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. 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 ...

Structure of eIF3b RNA Recognition Motif and Its Interaction with eIF3j

“…units and the PRT1-RRM, which stimulates binding of the eIF3 complex to 40 S subunits (10). In humans, eIF3j is also required for stable 40 S ribosome binding to the eIF3 complex; eIF3 lacking eIF3j binds only weakly to 40 S subunits, but tight binding can be restored in vitro upon the addition of eIF3j (8). All of these data indicate that the interaction between eIF3b and eIF3j is crucial for the eIF3 recruitment to the 40 S ribosome.…”

“…Although co-purification of human eIF3j with full-length eIF3b has been previously reported (8), there is no direct evidence that the RRM domain of human eIF3b provides the interaction surface for eIF3j binding. To test this potential direct interaction, we performed gel mobility shift assays ( Fig.…”

“…This motif in TIF32 is important for the interaction with the yeast eIF3b-RRM, whereas mutation of this motif in HCR1 alters its ability to compensate the defects caused by PRT1 (eIF3b) or TIF32 (eIF3a) mutations (10). Last, the 16 last amino acids of human eIF3j, which have been shown to be important for stable binding to the 40 S ribosomal subunit (8), also contain acidic residues, which can potentially complement the basic interacting surface of eIF3b-RRM.…”

“…We therefore decided to investigate the interaction with its candidate protein-binding partner, the eIF3j subunit. Human eIF3j is a 35-kDa protein and is essential for the stable recruitment of the eIF3 complex to 40 S subunits (8). Recombinant eIF3j was expressed in E. coli and purified.…”

“…Among the components of eIF3, eIF3b is considered to be the major scaffolding subunit within the eIF3 complex, interacting with eIF3a (7), eIF3g, eIF3i, and eIF3j (8). The yeast homologue of the eIF3b subunit, PRT1, interacts with the yeast homologues of eIF3a (TIF32), eIF3g (TIF35), eIF3i (TIF34), eIF3j (HCR1), and eIF3e (Pci8) (9 -11), highlighting the conserved role of eIF3b in the structural organization of the eIF3 complex.…”