Translation initiation in eukaryotes depends on the interplay of a large number of eukaryotic initiation factors (eIFs). At least twelve different eIFs form a dynamic network which stimulates recruitment of the initiator Met-tRNAiMet and mRNA to the 40S ribosomal subunit, and scanning of the mRNA until the first AUG start codon is reached (for review, see [69]). Regulating initiation, eIFs are the focal point of translational control of gene expression in response to stress, hormones and developmental regulatory factors [70, 71]. The largest eukaryotic initiation factor, eIF3, was one of the first initiation factors to be identified. Mammalian eIF3 is comprised of 13 subunits which are designated eIF3a-m [72-77]. The molecular architecture of the budding yeast eIF3 is much simpler, it contains only five orthologs of five mammalian eIF3 subunits eIF3a, eIF3b, eIF3c, eIF3g and eIF3i [78]. All five subunits of the yeast eIF3 complex have been found to be essential for translation in vivo [79-85]. Remaining associated to the 40S ribosomal subunit (RSU) throughout translation initiation, eIF3 forms the scaffold for the interactions of the 40S RSU and the eIFs. Current models assume most of the reactions in the initiation pathway to be stimulated by eIF3. First, the assembly of the eIF2●GTP●Met-tRNAiMet
ternary complex (TC) and its binding to the free 40S RSU is stimulated by eIF1 and eIF1A, eIF3 and eIF5, yielding the 43S pre-inititation complex (PIC) [86-90]. Recruitment of mRNA bound to the cap-binding protein eIF4E and its interaction partners in the eIF4 complex as well as the poly(A)-binding protein (PABP) is stimulated by interactions between eIF4A and the 43S PIC, yielding the 48S PIC. In mammals, interactions between eIF3 and eIF4G have been reported to be involved in this step [91-94]. In the subsequent scanning for the first AUG codon, eIF5 stimulates GTP hydrolysis by the TC while eIF1 promotes scanning [95] and prevents the release of inorganic phosphate from the eIF2●GDP●Pi complex at non-AUG codons [96]. Both eIF1 and eIF5 interact with eIF3 [72, 78, 79, 97], mutations in the N-terminal domain of eIF3c in yeast have been reported to alter the specificity of AUG scanning [98]. In mammals, eIF3 and the eIF4F●m7GTP complex seem to confine eIF5-dependent GTP hydrolysis in the 48S PIC in absence of an AUG start codon at the P-site [99]. Owing to its implications in scanning and AUG recognition, eIF3 is believed to function in the reinitiation of GCN4 and other gene-specific translational control mechanisms. As an example, eIF3 seems to be remaining bound to the 60S RSU via a binding of eIF3g to the ribosomal protein L18 which is mediated by the transactivator protein TAV of plant caulimoviruses [100, 101]. In this way, TAV●eIF3 might be
transferred back to the 40S RSU during translation termination and release of the 60S RSU. The immediate back transfer would enable it to regenerate a PIC which is capable of scanning the mRNA to the next AUG, a process during which TC is rebound [100, 101].
Recently, eIF3 has been proposed to provide a regulation point of translation initiation by acting as a docking site to the mammalian target of rapamycin (mTOR), a protein kinase.
Being recruited to eIF3, mTOR phosphorylates and activates S6 kinase 1 (S6K1) as a
Figure 11 Eukaryotic Cap-Dependent Translation Initiation and Its Regulation. eIFs 1, 1A, and 3 promote dissociation of 80S ribosomes and, together with eIF5 and TC (eIF2•GTP•Met-tRNAi), assemble the 43S PIC. In yeast, these eIFs form a multifactor complex (MFC), which could bind 40S subunits as a unit.
mRNA is activated by binding of eIF4F (eIF4E•eIF4G•eIF4A) to the cap and PABP to the poly(A) tail, circularizing the mRNA. The 43S PIC binds near the cap, facilitated by eIF3/eIF5 interactions with eIF4G/eIF4B, and scans the leader for AUG in an ATP-dependent (and possibly DED1-stimulated) reaction, with partial hydrolysis of the eIF2-bound GTP in the TC to eIF2•GDP•Pi. AUG recognition triggers eIF1 dissociation from the 40S platform (not depicted), allowing release of Pi and eIF2•GDP. Joining of the 60S subunits, with release of other eIFs, is catalyzed by eIF5B-GTP, and GTP hydrolysis triggers release of eIF5B•GDP and eIF1A, to yield the final 80S initiation complex. Under stress or starvation conditions, TC formation is reduced by eIF2α phosphorylation and eIF4F assembly is blocked by 4E-BP binding to eIF4E.
Phosphorylation by mTOR dissociates 4E-BP from eIF4E. mTOR also promotes eIF4G and eIF4B phosphorylation either directly or via S6Ks. Mitogens and growth factors promote these phosphorylation events by activating mTOR via PI3K/Akt signaling or RAS/MAPK signaling. Reprinted with permission from [102].
response to adequate nutrient levels or insulin signalling. Being phosphorylated, S6K1 is released from the PIC and in turn phosphorylates ribosomal protein S6 and eIF4B which promotes eIFB4 recruitment and translation [103].
Generally, the eIF3 complex does not seem to have intrinsic catalytic activity and might not directly interact with tRNAiMet
or decoding sites on the ribosome [69]. Yet it is versatile in functionality throughout the process of translational initiation. It is currently believed that eIF3 acts as a scaffold for recruitment and organization for several proteins and protein complexes during translation initiation [69]. A cryo-EM reconstruction of human eIF3 and eIF3 interacting with the internal ribosomal entry site (IRES) of the hepatitis C virus (HCV) has been used to model the position of eIF3 on the 40S RSU [104]
based on the position of the IRES determined independently in previous experiments [105]. In this model, human eIF3 binds on the solvent side of the 40S RSU [104]. The binding of mammalian eIF3 to the solvent side of the 40S RSU has previously been reported from low-resolution electron microscopy studies of a mammalian 40S●eIF3 complex [106]. In the binding model of mammalian eIF3, an arm-like feature of eIF3 is suggested to cover the small subunit protein S15/rpS13 [104]. An interaction between S15/rp13 to helix 34 (h34) of the large ribosomal subunit is one of two intersubunit contacts which have been reported to be critical for the assembly of the 80S ribosome [64, 107, 108], suggesting that eIF3 to inhibit intersubunit joining by covering the relevant binding site. In yeast, deletion studies have shown that binding of eIF3 to the 40S RSU is impaired by deleting the N-terminal or C-terminal domains of eIF3c and the N-terminal domain of eIF3a, even if the other components of eIF3 are intact [69]. It has been further shown that a subcomplex consisting of the N-terminal half of eIF3a, eIF3c and eIF5, which binds to the N-terminal domain of eIF3c, can bind the 40S RSU in vitro and in vivo.
Together with experiments showing that the C-terminal domain of eIF3a is required for binding if the eIF3-eIF5 connection is disrupted by mutation [69], these findings support the notion that the terminal and C-terminal domains of eIF3c together with the N-terminal domain of eIF3a are among regular binding sites in the interaction of yeast eIF3 and the 40S RSU. Taken together, there might be a multiplicity of binding sites which are accounting for a stable and functional interaction of eIF3, the 40S RSU and other factors.
Generally, little is known about the spatial distribution of subunits of both yeast and mammalian eIF3. Knowledge on locations of the subunits within the overall complexes is a prerequisite for the further understanding of structure and function of eIF3.