Translation initiation is a multistep process ensuring formation of the 80S elongation–competent complex composed of both ribosomal subunits with the P-site occupied by the methionyl initiator tRNA (Met-tRNAiMet
) base-paired with the mRNA's AUG start codon. In the first step, Met-tRNAiMet
is bound by eukaryotic initiation factor 2 (eIF2) in its GTP form to produce the ternary complex (TC). eIF3 together with eIFs 1, 1A and 5 then promotes TC recruitment to the small ribosomal subunit (40S) producing the 43S preinitiation complex (PIC). Subsequently, the 43S PIC interacts with the 5′-end of capped mRNA in a reaction promoted by eIF4F, eIF4B, PABP and eIF3 [reviewed in (1
)]. Thus formed 48S PIC then scans the mRNA leader until the AUG start codon is recognized. This step is controlled by GTP-hydrolysis on eIF2 stimulated by eIF5 and by the subsequent release of free Pi from the 48S PIC triggered by eIF1. The scanning-arrested 48S PIC can now join the large ribosomal subunit with the help of GTP-bound eIF5B, upon which most eIFs are ejected with the exception of eIF1A and eIF3 (2–4
). Finally, GTP hydrolysis on eIF5B triggers the release of eIF1A and eIF5B producing an active 80S ribosome poised for elongation.
One of the key facilitators of the eukaryotic initiation pathway is the multifunctional, multiprotein complex eIF3. Yeast S. cerevisiae
eIF3 is composed of 6 subunits (a/TIF32, b/PRT1, c/NIP1, i/TIF34, g/TIF35 and j/HCR1), all of which have corresponding orthologs in the 13-subunit mammalian eIF3 complex. Yeast eIF3, as part of a higher order ribosome-free structure called the multifactor complex (MFC) composed also of the TC and eIFs 1 and 5 (5
), was shown to enhance the efficiency of the 43S and 48S PIC assembly processes and also to stimulate the postassembly steps such as scanning and AUG recognition (6–15
). The importance of eIF3 as a pivotal coordinator of the PIC assembly most probably lies in the fact that whereas its body resides on the solvent-exposed side of the small ribosomal subunit, several of its flexible protein interaction domains are thought to reach out to the interface side (16–18
). Moreover, at least on the yeast 40S, the locations of both the mRNA entry and exit pores are thought to overlap with the eIF3 position. Thus, eIF3 is ideally suited for spatial distribution of other eIF3-interacting eIFs over the 40S surface as well as for mRNA loading.
To better understand the molecular details of the eIF3 role in the PIC assembly process, we have begun a systematic mapping of the positions of specific domains of various eIF3 subunits on the 40S (16
). We found that the N-terminal domain (NTD) of a/TIF32 forms a crucial intermolecular bridge between eIF3 and the 40S by interacting with small ribosomal protein RPS0 in the vicinity of the mRNA exit pore (3
). In addition, we observed that deleting the C-terminal domain (CTD) of a/TIF32 reduced the MFC association with the 40S when the connection between eIF3 and eIF5/TIF5 in the MFC was impaired by the tif5-7A
). Interestingly, the a/TIF32-CTD was found to interact with helices 16–18 of 18S rRNA (16
) and RPS2 and RPS3 (14
), all constituents of the mRNA entry channel (19
). Consistently, RPS2 also interacts with the CTD of j/HCR1; i.e. the direct binding partner of the a/TIF32-CTD (12
). The g/TIF35 subunit interacts with the 40S beak proteins RPS3 and RPS20 (13
); however, its contribution to the overall 40S-binding affinity of eIF3 is currently unknown. Conversely, the RNA recognition motif (RRM) of b/PRT1 plays a direct role in anchoring eIF3 to the ribosome (9
); however, its binding partner(s) remains to be identified. Finally, deletion of the C-terminal 240 residues of c/NIP1 completely eliminated binding of the mutant form of the complex to the 40S in vivo
when competing with the wild-type eIF3. This result suggested that the c/NIP1-CTD also represents an important intermolecular bridge between eIF3 and some component(s) of the small ribosome (16
Interestingly, the c/NIP1-CTD is formed by the bipartite PCI domain that is known to serve as the principal scaffold for the 26S proteasome lid, COP9 signalosome (CSN) and eIF3 [reviewed in (20
)]. The PCI domain is defined by a conserved arrangement of curved bihelical tetratricopeptide-like repeats followed by a globular winged helix (WH) subdomain (21
). Besides c/NIP1, yeast eIF3 contains only one additional PCI subunit in a/TIF32, whereas mammalian eIF3 is composed of a ‘canonical number’ of 6 PCI subunits (a, c, e, k, l, m). The PCI subunits are believed to regulate proper complex assembly via interactions between each other as well as with other protein partners. Despite the recent findings that several PCI proteins occur in complexes related to nucleic acid regulation (22
), they are not known to be capable of direct RNA binding.
Among all 33 small ribosomal proteins, there is one that deserves a special attention—the yeast ribosomal protein ASC1 and its mammalian ortholog RACK1. They are both members of the WD40 (Trp-Asp) repeat scaffold protein family that adopts a seven-bladed β-propeller structure. RACK1/ASC1 (designated as ASC1 thereafter) is located on the head of the 40S ribosomal subunit near the mRNA exit tunnel and makes extensive contacts with helices h39 and h40 of 18S rRNA and ribosomal proteins RPS16, 17, and 3 (19
). Importantly, ASC1 was also shown to interact with a number of signaling molecules on and off the ribosome and thus it is believed to play a crucial role in a multitude of biological processes and serve as a regulatory link between signaling and translation [reviewed in (24
)]. For example, RACK1 recruits activated protein kinase C to the ribosome, which leads to the stimulation of translation through the phosphorylation of initiation factor 6 (25
). In a ribosome-free form RACK1 associates with membrane-bound receptors (26
), indicating that it can promote the docking of ribosomes at sites where local translation is required, such as focal adhesions.
Despite being implicated in all these cellular processes, yeast ASC1
is not essential for growth. The asc1Δ
null strains were shown to impair cellular growth, to produce halfmer polysomes that could arise either from a defective subunit joining step or from a defect in 60S biogenesis, to impact translational rates in a transcript-specific manner, to deregulate GCN4
translational control under amino acid starvation and to increase sensitivity to drugs affecting cell wall biosynthesis (27–31
). The breadth of the observed phenotypes may seem consistent with the documented multitasking by RACK1; however, there is one caveat. The ASC1
gene contains an intron encoding a small nucleolar RNA U24 (SNR24
) that plays a critical role in biogenesis of the large ribosomal subunit (32
). Because all aforementioned asc1Δ
null strains were also deleted for SNR24
, it is unclear what phenotypes derive from the lack of what functional molecule and so what is the true contribution of the ASC1 protein to general translation.
In this study, we have focused on the role of the C-terminal PCI domain of the c/NIP1 subunit of eIF3 in promoting association of eIF3 and other MFC components with the 40S. We demonstrate that a short C-terminal deletion in nip1-Δ60 and a specific clustered alanine-scanning mutation (CAM) nip1–743A752 impinging into the WH subdomain of the PCI domain produce slow growth (Slg−) phenotypes and significantly reduce the amounts of 40S-bound MFC components in vivo, consistent with the idea that the c/NIP1-PCI forms an important intermolecular bridge between eIF3 and the 40S. Whereas the extreme C-terminal region of c/NIP1 was found to interact directly with blades 1–3 of ASC1, the PCI domain, the 3D structure of which was modeled in silico, shows strong but unspecific binding to RNA that is severely reduced by two of our CAM mutants. To our knowledge, this is the first report showing that the protein–protein interacting PCI domain is also capable of direct RNA binding. Finally, we demonstrate that the halfmer phenotype previously associated with the ASC1 deletion is in fact caused by deletion of its intron encoding U24, which has been previously accidentally overlooked. Importantly, however, the deletion of the only ASC1 coding region produced the Slg− phenotype and also reduced the eIF3-binding affinity toward the 40S subunit in vivo, ascribing RACK1/ASC1 the eIF3-docking role in general translation initiation. In addition, 40S-binding of eIF5 and the TC was also reduced, and, accordingly, the asc1Δ strain derepressed GCN4 translational control under non-starvation conditions producing the Gcd− phenotype suppressible by overexpressing TC. Together, we conclude that the c/NIP1-PCI domain promotes the PIC assembly process by linking eIF3 with the head region of the small ribosomal subunit.