Eukaryotic initiation factor 6, an evolutionarily conserved regulator of ribosome biogenesis and protein translation

We recently identified Receptor for Activated C Kinase 1 (RACK1) as one of the molecular links between abscisic acid (ABA) signaling and its regulation on protein translation. Moreover, we identified Eukaryotic Initiation Factor 6 (eIF6) as an interacting partner of RACK1. Because the interaction between RACK1 and eIF6 in mammalian cells is known to regulate the ribosome assembly step of protein translation initiation, it was hypothesized that the same process of protein translation in Arabidopsis is also regulated by RACK1 and eIF6. In this article, we analyzed the amino acid sequences of eIF6 in different species from different lineages and discovered some intriguing differences in protein phosphorylation sites that may contribute to its action in ribosome assembly and biogenesis. In addition, we discovered that, distinct from non-plant organisms in which eIF6 is encoded by a single gene, all sequenced plant genomes contain two or more copies of eIF6 genes. While one copy of plant eIF6 is expressed ubiquitously and might possess the conserved function in ribosome biogenesis and protein translation, the other copy seems to be only expressed in specific organs and therefore may have gained some new functions. We proposed some important studies that may help us better understand the function of eIF6 in plants.Key words: CK1, eIF6, PKC, protein translation, RACK1, ribosome assembly, ribosome biogenesisEukaryotic Initiation Factor 6 (eIF6) was originally purified from wheat germ¹ and was found to function as a ribosome dissociation factor through binding to the 60S ribosome subunit and preventing its association with the 40S ribosome subunit.² Its homologous proteins were later purified from rabbit reticulocyte lysates,³ calf liver⁴ and human cells.⁴ The action of eIF6 in preventing the ribosome subunits association was later found to involve another two proteins, the activated Protein Kinase C (PKC) and the Receptor for Activated C Kinase 1 (RACK1) in mammalian cells.⁵ PKC is a family of proteins that can be activated by elevated cellular concentration of Ca²⁺ or diacylglycerol and is involved in multiple signal transduction pathways in mammalian cells.⁶ RACK1 was identified as a receptor for activated PKC, anchoring PKC to the subcellular location where its substrate is present.^7,8 In this protein complex, RACK1 serves as a scaffold protein that simultaneously binds to eIF6 and activated PKC to bring these two proteins to close proximity. PKC then phosphorylates eIF6, leading to its dissociation from the 60S ribosome subunit and consequently allowing the association between the 40S and 60S ribosome subunits to assemble a functional 80S subunit to initiate protein translation⁵ (Fig. 1). Genetic studies supported the role of mammalian eIF6 in protein translation initiation as well as in cell growth.⁹ More recent structural studies supported a similar role of eIF6 in regulating 80S ribosome assembly in yeast.^10,11 Yeast eIF6 (Tif6p) was also known to regulate 60S ribosome biogenesis.^12,13 Very recently, eIF6 was identified as a component of a protein complex that interacts with the RNA-induced silencing complex and plays a role in microRNA-directed gene silencing.¹⁴ For a more comprehensive review of eIF6''s function in mammalian cells and in yeast, readers should refer to the following review article.¹⁵Open in a separate windowFigure 1A schematic presentation of the proposed molecular mode of action of eIF6, RACK1, PKC and CK1 in ribosome assembly and protein translation. Nuclear CK1 phosphorylates eIF6 at Serine 174 and Serine 175. This phosphorylation is required for the shuttling of eIF6 from nucleus into cytosol. eIF6 prevents joining of the cytosolic 60S ribosome subunit with the 40S subunit from forming a functional 80S ribosome. RACK1, via binding simultaneously to the eIF6 and the activated PKC, can facilitate the phosphorylation of eIF6 at Serine 235 by PKC. The Serine 235 phosphorylated eIF6 then disassociates from 60S ribosome, thus allowing the assembly of functional 80S ribosome and initiation of protein translation. CK1, Casein Kinase 1; 60S, 60S ribosome subunit; 40S, 40S ribosome subunit; eIF6, Eukaryotic Initiation Factor 6; RACK1, Receptor for Activated C Kinase 1; PKC, Protein Kinase C; pi, phosphate.Despite considerable progress that has been made in the identification of central components of plant hormone abscisic acid (ABA) signaling, little is known about the molecular mechanism of the long-recognized effect of ABA on protein translation. Our group has been working on the functional analysis of Arabidopsis RACK1 gene family,^16–19 and has identified RACK1 as a negative regulator of ABA responses.¹⁸ Recently, we discovered that RACK1 may play a role in ribosome assembly and 60S ribosome subunit biogenesis and therefore serve as one of the molecular links between ABA signaling and its control on protein translation.²⁰ In addition, we discovered that RACK1 physically interacts with eIF6 in a yeast two-hybrid assay and in a Bi-molecular Fluorescence Complementation assay in an Arabidopsis leaf mesophyll protoplast system. The conserved interaction between RACK1 and eIF6 in plants and in mammals implies an evolutionarily conserved role of eIF6 and RACK1 in ribosome biogenesis, assembly and protein translation.     

Hepatitis C virus internal ribosome entry site-dependent translation in Saccharomyces cerevisiae is independent of polypyrimidine tract-binding protein, poly(rC)-binding protein 2, and La protein

Translation initiation of some viral and cellular mRNAs occurs by ribosome binding to an internal ribosome entry site (IRES). Internal initiation mediated by the hepatitis C virus (HCV) IRES in Saccharomyces cerevisiae was shown by translation of the second open reading frame in a bicistronic mRNA. Introduction of a single base change in the HCV IRES, known to abrogate internal initiation in mammalian cells, abolished translation of the second open reading frame. Internal initiation mediated by the HCV IRES was independent of the nonsense-mediated decay pathway and the cap binding protein eIF4E, indicating that translation is not a result of mRNA degradation or 5'-end-dependent initiation. Human La protein binds the HCV IRES and is required for efficient internal initiation. Disruption of the S. cerevisiae genes that encode La protein orthologs and synthesis of wild-type human La protein in yeast had no effect on HCV IRES-dependent translation. Polypyrimidine tract-binding protein (Ptb) and poly-(rC)-binding protein 2 (Pcbp2), which may be required for HCV IRES-dependent initiation in mammalian cells, are not encoded within the S. cerevisiae genome. HCV IRES-dependent translation in S. cerevisiae was independent of human Pcbp2 protein and stimulated by the presence of human Ptb protein. These findings demonstrate that the genome of S. cerevisiae encodes all proteins necessary for internal initiation of translation mediated by the HCV IRES.     

Coupled release of eukaryotic translation initiation factors 5B and 1A from 80S ribosomes following subunit joining

The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.     

RACK1 mediates multiple hormone responsiveness and developmental processes in Arabidopsis

The scaffold protein RACK1 (Receptor for Activated C Kinase 1) serves as an integrative point for diverse signal transduction pathways. The Arabidopsis genome contains three RACK1 orthologues, however, little is known about their functions. It is reported here that one member of this gene family, RACK1A, previously identified as the Arabidopsis homologue of the tobacco arcA gene, mediates hormone responses and plays a regulatory role in multiple developmental processes. RACK1A expresses ubiquitously in Arabidopsis. Loss-of-function mutations in RACK1A confer defects in multiple developmental processes including seed germination, leaf production, and flowering. rack1a mutants displayed reduced sensitivity to gibberellin and brassinosteroid in seed germination, hypersensitivity to abscisic acid in seed germination and early seedling development, and hyposensitivity to auxin in adventitious and lateral root formation. These results provide the first genetic evidence that RACK1A is involved in multiple signal transduction pathways.     

Uncoupling of initiation factor eIF5B/IF2 GTPase and translational activities by mutations that lower ribosome affinity

Translation initiation factor eIF5B/IF2 is a GTPase that promotes ribosomal subunit joining. We show that eIF5B mutations in Switch I, an element conserved in all GTP binding domains, impair GTP hydrolysis and general translation but not eIF5B subunit joining function. Intragenic suppressors of the Switch I mutation restore general translation, but not eIF5B GTPase activity. These suppressor mutations reduce the ribosome affinity of eIF5B and increase AUG skipping/leaky scanning. The uncoupling of translation and eIF5B GTPase activity suggests a regulatory rather than mechanical function for eIF5B GTP hydrolysis in translation initiation. The translational defect suggests eIF5B stabilizes Met-tRNA(i)(Met) binding and that GTP hydrolysis by eIF5B is a checkpoint monitoring 80S ribosome assembly in the final step of translation initiation.     

The beta4 integrin interactor p27(BBP/eIF6) is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly

p27(BBP/eIF6) is an evolutionarily conserved protein that was originally identified as p27(BBP), an interactor of the cytoplasmic domain of integrin beta4 and, independently, as the putative translation initiation factor eIF6. To establish the in vivo function of p27(BBP/eIF6), its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing beta4 integrin, p27(BBP/eIF6) is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of beta4 integrin. Surprisingly, in the absence and in the presence of the beta4 integrin subunit, p27(BBP/eIF6) is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27(BBP/eIF6) homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit. Furthermore, human p27(BBP/eIF6) can rescue the lethal effect of the iihDelta yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27(BBP/eIF6) in ribosome biogenesis or assembly rather than in translation. A further function related to the beta4 integrin subunit may have evolved specifically in higher eukaryotic cells.     

Functional equivalence of translation factor eIF5B from Candida albicans and Saccharomyces cerevisiae

Eukaryotic translation initiation factor 5B (eIF5B) plays a role in recognition of the AUG codon in conjunction with translation factor eIF2, and promotes joining of the 60S ribosomal subunit. To see whether the eIF5B proteins of other organisms function in Saccharomyces cerevisiae, we cloned the corresponding genes from Oryza sativa, Arabidopsis thaliana, Aspergillus nidulans and Candida albican and expressed them under the control of the galactose-inducible GAL promoter in the fun12Delta strain of Saccharomyces cerevisiae. Expression of Candida albicans eIF5B complemented the slow-growth phenotype of the fun12Delta strain, but that of Aspergillus nidulance did not, despite the fact that its protein was expressed better than that of Candida albicans. The Arabidopsis thaliana protein was also not functional in Saccharomyces. These results reveal that the eIF5B in Candida albicans has a close functional relationship with that of Sacharomyces cerevisiae, as also shown by a phylogenetic analysis based on the amino acid sequences of the eIF5Bs.     

Identification of RCN1 and RSA3 as ethanol-tolerant genes in Saccharomyces cerevisiae using a high copy barcoded library

Saccharomyces cerevisiae (S.?cerevisiae) encounters a multitude of stresses during industrial processes such as wine fermentation including ethanol toxicity. High levels of ethanol reduce the viability of yeast and may prevent completion of fermentation. The identification of ethanol-tolerant genes is important for creating stress-resistant industrial yeast, and S.?cerevisiae genomic resources have been utilized for this purpose. We have employed a molecular barcoded yeast open reading frame (MoBY-ORF) high copy plasmid library to identify ethanol-tolerant genes in both the S.?cerevisiae S288C laboratory and M2 wine strains. We find that increased dosage of either RCN1 or RSA3 improves tolerance of S288C and M2 to toxic levels of ethanol. RCN1 is a regulator of calcineurin, whereas RSA3 has a role in ribosome maturation. Additional fitness advantages conferred upon overproduction of RCN1 and RSA3 include increased resistance to cell wall degradation, heat, osmotic and oxidative stress. We find that the M2 wine yeast strain is generally more tolerant of stress than S288C with the exception of translation inhibition, which affects M2 growth more severely than S288C. We conclude that regulation of ribosome biogenesis and ultimately translation is a critical factor for S.?cerevisiae survival during industrial-related environmental stress.     

Translational control by 80S formation and 60S availability: The central role of eIF6, a rate limiting factor in cell cycle progression and tumorigenesis

Ribosome biogenesis and translation can be simplified as the processes of generating ribosomes and their use for decoding mRNA into a protein. Ribosome biogenesis has been efficiently studied in unicellular organisms like the budding yeast, allowing us a deep and basic knowledge of this process in growing cells. Translation has been modeled in vitro and in unicellular organisms. These studies have given us an important insight into the mechanisms and evolutionarily conserved aspects of ribosome biology. However, we advocate the need of the direct study of these processes in multicellular organisms. Analysis of ribosome biogenesis and translation in vivo in Metazoa and mammalian models is emerging and unveils the unexpected consequences of perturbed ribosome biogenesis and translation. Here, we will describe how one factor, eIF6, plays a crucial role both in the generation of the large ribosomal subunit and its availability for translation. From there, we will make specific conclusions on the physiological relevance of eIF6 in 80S formation, cell cycle progression and disease, raising the point that the control of gene expression may occur at the unexpected level of the large ribosomal subunit. In the future, the modulation of eIF6 binding to the 60S may be pharmacologically exploited to reduce the growth of cancer cells or ameliorate the phenotype of SDS syndrome.     

A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry

Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotide-long 5' untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translation systems. Here, the mechanism of action of the RhPV IRES has been characterized by reconstitution of mammalian 48S initiation complexes on the IRES from purified components combined with the toeprint assay. There is an absolute requirement for the initiation factors eIF2 and eIF3 and the scanning factor eIF1 to form 48S complexes on the IRES. In addition, eIF1A, eIF4F (or the C-terminal fragment of eIF4G), and eIF4A strongly stimulated the assembly of this complex, whereas eIF4B had no effect. Although the eIF4-dependent pathway is dominant in the RhPV IRES-directed cell-free translation, omission of either eIF4G or eIF4A or both still allowed the assembly of 48S complexes from purified components with approximately 23% of maximum efficiency. Deletions of up to 100 nucleotides throughout the 5'-UTR sequence produced at most a marginal effect on the IRES activity, suggesting the absence of specific binding sites for initiation factors. Only deletion of the U-rich unstructured 380-nucleotide region proximal to the initiation codon resulted in a complete loss of the IRES activity. We suggest that the single-stranded nature of the RhPV IRES accounts for its strong but less selective potential to bind key mRNA recruiting components of the translation initiation apparatus from diverse origins.     

Plant initiation factor 3 subunit composition resembles mammalian initiation factor 3 and has a novel subunit

Eukaryotic initiation factor 3 (eIF3) is a multisubunit complex that is required for binding of mRNA to 40 S ribosomal subunits, stabilization of ternary complex binding to 40 S subunits, and dissociation of 40 and 60 S subunits. These functions and the complex nature of eIF3 suggest multiple interactions with many components of the translational machinery. Recently, the subunits of mammalian and Saccharomyces cerevisiae eIF3 were identified, and substantial differences in the subunit composition of mammalian and S. cerevisiae were observed. Mammalian eIF3 consists of 11 nonidentical subunits, whereas S. cerevisiae eIF3 consists of up to eight nonidentical subunits. Only five of the subunits of mammalian and S. cerevisiae are shared in common, and these five subunits comprise a "core" complex in S. cerevisiae. eIF3 from wheat consists of at least 10 subunits, but their relationship to either the mammalian or S. cerevisiae eIF3 subunits is unknown. Peptide sequences derived from purified wheat eIF3 subunits were used to correlate each subunit with mammalian and/or S. cerevisiae subunits. The peptide sequences were also used to identify Arabidopsis thaliana cDNAs for each of the eIF3 subunits. We report seven new cDNAs for A. thaliana eIF3 subunits. A. thaliana eIF3 was purified and characterized to confirm that the subunit composition and activity of wheat and A. thaliana eIF3 were similar. We report that plant eIF3 closely resembles the subunit composition of mammalian eIF3, having 10 out of 11 subunits in common. Further, we find a novel subunit in the plant eIF3 complex not present in either mammalian or S. cerevisiae eIF3. These results suggest that plant and mammalian eIF3 evolved similarly, whereas S. cerevisiae has diverged.     

Fission yeast receptor of activated C kinase (RACK1) ortholog Cpc2 regulates mitotic commitment through Wee1 kinase

In the fission yeast Schizosaccharomyces pombe, Wee1-dependent inhibitory phosphorylation of the highly conserved Cdc2/Cdk1 kinase determines the mitotic onset when cells have reached a defined size. The receptor of activated C kinase (RACK1) is a scaffolding protein strongly conserved among eukaryotes which binds to other proteins to regulate multiple processes in mammalian cells, including the modulation of cell cycle progression during G(1)/S transition. We have recently described that Cpc2, the fission yeast ortholog to RACK1, controls from the ribosome the activation of MAPK cascades and the cellular defense against oxidative stress by positively regulating the translation of specific genes whose products participate in the above processes. Intriguingly, mutants lacking Cpc2 display an increased cell size at division, suggesting the existence of a specific cell cycle defect at the G(2)/M transition. In this work we show that protein levels of Wee1 mitotic inhibitor are increased in cells devoid of Cpc2, whereas the levels of Cdr2, a Wee1 inhibitor, are down-regulated in the above mutant. On the contrary, the kinetics of G(1)/S transition was virtually identical both in control and Cpc2-less strains. Thus, our results suggest that in fission yeast Cpc2/RACK1 positively regulates from the ribosome the mitotic onset by modulating both the protein levels and the activity of Wee1. This novel mechanism of translational control of cell cycle progression might be conserved in higher eukaryotes.     

Composition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals

We have conducted a proteomic analysis of the 80S cytosolic ribosome from the eukaryotic green alga Chlamydomonas reinhardtii, and accompany this with a cryo-electron microscopy structure of the ribosome. Proteins homologous to all but one rat 40S subunit protein, including a homolog of RACK1, and all but three rat 60S subunit proteins were identified as components of the C. reinhardtii ribosome. Expressed Sequence Tag (EST) evidence and annotation of the completed C. reinhardtii genome identified genes for each of the four proteins not identified by proteomic analysis, showing that algae potentially have a complete set of orthologs to mammalian 80S ribosomal proteins. Presented at 25A, the algal 80S ribosome is very similar in structure to the yeast 80S ribosome, with only minor distinguishable differences. These data show that, although separated by billions of years of evolution, cytosolic ribosomes from photosynthetic organisms are highly conserved with their yeast and animal counterparts.     

Bovine adenovirus‐3 protein VIII associates with eukaryotic initiation factor‐6 during infection

Adenovirus protein VIII appears to connect core with the inner surface of the adenovirus capsid. Because protein–protein interactions are central to virus replication, identification of proteins interacting with protein VIII may help in understanding their role in adenovirus infection. Our yeast 2‐hybrid assay indicated that protein VIII interacts with eukaryotic initiation factor 6 (eIF6). These findings were confirmed by Glutathione S‐transferase‐pull down assay, bimolecular fluorescent complementation assay, and coimmunoprecipitation assay in plasmid DNA transfected and bovine adenovirus‐3 (BAdV‐3) infected cells. The C‐terminus amino acids 147 to 174 of protein VIII and N‐terminus amino acids 44 to 97 of eIF6 are involved in these interactions. Polysome analysis demonstrated increased level of 60S ribosomal subunit and decreased level of 80S complex in protein VIII expressing cells or BAdV‐3 infected cells. Our results suggest that formation of functional 80S ribosome appears impaired in the presence of protein VIII at late times post infection. We speculate that this impaired ribosome assembly may be responsible for the inhibition of cellular mRNA translation observed late in adenovirus infected cells. Moreover, analysis of recombinant BAdV‐3 expressing mutant protein VIII (deletion of eIF6 interacting domain) suggests that interaction of protein VIII and eIF6 may help in preferential translation of adenovirus genes during late phase of adenovirus infection.     

Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast

Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo.     

Translation initiation factor eIF4G-1 binds to eIF3 through the eIF3e subunit

eIF3 in mammals is the largest translation initiation factor ( approximately 800 kDa) and is composed of 13 nonidentical subunits designated eIF3a-m. The role of mammalian eIF3 in assembly of the 48 S complex occurs through high affinity binding to eIF4G. Interactions of eIF4G with eIF4E, eIF4A, eIF3, poly(A)-binding protein, and Mnk1/2 have been mapped to discrete domains on eIF4G, and conversely, the eIF4G-binding sites on all but one of these ligands have been determined. The only eIF4G ligand for which this has not been determined is eIF3. In this study, we have sought to identify the mammalian eIF3 subunit(s) that directly interact(s) with eIF4G. Established procedures for detecting protein-protein interactions gave ambiguous results. However, binding of partially proteolyzed HeLa eIF3 to the eIF3-binding domain of human eIF4G-1, followed by high throughput analysis of mass spectrometric data with a novel peptide matching algorithm, identified a single subunit, eIF3e (p48/Int-6). In addition, recombinant FLAG-eIF3e specifically competed with HeLa eIF3 for binding to eIF4G in vitro. Adding FLAG-eIF3e to a cell-free translation system (i) inhibited protein synthesis, (ii) caused a shift of mRNA from heavy to light polysomes, (iii) inhibited cap-dependent translation more severely than translation dependent on the HCV or CSFV internal ribosome entry sites, which do not require eIF4G, and (iv) caused a dramatic loss of eIF4G and eIF2alpha from complexes sedimenting at approximately 40 S. These data suggest a specific, direct, and functional interaction of eIF3e with eIF4G during the process of cap-dependent translation initiation, although they do not rule out participation of other eIF3 subunits.     

Regulation of eukaryotic translation by the RACK1 protein: a platform for signalling molecules on the ribosome

The receptor for activated C-kinase (RACK1) is a scaffold protein that is able to interact simultaneously with several signalling molecules. It binds to protein kinases and membrane-bound receptors in a regulated fashion. Interestingly, RACK1 is also a constituent of the eukaryotic ribosome, and a recent cryo-electron microscopy study localized it to the head region of the 40S subunit in the vicinity of the messenger RNA (mRNA) exit channel. RACK1 recruits activated protein kinase C to the ribosome, which leads to the stimulation of translation through the phosphorylation of initiation factor 6 and, potentially, of mRNA-associated proteins. RACK1 therefore links signal-transduction pathways directly to the ribosome, which allows translation to be regulated in response to cell stimuli. In addition, the fact that RACK1 associates with membrane-bound receptors indicates that it promotes the docking of ribosomes at sites where local translation is required, such as focal adhesions.