Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3)

California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2011; 108(51):20473-8. DOI: 10.1073/pnas.1116821108
Source: PubMed

ABSTRACT Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli, revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.

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    • "Furthermore, RACK1 was shown to associate with one of the eIF3 subunits in order to assemble a translation preinitiation complex in yeast (Hashem et al., 2013a; Kouba et al., 2012). Although our understanding of the molecular structure of the core of the 13 subunits eIF3 complex has progressed remarkably in recent years (e.g., Hashem et al., 2013b; Sun et al., 2011), the role of the noncore subunits remains essentially untested in an- imals. "
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    ABSTRACT: Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in devel-opment of resistance. Viruses depend on cellular molecules—which are attractive alternative tar-gets—for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we iden-tify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general transla-tion. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and un-cover a target for the development of broad antiviral intervention. INTRODUCTION
    Cell 11/2014; 159(5):1086 - 1095. DOI:10.1016/j.cell.2014.10.041 · 32.24 Impact Factor
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    • "Despite many attempts of several groups, the overall structure of eIF3 remains somewhat obscure (7, 14, 15, 44). It seems evident that the eIF3 core is formed by the PCI/MPN octamer to which other subunits bind (14–16). "
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    ABSTRACT: The main role of the translation initiation factor 3 (eIF3) is to orchestrate formation of 43S-48S pre-initiation complexes (PICs). Until now, most of our knowledge on eIF3 functional contribution to regulation of gene expression comes from yeast studies. Hence, here we developed several novel in vivo assays to monitor integrity of all 13 subunits of human eIF3, defects in assembly of 43S PICs, efficiency of mRNA recruitment, and in post-assembly events such as AUG recognition. We knocked-down expression of the PCI/MPN octameric eIF3c and eIF3a subunits, and of eIF3j, in human HeLa and HEK293 cells and analyzed the consequences. Whereas eIF3j down-regulation had barely any effect and eIF3a knock-down disintegrated entire eIF3, eIF3c knock-down produced a separate assembly of the a-b-g-i subunits (closely resembling the yeast evolutionary conserved eIF3 core), which preserved relatively high 40S-binding affinity, an ability to promote mRNA recruitment to 40S subunits, and displayed defects in AUG recognition. Both eIF3c and eIF3a knock-downs also severely reduced protein but not mRNA levels of many other eIF3 subunits and indeed shut off translation. We propose that eIF3a and eIF3c control abundance and assembly of entire eIF3 and thus represent its crucial scaffolding elements critically required for formation of PICs.
    Molecular and Cellular Biology 06/2014; 34(16). DOI:10.1128/MCB.00663-14 · 4.78 Impact Factor
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    • "eIF4F recruits ribosomes indirectly through bridging interactions with a 40S ribosome-associated complex, eIF3 (Fig. 1A; Hinnebusch 2006). Mammalian eIF3 consists of 10–13 subunits (a–m) with a core comprised of five to eight subunits, of which a, b, c, g, and i have yeast homologs (Zhou et al. 2008; Sun et al. 2011; Querol-Audi et al. 2013), although a ''functional core'' of subunits a, b, c, e, f, and h has been suggested (Masutani et al. 2007). As a translation initiation factor, eIF3 stimulates ternary complex (TC) recruitment to the 40S ribosome and prevents premature 60S ribosome joining, both of which require noncore eIF3 subunits (Hinnebusch 2006). "
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    ABSTRACT: 40S ribosomes are loaded onto capped mRNAs via the multisubunit translation initiation factors eIF3 and eIF4F. While eIF4E is the eIF4F cap recognition component, the eIF4G subunit associates with 40S-bound eIF3. How this intricate process is coordinated remains poorly understood. Here, we identify an eIF3 subunit that regulates eIF4F modification and show that eIF3e is required for inducible eIF4E phosphorylation. Significantly, recruitment of the eIF4E kinase Mnk1 (MAPK signal-integrating kinase 1) to eIF4F depended on eIF3e, and eIF3e was sufficient to promote Mnk1-binding to eIF4G. This establishes a mechanism by which 40S ribosome loading imparts a phosphorylation mark on the cap-binding eIF4F complex that regulates selective mRNA translation and is synchronized by a specific eIF3 subunit.
    Genes & development 04/2014; 28(8):835-40. DOI:10.1101/gad.236752.113 · 10.80 Impact Factor
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