The j-subunit of human translation initiation factor eIF3 is required for the stable binding of eIF3 and its subcomplexes to 40 S ribosomal subunits in vitro
ABSTRACT 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.
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ABSTRACT: P311, a conserved, 8-kDa, intracellular protein expressed in brain, smooth muscle, regenerating tissues, and malignant glioblastomas, represents the first-documented pan-stimulator of TGF-β1-3 translation in vitro and in vivo. Here, we initiated efforts to define the mechanism underlying P311 function. PONDR analysis suggested and circular dichroism (CD) confirmed that P311 is an intrinsically disordered protein, requiring therefore an interacting partner to acquire tertiary structure and function. Immunoprecipitation, coupled with mass spectroscopy, identified eukaryotic translation initiation factor 3 subunit b (eIF3b) as a novel P311 binding partner. Immunohistochemical co-localization, GST pulldown, and surface plasmon resonance (SPR) studies revealed that P311-eIF3b interaction is direct and has a Kd of 1.26 uM. Binding sites were mapped to the non-canonical RNA recognition motif (RRM) of eIF3b and a central 11 amino acid-long region of P311, here referred to as eIF3b binding motif (EBM). Disruption of P311-eIF3b binding inhibited translation of TGF-β1, 2 and 3, as indicated by luciferase reporter assays, polysome fractionation studies, and western blot analysis. RIP assays after UV-crosslinking and RNA-protein EMSA demonstrated that P311 directly binds to TGF-β 5'UTRs mRNAs through a previously unidentified RRM-like motif. Our results demonstrate that P311 is a novel RNA-binding protein that by interacting with TGF-βs 5'UTRs and eIF3b stimulates the translation of TGF-β1, 2 and 3.Journal of Biological Chemistry 10/2014; 289(49). DOI:10.1074/jbc.M114.609495 · 4.60 Impact Factor
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ABSTRACT: Selenium nanoparticles have been recently proposed as a potential chemotherapeutic agent due to its low toxicity and its ability to arrest the cell cycle of cancer cells. However, the biochemical mechanisms associated to this effect have not yet been uncovered. We evaluate here the potential of chitosan-stabilized selenium nanoparticles to induce cell cycle arrest and to inhibit in-vitro invasiveness in HepG2 cells. In addition, we use a quantitative proteomic approach to identify potential protein targets involved in the mechanisms associated to selenium nanoparticles exposure. Our data suggest that the induction of the cell cycle arrest at the S phase is mediated by de-regulation of the eIF3 protein complex. We found additional de-regulated proteins upon selenium nanoparticles exposure that could also be involved in the overall inhibition of cell proliferation. These findings not only support the potential of chitosan-stabilized selenium nanoparticles as anti-cancer therapy but also provide a deeper insight into the mechanisms associated to their chemotherapeutic effects.Journal of Nanomedicine & Nanotechnology 09/2014; 5(5):1. DOI:10.4172/2157-7439.1000226 · 5.72 Impact Factor