The eucaryotic translation initiation factor 4B (eIF4B) stimulates the helicase activity of the DEAD box protein eIF4A to unwind inhibitory secondary structure in the 5 0 untranslated region of eucaryotic mRNAs. Here, using phosphopeptide mapping and a phosphospecific antiserum, we identify a serum-responsive eIF4B phosphorylation site, Ser422, located in an RNA-binding region required for eIF4A helicase-promoting activity. Ser422 phosphorylation appears to be regulated by the S6Ks: (a) Ser422 phosphorylation is sensitive to pharmacological inhibitors of phosphoinositide-3 kinase and the mammalian target of rapamycin; (b) S6K1/S6K2 specifically phosphorylate Ser422 in vitro; and (c) rapamycin-resistant S6Ks confer rapamycin resistance upon Ser422 phosphorylation in vivo. Substitution of Ser422 with Ala results in a loss of activity in an in vivo translation assay, indicating that phosphorylation of this site plays an important role in eIF4B function. We therefore propose that eIF4B may mediate some of the effects of the S6Ks on translation.

In mammalian cells, nonsense-mediated mRNA decay (NMD) generally requires that translation terminates sufficiently upstream of a post-splicing exon junction complex (EJC) during a pioneer round of translation. The subsequent binding of Upf1 to the EJC triggers Upf1 phosphorylation. We provide evidence that phospho-Upf1 functions after nonsense codon recognition during steps that involve the translation initiation factor eIF3 and mRNA decay factors. Phospho-Upf1 interacts directly with eIF3 and inhibits the eIF3-dependent conversion of 40S/Met-tRNA(i)(Met)/mRNA to translationally competent 80S/Met-tRNA(i)(Met)/mRNA initiation complexes to repress continued translation initiation. Consistent with phospho-Upf1 impairing eIF3 function, NMD fails to detectably target nonsense-containing transcripts that initiate translation independently of eIF3 from the CrPV IRES. There is growing evidence that translational repression is a key transition that precedes mRNA delivery to the degradation machinery. Our results uncover a critical step during NMD that converts a pioneer translation initiation complex to a translationally compromised mRNP.

Eukaryotic initiation factor 3 (eIF3) is a 12-subunit protein complex that plays a central role in binding of initiator methionyl-tRNA and mRNA to the 40 S ribosomal subunit to form the 40 S initiation complex. The molecular mechanisms by which eIF3 exerts these functions are poorly understood. To learn more about the structure and function of eIF3 we have expressed and purified individual human eIF3 subunits or complexes of eIF3 subunits using baculovirus-infected Sf9 cells. The results indicate that the subunits of human eIF3 that have homologs in Saccharomyces cerevisiae form subcomplexes that reflect the subunit interactions seen in the yeast eIF3 core complex. In addition, we have used an in vitro 40 S ribosomal subunit binding assay to investigate subunit requirements for efficient association of the eIF3 subcomplexes to the 40 S ribosomal subunit. eIF3j alone binds to the 40 S ribosomal subunit, and its presence is required for stable 40 S binding of an eIF3bgi subcomplex. Furthermore, purified eIF3 lacking eIF3j binds 40 S ribosomal subunits weakly, but binds tightly when eIF3j is added. Cleavage of a 16-residue C-terminal peptide from eIF3j by caspase-3 significantly reduces the affinity of eIF3j for the 40 S ribosomal subunit, and the cleaved form provides substantially less stabilization of purified eIF3-40S complexes. These results indicate that eIF3j, and especially its C terminus, play an important role in the recruitment of eIF3 to the 40 S ribosomal subunit. Eukaryotic initiation factor 3 (eIF3)1 was first isolated and purified as a high molecular weight complex from rabbit reticulocytes (1-3). The mammalian factor possesses a molecular mass of about 600 kDa and contains at least 12 nonidentical protein subunits, named in order of decreasing molecular weight as recommended (4): eIF3a, eIF3b, eIF3c, eIF3d, eIF3l, eIF3e, eIF3f, eIF3g, eIF3h, eIF3i, eIF3j, and eIF3k (5, 6). Specific functions for mammalian eIF3 have been identified by a variety of in vitro experiments. It binds directly to 40 S ribosomal subunits in the absence of other initiation components (1), and affects the association/dissociation of ribosomes (7-10). It promotes the binding of Met-tRNA i and mRNA to the 40 S ribosomal subunit (5), and binds directly to eIF1 (11), eIF4B (12), eIF4G (13,14), and eIF5 (15). Clearly, eIF3 plays a central role in the initiation pathway, perhaps structurally organizing other translational components on the surface of the 40 S ribosomal subunit.An eIF3 complex was first identified and isolated from Saccharomyces cerevisiae by employing either of two assay systems: stimulation of methionyl-puromycin synthesis based on mammalian assay components (16) and stimulation of protein synthesis in a heat-inactivated yeast lysate derived from a conditional mutant of eIF3b (17). Purification of eIF3 using an oligohistidine-tagged eIF3b identified a core of five subunits associated with eIF5 (18). The five core subunits, eIF3a, eIF3b, eIF3c, eIF3g, and eIF3i, are all essential for yeast growth and are conserved ...

Protein synthesis in mammalian cells requires initiation factor eIF3, an ϳ800-kDa protein complex that plays a central role in binding of initiator methionyl-tRNA and mRNA to the 40 S ribosomal subunit to form the 48 S initiation complex. The eIF3 complex also prevents premature association of the 40 and 60 S ribosomal subunits and interacts with other initiation factors involved in start codon selection. The molecular mechanisms by which eIF3 exerts these functions are poorly understood. Since its initial characterization in the 1970s, the exact size, composition, and post-translational modifications of mammalian eIF3 have not been rigorously determined. Two powerful mass spectrometric approaches were used in the present study to determine post-translational modifications that may regulate the activity of eIF3 during the translation initiation process and to characterize the molecular structure of the human eIF3 protein complex purified from HeLa cells. In the first approach, the bottom-up analysis of eIF3 allowed for the identification of a total of 13 protein components (eIF3a-m) with a sequence coverage of ϳ79%. Furthermore 29 phosphorylation sites and several other post-translational modifications were unambiguously identified within the eIF3 complex. The second mass spectrometric approach, involving analysis of intact eIF3, allowed the detection of a complex with each of the 13 subunits present in stoichiometric amounts. Using tandem mass spectrometry four eIF3 subunits (h, i, k, and m) were found to be most easily dissociated and therefore likely to be on the periphery of the complex. It is noteworthy that none of these four subunits were found to be phosphorylated. These data raise interesting questions about the function of phosphorylation as it relates to the core subunits of the complex. Molecular & Cellular Proteomics 6:1135-1146, 2007.The initiation phase of eukaryotic protein synthesis involves formation of an 80 S ribosomal complex containing the initiator methionyl-tRNA i bound to the initiation codon in the mRNA. This is a multistep process promoted by proteins called eukaryotic initiation factors (eIFs).1 Currently at least 12 eIFs, composed of at least 29 distinct subunits, have been identified (1). Mammalian eIF3, the largest initiation factor, is a multisubunit complex with an apparent molecular mass of ϳ800 kDa. This protein complex plays an essential role in translation by binding directly to the 40 S ribosomal subunit and promoting formation of the 43 S preinitiation complex consisting of the Met-tRNA i ⅐eIF2⅐GTP ternary complex, eIF1, eIF1A, and the 40 S ribosomal subunit (2, 3). The ability of eIF3 to bind to 40 S subunits in the absence of other initiation factors is enhanced by the presence of its loosely associated eIF3j subunit (4). The eIF3 complex also promotes binding of 5Ј-m 7 G-capped mRNA through its interaction with eIF4G, the largest member of the eIF4F cap-binding complex (5, 6). The 43 S preinitiation complex then scans the mRNA (together forming the 48 S complex) in a 5Ј ...

The androgen receptor (AR) dynamically assembles and disassembles multicomponent receptor complexes in order to respond rapidly and reversibly to fluctuations in androgen levels. We are interested in identifying the basal factors that compose the AR aporeceptor and holoreceptor complexes and impact the transcriptional process. Using tandem mass spectroscopy analysis, we identified the trimeric DNA-dependent protein kinase (