic translation initiation factor 4B (eIF4B) is a cofactor for eIF4A but also might function independently of eIF4A. Ribosome profiling of mutants lacking eIF4B or with impaired eIF4A or Ded1 activity revealed that eliminating eIF4B reduces the relative translational efficiencies of many more genes than does inactivation of eIF4A, despite comparable reductions in bulk translation, and few genes display unusually strong requirements for both factors. However, either eliminating eIF4B or inactivating eIF4A preferentially impacts mRNAs with longer, more structured 5′ untranslated regions (UTRs). These findings reveal an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor in promoting PIC attachment or scanning on structured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a fashion mitigated by the ability to form closed-loop messenger ribonucleoprotein particles (mRNPs) via eIF4F–poly(A)-binding protein 1 (Pab1) association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions. Remarkably, depleting eukaryotic translation initiation factor 4G (eIF4G), the scaffold subunit of eukaryotic translation initiation factor 4F (eIF4F), preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5′ UTRs, exactly the opposite features associated with hyperdependence on the eIF4B/helicases. We propose that short, highly efficient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly.
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Morino, Shigenobu, Hiroaki Imataka, Yuri V. Svitkin, Tatyana V. Pestova, and Nahum Sonenberg. "Eukaryotic Translation Initiation Factor 4E (eIF4E) Binding Site and the Middle One-Third of eIF4GI Constitute the Core Domain for Cap-Dependent Translation, and the C-Terminal One-Third Functions as a Modulatory Region." Molecular and Cellular Biology 20, no. 2 (January 15, 2000): 468–77. http://dx.doi.org/10.1128/mcb.20.2.468-477.2000.
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ABSTRACT The mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three roughly equal regions; an amino-terminal one-third (amino acids [aa] 1 to 634), which contains the poly(A) binding protein (PABP) and eIF4E binding sites; a middle third (aa 635 to 1039), which binds eIF4A and eIF3; and a carboxy-terminal third (aa 1040 to 1560), which harbors a second eIF4A binding site and a docking sequence for the Ser/Thr kinase Mnk1. Previous reports demonstrated that the middle one-third of eIF4GI is sufficient for cap-independent translation. To delineate the eIF4GI core sequence required for cap-dependent translation, various truncated versions of eIF4GI were examined in an in vitro ribosome binding assay with β-globin mRNA. A sequence of 540 aa encompassing aa 550 to 1090, which contains the eIF4E binding site and the middle region of eIF4GI, is the minimal sequence required for cap-dependent translation. In agreement with this, a point mutation in eIF4GI which abolished eIF4A binding in the middle region completely inhibited ribosomal binding. However, the eIF4GI C-terminal third region, which does not have a counterpart in yeast, modulates the activity of the core sequence. When the eIF4A binding site in the C-terminal region of eIF4GI was mutated, ribosome binding was decreased three- to fourfold. These data indicate that the interaction of eIF4A with the middle region of eIF4GI is necessary for translation, whereas the interaction of eIF4A with the C-terminal region plays a modulatory role.
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Kuyumcu-Martinez, N. Muge, Marc E. Van Eden, Patrick Younan, and Richard E. Lloyd. "Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Protease Inhibits Host Cell Translation: a Novel Mechanism for Host Translation Shutoff." Molecular and Cellular Biology 24, no. 4 (February 15, 2004): 1779–90. http://dx.doi.org/10.1128/mcb.24.4.1779-1790.2004.
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ABSTRACT Cleavage of eukaryotic translation initiation factor 4GI (eIF4GI) by viral 2A protease (2Apro) has been proposed to cause severe translation inhibition in poliovirus-infected cells. However, infections containing 1 mM guanidine-HCl result in eIF4GI cleavage but only partial translation shutoff, indicating eIF4GI cleavage is insufficient for drastic translation inhibition. Viral 3C protease (3Cpro) cleaves poly(A)-binding protein (PABP) and removes the C-terminal domain (CTD) that interacts with several translation factors. In HeLa cell translation extracts that exhibit cap-poly(A) synergy, partial cleavage of PABP by 3Cpro inhibited translation of endogenous mRNAs and reporter RNA as effectively as complete cleavage of eIF4GI and eIF4GII by 2Apro. 3Cpro-mediated translation inhibition was poly(A) dependent, and addition of PABP to extracts restored translation. Expression of 3Cpro in HeLa cells resulted in partial PABP cleavage and similar inhibition of translation. PABP cleavage did not affect eIF4GI-PABP interactions, and the results of kinetics experiments suggest that 3Cpro might inhibit late steps in translation or ribosome recycling. The data illustrate the importance of the CTD of PABP in poly(A)-dependent translation in mammalian cells. We propose that enteroviruses use a dual strategy for host translation shutoff, requiring cleavage of PABP by 3Cpro and of eIF4G by 2Apro.
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Gradi, Alessandra, Yuri V. Svitkin, Wolfgang Sommergruber, Hiroaki Imataka, Shigenobu Morino, Tim Skern, and Nahum Sonenberg. "Human Rhinovirus 2A Proteinase Cleavage Sites in Eukaryotic Initiation Factors (eIF) 4GI and eIF4GII Are Different." Journal of Virology 77, no. 8 (April 15, 2003): 5026–29. http://dx.doi.org/10.1128/jvi.77.8.5026-5029.2003.
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ABSTRACT Several picornaviruses shut down host cellular protein synthesis by proteolytic cleavage of the eukaryotic initiation factor (eIF) 4GI and eIF4GII isoforms. Viral RNA translation is maintained by a cap-independent mechanism. Here, we identify the human rhinovirus 2 2Apro cleavage site in eIF4GII in vitro as PLLNV699*GSR; this sequence lies seven amino acids C-terminal to the cleavage site previously identified in eIF4GI (LSTR681*GPP).
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Yang, Hsin-Sheng, Aaron P. Jansen, Anton A. Komar, Xiaojing Zheng, William C. Merrick, Sylvain Costes, Stephen J. Lockett, Nahum Sonenberg, and Nancy H. Colburn. "The Transformation Suppressor Pdcd4 Is a Novel Eukaryotic Translation Initiation Factor 4A Binding Protein That Inhibits Translation." Molecular and Cellular Biology 23, no. 1 (January 1, 2003): 26–37. http://dx.doi.org/10.1128/mcb.23.1.26-37.2003.
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ABSTRACT Pdcd4 is a novel transformation suppressor that inhibits tumor promoter-induced neoplastic transformation and the activation of AP-1-dependent transcription required for transformation. A yeast two-hybrid analysis revealed that Pdcd4 associates with the eukaryotic translation initiation factors eIF4AI and eIF4AII. Immunofluorescent confocal microscopy showed that Pdcd4 colocalizes with eIF4A in the cytoplasm. eIF4A is an ATP-dependent RNA helicase needed to unwind 5′ mRNA secondary structure. Recombinant Pdcd4 specifically inhibited the helicase activity of eIF4A and eIF4F. In vivo translation assays showed that Pdcd4 inhibited cap-dependent but not internal ribosome entry site (IRES)-dependent translation. In contrast, Pdcd4D418A, a mutant inactivated for binding to eIF4A, failed to inhibit cap-dependent or IRES-dependent translation or AP-1 transactivation. Recombinant Pdcd4 prevented eIF4A from binding to the C-terminal region of eIF4G (amino acids 1040 to 1560) but not to the middle region of eIF4G(amino acids 635 to 1039). In addition, both Pdcd4 and Pdcd4D418A bound to the middle region of eIF4G. The mechanism by which Pdcd4 inhibits translation thus appears to involve inhibition of eIF4A helicase, interference with eIF4A association-dissociation from eIF4G, and inhibition of eIF4A binding to the C-terminal domain of eIF4G. Pdcd4 binding to eIF4A is linked to its transformation-suppressing activity, as Pdcd4-eIF4A binding and consequent inhibition of translation are required for Pdcd4 transrepression of AP-1.
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Alard, Amandine, Bertrand Fabre, Rodica Anesia, Catherine Marboeuf, Philippe Pierre, Christiane Susini, Corinne Bousquet, and Stéphane Pyronnet. "NAD(P)H Quinone-Oxydoreductase 1 Protects Eukaryotic Translation Initiation Factor 4GI from Degradation by the Proteasome." Molecular and Cellular Biology 30, no. 4 (December 22, 2009): 1097–105. http://dx.doi.org/10.1128/mcb.00868-09.
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ABSTRACT The eukaryotic translation initiation factor 4GI (eIF4GI) serves as a central adapter in cap-binding complex assembly. Although eIF4GI has been shown to be sensitive to proteasomal degradation, how the eIF4GI steady-state level is controlled remains unknown. Here, we show that eIF4GI exists in a complex with NAD(P)H quinone-oxydoreductase 1 (NQO1) in cell extracts. Treatment of cells with dicumarol (dicoumarol), a pharmacological inhibitor of NQO1 known to preclude NQO1 binding to its protein partners, provokes eIF4GI degradation by the proteasome. Consistently, the eIF4GI steady-state level also diminishes upon the silencing of NQO1 (by transfection with small interfering RNA), while eIF4GI accumulates upon the overexpression of NQO1 (by transfection with cDNA). We further reveal that treatment of cells with dicumarol frees eIF4GI from mRNA translation initiation complexes due to strong activation of its natural competitor, the translational repressor 4E-BP1. As a consequence of cap-binding complex dissociation and eIF4GI degradation, protein synthesis is dramatically inhibited. Finally, we show that the regulation of eIF4GI stability by the proteasome may be prominent under oxidative stress. Our findings assign NQO1 an original role in the regulation of mRNA translation via the control of eIF4GI stability by the proteasome.
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Imataka, H., and N. Sonenberg. "Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A." Molecular and Cellular Biology 17, no. 12 (December 1997): 6940–47. http://dx.doi.org/10.1128/mcb.17.12.6940.
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Mammalian translation initiation factor 4F (eIF4F) consists of three subunits, eIF4A, eIF4E, and eIF4G. eIF4G interacts directly with both eIF4A and eIF4E. The binding site for eIF4E is contained in the amino-terminal third of eIF4G, while the binding site for eIF4A was mapped to the carboxy-terminal third of the molecule. Here we show that human eIF4G possesses two separate eIF4A binding domains in the middle third (amino acids [aa] 478 to 883) and carboxy-terminal third (aa 884 to 1404) of the molecule. The amino acid sequence of the middle portion of eIF4G is well conserved between yeasts and humans. We show that mutations of conserved amino acid stretches in the middle domain abolish or reduce eIF4A binding as well as eIF3 binding. In addition, a separate and nonoverlapping eIF4A binding domain exists in the carboxy-terminal third (aa 1045 to 1404) of eIF4G, which is not present in yeast. The C-terminal two-thirds region (aa 457 to 1404) of eIF4G, containing both eIF4A binding sites, is required for stimulating translation. Neither one of the eIF4A binding domains alone activates translation. In contrast to eIF4G, human p97, a translation inhibitor with homology to eIF4G, binds eIF4A only through the amino-terminal proximal region, which is homologous to the middle domain of eIF4G.
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Martínez-Alonso, Emma, Natalia Guerra-Pérez, Alejandro Escobar-Peso, Lorena Peracho, Rocío Vera-Lechuga, Antonio Cruz-Culebras, Jaime Masjuan, and Alberto Alcázar. "Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1147 Is Specific for eIF4G1 Bound to eIF4E in Delayed Neuronal Death after Ischemia." International Journal of Molecular Sciences 23, no. 3 (February 6, 2022): 1830. http://dx.doi.org/10.3390/ijms23031830.
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Ischemic strokes are caused by a reduction in cerebral blood flow and both the ischemic period and subsequent reperfusion induce brain injury, with different tissue damage depending on the severity of the ischemic insult, its duration, and the particular areas of the brain affected. In those areas vulnerable to cerebral ischemia, the inhibition of protein translation is an essential process of the cellular response leading to delayed neuronal death. In particular, translation initiation is rate-limiting for protein synthesis and the eukaryotic initiation factor (eIF) 4F complex is indispensable for cap-dependent protein translation. In the eIF4F complex, eIF4G is a scaffolding protein that provides docking sites for the assembly of eIF4A and eIF4E, binding to the cap structure of the mRNA and stabilizing all proteins of the complex. The eIF4F complex constituents, eIF4A, eIF4E, and eIF4G, participate in translation regulation by their phosphorylation at specific sites under cellular stress conditions, modulating the activity of the cap-binding complex and protein translation. This work investigates the phosphorylation of eIF4G1 involved in the eIF4E/eIF4G1 association complex, and their regulation in ischemia-reperfusion (IR) as a stress-inducing condition. IR was induced in an animal model of transient cerebral ischemia and the results were studied in the resistant cortical region and in the vulnerable hippocampal CA1 region. The presented data demonstrate the phosphorylation of eIF4G1 at Ser1147, Ser1185, and Ser1231 in both brain regions and in control and ischemic conditions, being the phosphorylation of eIF4G1 at Ser1147 the only one found in the eIF4E/eIF4G association complex from the cap-containing matrix (m7GTP-Sepharose). In addition, our work reveals the specific modulation of the phosphorylation of eIF4G1 at Ser1147 in the vulnerable region, with increased levels and colocalization with eIF4E in response to IR. These findings contribute to elucidate the molecular mechanism of protein translation regulation that underlies in the balance of cell survival/death during pathophysiological stress, such as cerebral ischemia.
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Kempf, Brian J., and David J. Barton. "Poliovirus 2APro Increases Viral mRNA and Polysome Stability Coordinately in Time with Cleavage of eIF4G." Journal of Virology 82, no. 12 (April 9, 2008): 5847–59. http://dx.doi.org/10.1128/jvi.01514-07.
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ABSTRACT Poliovirus (PV) 2A protease (2APro) cleaves eukaryotic initiation factors 4GI and 4GII (eIF4GI and eIF4GII) within virus-infected cells, effectively halting cap-dependent mRNA translation. PV mRNA, which does not possess a 5′ cap, is translated via cap-independent mechanisms within viral protease-modified messenger ribonucleoprotein (mRNP) complexes. In this study, we determined that 2APro activity was required for viral polysome formation and stability. 2APro cleaved eIF4GI and eIF4GII as PV polysomes assembled. A 2ACys109Ser (2APro with a Cys109Ser mutation) protease active site mutation that prevented cleavage of eIF4G coordinately inhibited the de novo formation of viral polysomes, the stability of viral polysomes, and the stability of PV mRNA within polysomes. 2ACys109Ser-associated defects in PV mRNA and polysome stability correlated with defects in PV mRNA translation. 3CPro activity was not required for viral polysome formation or stability. 2APro-mediated cleavage of eIF4G along with poly(rC) binding protein binding to the 5′ terminus of uncapped PV mRNA appear to be concerted mechanisms that allow PV mRNA to form mRNP complexes that evade cellular mRNA degradation machinery.
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Glaser, Walter, Andrea Triendl, and Tim Skern. "The Processing of eIF4GI by Human Rhinovirus Type 2 2Apro: Relationship to Self-Cleavage and Role of Zinc." Journal of Virology 77, no. 8 (April 15, 2003): 5021–25. http://dx.doi.org/10.1128/jvi.77.8.5021-5025.2003.
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ABSTRACT The 2A proteinase (2Apro) of human rhinoviruses (HRVs) is a cysteine protease containing a structurally important zinc ion. In the viral polyprotein, the enzyme cleaves between the C terminus of VP1 and its own N terminus. 2Apro also processes the two isoforms of the cellular protein, eukaryotic initiation factor 4G (eIF4G). We have shown that mature HRV2 2Apro, when translated in vitro in rabbit reticulocyte lysates, efficiently cleaves eIF4GI, although the enzyme was not immediately active upon synthesis. Here, we examine the relationship between self-processing and eIF4GI cleavage. The onset of both reactions first occurred at least 10 min after initiation of protein synthesis. Furthermore, when self-processing was prevented by a specific mutation between VP1 and 2Apro, the VP1-2Apro precursor was essentially unable to cleave eIF4GI, implying that self-processing is a prerequisite for eIF4GI cleavage. 2Apro synthesized in the presence of a potent zinc chelator is inactive; however, upon addition of excess zinc, HRV2 2Apro rapidly gained activity. Finally, the presence of the zinc chelator in the culture medium can protect HeLa cells from HRV infection.
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Haizel, Solomon A., Usha Bhardwaj, Ruben L. Gonzalez, Somdeb Mitra, and Dixie J. Goss. "5′-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs." Journal of Biological Chemistry 295, no. 33 (June 22, 2020): 11693–706. http://dx.doi.org/10.1074/jbc.ra120.013678.
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During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4GI (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5′ UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5′ UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5′ UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.
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Montero, Hilda, Carlos F. Arias, and Susana Lopez. "Rotavirus Nonstructural Protein NSP3 Is Not Required for Viral Protein Synthesis." Journal of Virology 80, no. 18 (September 15, 2006): 9031–38. http://dx.doi.org/10.1128/jvi.00437-06.
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ABSTRACT Initiation is the rate-limiting step in protein synthesis and therefore an important target for regulation. For the initiation of translation of most cellular mRNAs, the cap structure at the 5′ end is bound by the translation factor eukaryotic initiation factor 4E (eIF4E), while the poly(A) tail, at the 3′ end, is recognized by the poly(A)-binding protein (PABP). eIF4G is a scaffold protein that brings together eIF4E and PABP, causing the circularization of the mRNA that is thought to be important for an efficient initiation of translation. Early in infection, rotaviruses take over the host translation machinery, causing a severe shutoff of cell protein synthesis. Rotavirus mRNAs lack a poly(A) tail but have instead a consensus sequence at their 3′ ends that is bound by the viral nonstructural protein NSP3, which also interacts with eIF4GI, using the same region employed by PABP. It is widely believed that these interactions lead to the translation of rotaviral mRNAs, impairing at the same time the translation of cellular mRNAs. In this work, the expression of NSP3 in infected cells was knocked down using RNA interference. Unexpectedly, under these conditions the synthesis of viral proteins was not decreased, while the cellular protein synthesis was restored. Also, the yield of viral progeny increased, which correlated with an increased synthesis of viral RNA. Silencing the expression of eIF4GI further confirmed that the interaction between eIF4GI and NSP3 is not required for viral protein synthesis. These results indicate that NSP3 is neither required for the translation of viral mRNAs nor essential for virus replication in cell culture.
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Willcocks, Margaret M., Michael J. Carter, and Lisa O. Roberts. "Cleavage of eukaryotic initiation factor eIF4G and inhibition of host-cell protein synthesis during feline calicivirus infection." Journal of General Virology 85, no. 5 (May 1, 2004): 1125–30. http://dx.doi.org/10.1099/vir.0.19564-0.
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Caliciviruses are small, non-enveloped, positive-stranded RNA viruses that are pathogenic for both animals and man. Although their capsid structure and genomic organization are distinct from picornaviruses, they have similarities to these viruses in their non-structural proteins. Picornaviruses induce a rapid inhibition of host-cell cap-dependent protein synthesis and this is mainly achieved through cleavage of eIF4G and/or dephosphorylation of 4E-BP1. In this study, the effect of calicivirus infection was examined on host-cell protein synthesis in order to determine whether they also induce host shut-off. We report that infection of cells with feline calicivirus (FCV) leads to the inhibition of cellular protein synthesis. This is accompanied by the cleavage of the eukaryotic translation initiation factors eIF4GI and eIF4GII in a manner reminiscent of that induced by picornaviruses. However, the cleavages occur at different sites. The potential mechanisms of these cleavage events and the implications for the translation of calicivirus mRNA are discussed.
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Gradi, Alessandra, Nicole Foeger, Rebecca Strong, Yuri V. Svitkin, Nahum Sonenberg, Tim Skern, and Graham J. Belsham. "Cleavage of Eukaryotic Translation Initiation Factor 4GII within Foot-and-Mouth Disease Virus-Infected Cells: Identification of the L-Protease Cleavage Site In Vitro." Journal of Virology 78, no. 7 (April 1, 2004): 3271–78. http://dx.doi.org/10.1128/jvi.78.7.3271-3278.2004.
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ABSTRACT Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (Lpro) alone, generating cleavage products similar to those induced by enterovirus and rhinovirus 2A protease (2Apro). By the use of a fusion protein containing residues 445 to 744 of human eIF4GII, it was demonstrated that the FMDV Lpro specifically cleaves this protein between residues G700 and S701, immediately adjacent to the site (V699/G700) cleaved by rhinovirus 2Apro in vitro. The G700/S701 cleavage site does not correspond, by amino acid sequence alignment, to that cleaved in eIF4GI by the FMDV Lpro in vitro. Knowledge of the cleavage sites and the three-dimensional structures of the FMDV Lpro and rhinovirus 2Apro enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.
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Li, Lei, and Ching C. Wang. "Identification in the Ancient Protist Giardia lamblia of Two Eukaryotic Translation Initiation Factor 4E Homologues with Distinctive Functions." Eukaryotic Cell 4, no. 5 (May 2005): 948–59. http://dx.doi.org/10.1128/ec.4.5.948-959.2005.
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ABSTRACT Eukaryotic translation initiation factor 4E (eIF4E) binds to the m7GTP of capped mRNAs and is an essential component of the translational machinery that recruits the 40S small ribosomal subunit. We describe here the identification and characterization of two eIF4E homologues in an ancient protist, Giardia lamblia. Using m7GTP-Sepharose affinity column chromatography, a specific binding protein was isolated and identified as Giardia eIF4E2. The other homologue, Giardia eIF4E1, bound only to the m2,2,7GpppN structure. Although neither homologue can rescue the function of yeast eIF4E, a knockdown of eIF4E2 mRNA in Giardia by a virus-based antisense ribozyme decreased translation, which was shown to use m7GpppN-capped mRNA as a template. Thus, eIF4E2 is likely the cap-binding protein in a translation initiation complex. The same knockdown approach indicated that eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of both homologues in the cytoplasm. But eIF4E1 was also found concentrated and colocalized with the m2,2,7GpppN cap, 16S-like rRNA, and fibrillarin in the nucleolus-like structure in the nucleus. eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein was bound to Giardia small nuclear RNAs D and H known to have an m2,2,7GpppN cap, thus suggesting a novel function not yet observed among other eIF4Es in eukaryotes.
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Zamora, Miguel, Wilfred E. Marissen, and Richard E. Lloyd. "Multiple eIF4GI-Specific Protease Activities Present in Uninfected and Poliovirus-Infected Cells." Journal of Virology 76, no. 1 (January 1, 2002): 165–77. http://dx.doi.org/10.1128/jvi.76.1.165-177.2002.
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ABSTRACT Cleavage of eukaryotic translation initiation factor 4GI (eIF4GI) is required for shutoff of host cell translation during poliovirus (PV) infection of HeLa cells. Reports published by several groups have led to confusion whether this cleavage is mediated by viral 2A protease (2Apro) or a putative cellular enzyme (termed eIF4Gase) which is activated by 2Apro or other aspects of viral infection. Here we have further investigated eIF4Gase activities in PV-infected cells. Column purification of eIF4GI cleavage activity separated two activities which generated N-terminal cleavage products of different lengths. Both activities were detected using either native eIF4G or radiolabeled recombinant eIF4G as the substrate. Analysis of cleavage products formed by each activity on native and mutant substrates suggests that one activity cleaves eIF4G1 at or very near the 2Apro cleavage site and the other activity cleaves approximately 40 residues upstream of the 2Apro cleavage site. When PV infections in HeLa cells were supplemented with 2 mM guanidine, which indirectly limits expression of 2Apro, two distinct C-terminal cleavage fragments of eIF4GI were detected. These C-terminal cleavage fragments of eIF4GI were purified from infected cells, and a new eIF4GI cleavage site was mapped to a unique site 43 amino acids upstream of the known 2Apro cleavage site. Further, eIF4GI cleavage in vivo could be blocked by addition of zVAD to PV-guanidine infections. zVAD is a broad-spectrum caspase inhibitor which had no effect on 2Apro cleavage activity or PV polyprotein processing. Lastly, similar types of eIF4Gase cleavage activities were also detected in uninfected cells under various conditions, including early apoptosis or during cell cycle transit. The data suggest that the same types of eIF4GI cleavage activities which are generated in PV-infected cells can also be generated in the absence of virus. Taken together, the data support a model in which multiple cellular activities process eIF4GI in PV-infected cells, in addition to 2Apro.
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Kamenska, Anastasiia, Clare Simpson, and Nancy Standart. "eIF4E-binding proteins: new factors, new locations, new roles." Biochemical Society Transactions 42, no. 4 (August 1, 2014): 1238–45. http://dx.doi.org/10.1042/bst20140063.
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The cap-binding translation initiation factor eIF4E (eukaryotic initiation factor 4E) is central to protein synthesis in eukaryotes. As an integral component of eIF4F, a complex also containing the large bridging factor eIF4G and eIF4A RNA helicase, eIF4E enables the recruitment of the small ribosomal subunit to the 5′ end of mRNAs. The interaction between eIF4E and eIF4G via a YXXXXLϕ motif is regulated by small eIF4E-binding proteins, 4E-BPs, which use the same sequence to competitively bind eIF4E thereby inhibiting cap-dependent translation. Additional eIF4E-binding proteins have been identified in the last 10–15 years, characterized by the YXXXXLϕ motif, and by interactions (many of which remain to be detailed) with RNA-binding proteins, or other factors in complexes that recognize the specific mRNAs. In the present article, we focus on the metazoan 4E-T (4E-transporter)/Cup family of eIF4E-binding proteins, and also discuss very recent examples in yeast, fruitflies and humans, some of which predictably inhibit translation, while others may result in mRNA decay or even enhance translation; altogether considerably expanding our understanding of the roles of eIF4E-binding proteins in gene expression regulation.
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DOMINGUEZ, Diana, Elisabeth KISLIG, Michael ALTMANN, and Hans TRACHSEL. "Structural and functional similarities between the central eukaryotic initiation factor (eIF)4A-binding domain of mammalian eIF4G and the eIF4A-binding domain of yeast eIF4G." Biochemical Journal 355, no. 1 (February 26, 2001): 223–30. http://dx.doi.org/10.1042/bj3550223.
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The translation eukaryotic initiation factor (eIF)4G of the yeast Saccharomyces cerevisiae interacts with the RNA helicase eIF4A (a member of the DEAD-box protein family; where DEAD corresponds to Asp-Glu-Ala-Asp) through a C-terminal domain in eIF4G (amino acids 542–883). Mammalian eIF4G has two interaction domains for eIF4A, a central domain and a domain close to the C-terminus. This raises the question of whether eIF4A binding to eIF4G is conserved between yeast and mammalian cells or whether it is different. We isolated eIF4G1 mutants defective in eIF4A binding and showed that these mutants are strongly impaired in translation and growth. Extracts from mutants displaying a temperature-sensitive phenotype for growth have low in vitro translation activity, which can be restored by addition of the purified eIF4G1–eIF4E complex, but not by eIF4E alone. Analysis of mutant eIF4G542–883 proteins defective in eIF4A binding shows that the interaction of yeast eIF4A with eIF4G1 depends on amino acid motifs that are conserved between the yeast eIF4A-binding site and the central eIF4A-binding domain of mammalian eIF4G. We show that mammalian eIF4A binds tightly to yeast eIF4G1 and, furthermore, that mutant yeast eIF4G542–883 proteins, which do not bind yeast eIF4A, do not interact with mammalian eIF4A. Despite the conservation of the eIF4A-binding site in eIF4G and the strong sequence conservation between yeast and mammalian eIF4A (66% identity; 82% similarity at the amino acid level) mammalian eIF4A does not substitute for the yeast factor in vivo and is not functional in a yeast in vitro translation system.
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Mokas, Sophie, John R. Mills, Cristina Garreau, Marie-Josée Fournier, Francis Robert, Prabhat Arya, Randal J. Kaufman, Jerry Pelletier, and Rachid Mazroui. "Uncoupling Stress Granule Assembly and Translation Initiation Inhibition." Molecular Biology of the Cell 20, no. 11 (June 2009): 2673–83. http://dx.doi.org/10.1091/mbc.e08-10-1061.
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Cytoplasmic stress granules (SGs) are specialized regulatory sites of mRNA translation that form under different stress conditions known to inhibit translation initiation. The formation of SG occurs via two pathways; the eukaryotic initiation factor (eIF) 2α phosphorylation-dependent pathway mediated by stress and the eIF2α phosphorylation-independent pathway mediated by inactivation of the translation initiation factors eIF4A and eIF4G. In this study, we investigated the effects of targeting different translation initiation factors and steps in SG formation in HeLa cells. By depleting eIF2α, we demonstrate that reduced levels of the eIF2.GTP.Met-tRNAiMet ternary translation initiation complexes is sufficient to induce SGs. Likewise, reduced levels of eIF4B, eIF4H, or polyA-binding protein, also trigger SG formation. In contrast, depletion of the cap-binding protein eIF4E or preventing its assembly into eIF4F results in modest SG formation. Intriguingly, interfering with the last step of translation initiation by blocking the recruitment of 60S ribosome either with 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamideis or through depletion of the large ribosomal subunits protein L28 does not induce SG assembly. Our study identifies translation initiation steps and factors involved in SG formation as well as those that can be targeted without induction of SGs.
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Gallie, Daniel R. "Cap-Independent Translation Conferred by the 5′ Leader of Tobacco Etch Virus Is Eukaryotic Initiation Factor 4G Dependent." Journal of Virology 75, no. 24 (December 15, 2001): 12141–52. http://dx.doi.org/10.1128/jvi.75.24.12141-12152.2001.
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ABSTRACT 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.
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Kommaraju, Sai Shilpa, Julieta Aulicino, Shruthi Gobbooru, Jing Li, Mingzhao Zhu, Daniel Romo, and Woon-Kai Low. "Investigation of the mechanism of action of a potent pateamine A analog, des-methyl, des-amino pateamine A (DMDAPatA)." Biochemistry and Cell Biology 98, no. 4 (August 2020): 502–10. http://dx.doi.org/10.1139/bcb-2019-0307.
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The natural product pateamineA (PatA) is a highly potent antiproliferative agent. PatA and the simplified analog desmethyl, desamino pateamineA (DMDAPatA) have exhibited cytotoxicity selective for rapidly proliferating cells, and have been shown to inhibit cap-dependent translation initiation through binding to eIF4A (eukaryotic initiation factor 4A) of the eIF4F complex. PatA and DMDAPatA are both known to stimulate the RNA-dependent ATPase, and ATP-dependent RNA helicase activities of eIF4A. The impact of other eIF4F components, eIF4E and eIF4G, on DMDAPatA action were investigated in vitro and in cultured mammalian cells. The perturbation of the eIF4A–eIF4G association was found to be eIF4E- and mRNA cap-dependent. An inhibitory effect on helicase activity of eIF4A was observed when it was part of a complex that mimicked the eIF4F complex. We propose a model of action for DMDAPatA (and by supposition PatA) where the cellular activity of the compound is dependent on an “active” eIF4F complex.
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Petegnief, Valérie, Míriam Font-Nieves, M. Elena Martín, Matilde Salinas, and Anna M. Planas. "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." Biochemical Journal 411, no. 3 (April 14, 2008): 667–77. http://dx.doi.org/10.1042/bj20071060.
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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) 2α 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 eIF2α 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.
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van Breukelen, Frank, Nahum Sonenberg, and Sandra L. Martin. "Seasonal and state-dependent changes of eIF4E and 4E-BP1 during mammalian hibernation: implications for the control of translation during torpor." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 287, no. 2 (August 2004): R349—R353. http://dx.doi.org/10.1152/ajpregu.00728.2003.
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Mammalian hibernation involves cessation of energetically costly processes typical of homeostatic regulation including protein synthesis. To further elucidate the mechanisms employed in depressing translation, we surveyed key eukaryotic initiation factors [eIF2, eIF4B, eIF4E, eIF4GI and -II, and 4E-binding protein-1 (4E-BP1), -2, and -3] for their availability and phosphorylation status in the livers of golden-mantled ground squirrels (Spermophilus lateralis) across the hibernation cycle. Western blot analyses indicated only one significant locus for regulation of translational initiation in ground squirrel liver: control of eIF4E. We found seasonal variation in a potent regulator of eIF4E activity, 4E-BP1. Summer squirrels lack 4E-BP1 and apparently control eIF4E activity through direct phosphorylation. In winter, eIF4E is regulated through binding with 4E-BP1. During the euthermic periods that separate bouts of torpor (interbout arousal), 4E-BP1 is hyperphosphorylated to promote initiation. However, during torpor, 4E-BP1 is hypophosphorylated and cap-dependent initiation of translation is restricted. The regulation of cap-dependent initiation of translation may allow for the differential expression of proteins directed toward enhancing survivorship.
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Svitkin, Yuri V., Barbara Herdy, Mauro Costa-Mattioli, Anne-Claude Gingras, Brian Raught, and Nahum Sonenberg. "Eukaryotic Translation Initiation Factor 4EAvailability Controls the Switch between Cap-Dependent andInternal Ribosomal Entry Site-MediatedTranslation." Molecular and Cellular Biology 25, no. 23 (December 1, 2005): 10556–65. http://dx.doi.org/10.1128/mcb.25.23.10556-10565.2005.
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ABSTRACT Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5′ end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.
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Chen, Sunrui, Cui Feng, Yan Fang, Xinying Zhou, Lei Xu, Wenshi Wang, Xiangdong Kong, Maikel P. Peppelenbosch, Qiuwei Pan, and Yuebang Yin. "The Eukaryotic Translation Initiation Factor 4F Complex Restricts Rotavirus Infection via Regulating the Expression of IRF1 and IRF7." International Journal of Molecular Sciences 20, no. 7 (March 29, 2019): 1580. http://dx.doi.org/10.3390/ijms20071580.
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The eIF4F complex is a translation initiation factor that closely regulates translation in response to a multitude of environmental conditions including viral infection. How translation initiation factors regulate rotavirus infection remains poorly understood. In this study, the knockdown of the components of the eIF4F complex using shRNA and CRISPR/Cas9 were performed, respectively. We have demonstrated that loss-of-function of the three components of eIF4F, including eIF4A, eIF4E and eIF4G, remarkably promotes the levels of rotavirus genomic RNA and viral protein VP4. Consistently, knockdown of the negative regulator of eIF4F and programmed cell death protein 4 (PDCD4) inhibits the expression of viral mRNA and the VP4 protein. Mechanically, we confirmed that the silence of the eIF4F complex suppressed the protein level of IRF1 and IRF7 that exert potent antiviral effects against rotavirus infection. Thus, these results demonstrate that the eIF4F complex is an essential host factor restricting rotavirus replication, revealing new targets for the development of new antiviral strategies against rotavirus infection.
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Jagus, Rosemary, Tsvetan R. Bachvaroff, Bhavesh Joshi, and Allen R. Place. "Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists." Comparative and Functional Genomics 2012 (2012): 1–21. http://dx.doi.org/10.1155/2012/134839.
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The greatest diversity of eukaryotic species is within the microbial eukaryotes, the protists, with plants and fungi/metazoa representing just two of the estimated seventy five lineages of eukaryotes. Protists are a diverse group characterized by unusual genome features and a wide range of genome sizes from 8.2 Mb in the apicomplexan parasiteBabesia bovisto 112,000-220,050 Mb in the dinoflagellateProrocentrum micans. Protists possess numerous cellular, molecular and biochemical traits not observed in “text-book” model organisms. These features challenge some of the concepts and assumptions about the regulation of gene expression in eukaryotes. Like multicellular eukaryotes, many protists encode multiple eIF4Es, but few functional studies have been undertaken except in parasitic species. An earlier phylogenetic analysis of protist eIF4Es indicated that they cannot be grouped within the three classes that describe eIF4E family members from multicellular organisms. Many more protist sequences are now available from which three clades can be recognized that are distinct from the plant/fungi/metazoan classes. Understanding of the protist eIF4Es will be facilitated as more sequences become available particularly for the under-represented opisthokonts and amoebozoa. Similarly, a better understanding of eIF4Es within each clade will develop as more functional studies of protist eIF4Es are completed.
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El-Kadi, Samer W., Ying Chen, Sydney R. McCauley, Kacie A. Seymour, Sally E. Johnson, and Robert P. Rhoads. "Decreased abundance of eIF4F subunits predisposes low birth weight neonatal pigs to reduced muscle hypertrophy." Journal of Applied Physiology 125, no. 4 (October 1, 2018): 1171–82. http://dx.doi.org/10.1152/japplphysiol.00704.2017.
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Muscle hypertrophy is limited in low birth weight (LBWT) neonates, suggesting a reduction in protein synthesis and increased protein degradation. Sixteen pairs of one-day-old normal birth weight (NBWT) and LBWT littermates ( n = 16) were euthanized, and the longissimus dorsi (LD) was sampled for protein abundance and kinase phosphorylation profile measures. Eukaryotic initiation factor (eIF)4E and eIF4G abundance, and assembly of the active eIF4E-eIF4G complex, was less for LBWT than for NBWT pig muscles. Similarly, eIF3f abundance was reduced in the muscle of LBWT compared with NBWT pigs and was associated with diminished ribosomal protein S6 kinase 1 phosphorylation. This decrease was linked to a lower phosphorylation of programmed cell death protein 4 (PDCD4) in LBWT pig muscle. By contrast, PDCD4 abundance was greater in the muscle of the LBWT than NBWT group, suggesting lower release and availability of eIF4A from the PDCD4-eIF4A complex. Moreover, protein abundance of eIF4A was lower in LBWT muscle, which is expected to further impair the formation of eIF4F translation initiation complex. Microtubule-associated light chain 3 (LC3) II to total LC3 ratio was greater in LBWT LD lysates, yet P62 abundance was similar between the two groups, suggesting no difference in autophagy. Muscle atrophy F-box (atrogin-1) abundance was less in LBWT LD lysates, suggesting decreased degradation through the ubiquitin-proteasome system. In conclusion, limited eIF4F subunit abundance and downregulated translation initiation are plausible mechanisms for diminished muscle growth in LBWT compared with NBWT neonatal pigs. NEW & NOTEWORTHY We demonstrated that eukaryotic initiation factor (eIF)4E, eIF4G, and eIF4A abundance, and assembly of the active eIF4E-eIF4G complex, were reduced in low birth weight (LBWT) compared with normal birth weight pig muscle. In contrast, our data indicated that protein degradation signaling does not seem to affect protein turnover in LBWT pig muscle. Thus, downregulated translation initiation is likely the key contributor that predisposes LBWT neonatal pigs to slower postnatal muscle growth.
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Skofler, Christina, Florian Kleinegger, Stefanie Krassnig, Anna Maria Birkl-Toeglhofer, Georg Singer, Holger Till, Martin Benesch, et al. "Eukaryotic Translation Initiation Factor 4AI: A Potential Novel Target in Neuroblastoma." Cells 10, no. 2 (February 2, 2021): 301. http://dx.doi.org/10.3390/cells10020301.
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Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Children suffering from high-risk and/or metastatic NB often show no response to therapy, and new therapeutic approaches are urgently needed. Malignant tumor development has been shown to be driven by the dysregulation of eukaryotic initiation factors (eIFs) at the translation initiation. Especially the activity of the heterotrimeric eIF4F complex is often altered in malignant cells, since it is the direct connection to key oncogenic signaling pathways such as the PI3K/AKT/mTOR-pathway. A large body of literature exists that demonstrates targeting the translational machinery as a promising anti-neoplastic approach. The objective of this study was to determine whether eIF4F complex members are aberrantly expressed in NB and whether targeting parts of the complex may be a therapeutic strategy against NB. We show that eIF4AI is overexpressed in NB patient tissue using immunohistochemistry, immunoblotting, and RT-qPCR. NB cell lines exhibit decreased viability, increased apoptosis rates as well as changes in cell cycle distribution when treated with the synthetic rocaglate CR-1-31-B, which clamps eIF4A and eIF4F onto mRNA, resulting in a translational block. Additionally, this study reveals that CR-1-31-B is effective against NB cell lines at low nanomolar doses (≤20 nM), which have been shown to not affect non-malignant cells in previous studies. Thus, our study provides information of the expression status on eIF4AI in NB and offers initial promising insight into targeting translation initiation as an anti-tumorigenic approach for NB.
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Lu, Wei-Ting, Anna Wilczynska, Ewan Smith, and Martin Bushell. "The diverse roles of the eIF4A family: you are the company you keep." Biochemical Society Transactions 42, no. 1 (January 23, 2014): 166–72. http://dx.doi.org/10.1042/bst20130161.
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The eIF4A (eukaryotic initiation factor 4A) proteins belong to the extensive DEAD-box RNA helicase family, the members of which are involved in many aspects of RNA metabolism by virtue of their RNA-binding capacity and ATPase activity. Three eIF4A proteins have been characterized in vertebrates: eIF4A1 and eIF4A2 are cytoplasmic, whereas eIF4A3 is nuclear-localized. Although highly similar, they have been shown to possess rather diverse roles in the mRNA lifecycle. Their specific and diverse functions are often regulated and dictated by interacting partner proteins. The key differences between eIF4A family members are discussed in the present review.
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Cosentino, Gregory P., Tobias Schmelzle, Ashkan Haghighat, Stephen B. Helliwell, Michael N. Hall, and Nahum Sonenberg. "Eap1p, a Novel Eukaryotic Translation Initiation Factor 4E-Associated Protein in Saccharomyces cerevisiae." Molecular and Cellular Biology 20, no. 13 (July 1, 2000): 4604–13. http://dx.doi.org/10.1128/mcb.20.13.4604-4613.2000.
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ABSTRACT Ribosome binding to eukaryotic mRNA is a multistep process which is mediated by the cap structure [m7G(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 theEAP1 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.
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Goldstaub, Dan, Alessandra Gradi, Zippi Bercovitch, Zehava Grosmann, Yaron Nophar, Sylvie Luria, Nahum Sonenberg, and Chaim Kahana. "Poliovirus 2A Protease Induces Apoptotic Cell Death." Molecular and Cellular Biology 20, no. 4 (February 15, 2000): 1271–77. http://dx.doi.org/10.1128/mcb.20.4.1271-1277.2000.
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ABSTRACT A cell line was generated that expresses the poliovirus 2A protease in an inducible manner. Tightly controlled expression was achieved by utilizing the muristerone A-regulated expression system. Upon induction, cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI) and eIF4GII is observed, with the latter being cleaved in a somewhat slower kinetics. eIF4G cleavage was accompanied by a severe inhibition of protein synthesis activity. Upon induction of the poliovirus 2A protease, the cells displayed fragmented nuclei, chromatin condensation, oligonucleosome-size DNA ladder, and positive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining; hence, their death can be characterized as apoptosis. These results indicate that the expression of the 2A protease in mammalian cells is sufficient to induce apoptosis. We suggest that the poliovirus 2A protease induces apoptosis either by arresting cap-dependent translation of some cellular mRNAs that encode proteins required for cell viability, by preferential cap-independent translation of cellular mRNAs encoding apoptosis inducing proteins, or by cleaving other, yet unidentified cellular target proteins.
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Lachance, Pascal E. D., Mathieu Miron, Brian Raught, Nahum Sonenberg, and Paul Lasko. "Phosphorylation of Eukaryotic Translation Initiation Factor 4E Is Critical for Growth." Molecular and Cellular Biology 22, no. 6 (March 15, 2002): 1656–63. http://dx.doi.org/10.1128/mcb.22.6.1656-1663.2002.
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ABSTRACT Eukaryotic translation initiation factor 4E (eIF4E) binds to the cap structure at the 5′ end of mRNAs and is a critical target for the control of protein synthesis. eIF4E is phosphorylated in many systems in response to extracellular stimuli, but biochemical evidence to date has been equivocal as to the biological significance of this modification. Here we use a genetic approach to this problem. We show that, in Drosophila melanogaster, homozygous eIF4E mutants arrest growth during larval development. In Drosophila eIF4EI, Ser251 corresponds to Ser209 of mammalian eIF4E, which is phosphorylated in response to extracellular signals. We find that, in vivo, eIF4EI Ser251 mutants cannot incorporate labeled phosphate. Furthermore, transgenic Drosophila organisms expressing eIF4ESer251Ala in an eIF4E mutant background have reduced viability. Escapers develop more slowly than control siblings and are smaller. These genetic data provide evidence that eIF4E phosphorylation is biologically significant and is essential for normal growth and development.
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Jaiswal, Praveen K., Sweaty Koul, Saikolappan Sankaralingam, and Hari K. Koul. "Abstract 396: Therapeutic targeting of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in enzalutamide resistant (ENZR) prostate cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 396. http://dx.doi.org/10.1158/1538-7445.am2022-396.
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Abstract Objective: Deregulation of the translation initiation machinery is one of the critical steps for oncogenic mRNA translation in tumor cells. Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is a crucial scaffold of translation initiation complex (EIF4F). Our recent studies suggest a role for EIF4G1 in multiple tumor types including prostate cancer progression. In the present study, we tested the unknown function of EIF4G1 in Enzalutamide resistant prostate cancer cells. Methods: We used enzalutamide resistant C4-2B (C4-2BENZR) and parental C4-2B cells for the current study. For the functional studies, we used shRNA targeting the EIF4G1 and pharmacological EIF4G-EIF4E complex inhibitor (4EGI-1). The functional assays such as clonogenic, cell proliferation, cell viability, trans-well migration/invasion, and prostasphere formation were performed. To further expand in vitro findings we employed, in-vivo tumor xenograft model derived from enzalutamide resistance C4-2B cells. Results: We found a higher level of EIF4G1 protein in ENZ resistance (C4-2B ENZR) cell lines as compared to parental C4-2B cell lines. Furthermore, knockdown of EIF4G1 by shRNA or treatment with 4EGI-1 inhibitor impaired the clonogenic potential, prostasphere formation, cell viability/proliferation, and cell migration/invasion in C4-2B ENZR cell line, suggesting a critical role of EIF4G1 in modulating enzalutamide resistance in CRPC cells. C4-2BENZR subcutaneous tumor xenograft treated with
combination of 4EGI-1 and ENZ were significantly reduced the tumor volume. Moreover 4EGI-1 sensitized ENZ Resistance C4-2B cells to Enzalutamide treatment in tumor xenograft model. Conclusions: Overall, the findings suggest that EIF4G1 could serve as a novel therapeutic target in overcoming therapeutic resistance to current treatments in PCa. Citation Format: Praveen K. Jaiswal, Sweaty Koul, Saikolappan Sankaralingam, Hari K. Koul. Therapeutic targeting of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in enzalutamide resistant (ENZR) prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 396.
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Sugiyama, Hayami, Kazutoshi Takahashi, Takuya Yamamoto, Mio Iwasaki, Megumi Narita, Masahiro Nakamura, Tim A. Rand, Masato Nakagawa, Akira Watanabe, and Shinya Yamanaka. "Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells." Proceedings of the National Academy of Sciences 114, no. 2 (December 21, 2016): 340–45. http://dx.doi.org/10.1073/pnas.1617234114.
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Novel APOBEC1 target 1 (Nat1) (also known as “p97,” “Dap5,” and “Eif4g2”) is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil–containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1. Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.