Archaeal translation initiation factor aIF2 can substitute for eukaryotic eIF2 in ribosomal scanning during mammalian 48S complex formation

Heterotrimeric translation initiation factor (IF) a/eIF2 (archaeal/eukaryotic IF 2) is present in both Eukarya and Archaea. Despite strong structural similarity between a/eIF2 orthologs from the two domains of life, their functional relationship is obscure. Here, we show that aIF2 from Sulfolobus solfataricus can substitute for its mammalian counterpart in the reconstitution of eukaryotic 48S initiation complexes from purified components. aIF2 is able to correctly place the initiator Met-tRNA_i into the P-site of the 40S ribosomal subunit and accompany the entire set of eukaryotic translation IFs in the process of cap-dependent scanning and AUG codon selection. However, it seems to be unable to participate in the following step of ribosomal subunit joining. In accordance with this, aIF2 inhibits rather than stimulates protein synthesis in mammalian cell-free system. The ability of recombinant aIF2 protein to direct ribosomal scanning suggests that some archaeal mRNAs may utilize this mechanism during translation initiation.     

The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity

The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. Here we demonstrate that the p90 ribosomal protein S6 kinase (RSK) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3.     

Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and implications for ribosome evolution

Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter thermautotrophicus in complex with archaeal IF6 at 6.6?? resolution using cryo-electron microscopy (EM). The structure provides detailed architectural insights into the 50S ribosomal subunit from a methanogenic archaeon through identification of the rRNA expansion segments and ribosomal proteins that are shared between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea.     

Archaeal aIF2B Interacts with Eukaryotic Translation Initiation Factors eIF2α and eIF2Bα: Implications for aIF2B Function and eIF2B Regulation

Translation initiation is down-regulated in eukaryotes by phosphorylation of the α-subunit of eIF2 (eukaryotic initiation factor 2), which inhibits its guanine nucleotide exchange factor, eIF2B. The N-terminal S1 domain of phosphorylated eIF2α interacts with a subcomplex of eIF2B formed by the three regulatory subunits α/GCN3, β/GCD7, and δ/GCD2, blocking the GDP-GTP exchange activity of the catalytic ?-subunit of eIF2B. These regulatory subunits have related sequences and have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO₂ fixation. Our sequence analyses however predicted that members of one phylogenetically distinct and coherent group of these archaeal proteins [designated aIF2Bs (archaeal initiation factor 2Bs)] are functional homologs of the α, β, and δ subunits of eIF2B. Three of these proteins, from different archaea, have been shown to bind in vitro to the α-subunit of the archaeal aIF2 from the cognate archaeon. In one case, the aIF2B protein was shown further to bind to the S1 domain of the α-subunit of yeast eIF2 in vitro and to interact with eIF2Bα/GCN3 in vivo in yeast. The aIF2B-eIF2α interaction was however independent of eIF2α phosphorylation. Mass spectrometry has identified several proteins that co-purify with aIF2B from Thermococcus kodakaraensis, and these include aIF2α, a sugar-phosphate nucleotidyltransferase with sequence similarity to eIF2B?, and several large-subunit (50S) ribosomal proteins. Based on this evidence that aIF2B has functions in common with eIF2B, the crystal structure established for an aIF2B was used to construct a model of the eIF2B regulatory subcomplex. In this model, the evolutionarily conserved regions and sites of regulatory mutations in the three eIF2B subunits in yeast are juxtaposed in one continuous binding surface for phosphorylated eIF2α.     

HCV and CSFV IRES domain II mediate eIF2 release during 80S ribosome assembly

Internal ribosome entry site (IRES) RNAs from the hepatitis C virus (HCV) and classical swine fever virus (CSFV) coordinate cap-independent assembly of eukaryotic 48S initiation complexes, consisting of the 40S ribosomal subunit, eukaryotic initiation factor (eIF) 3 and the eIF2/GTP/Met-tRNA(i)(Met) ternary complex. Here, we report that these IRESes also play a functional role during 80S ribosome assembly downstream of 48S complex formation, in promoting eIF5-induced GTP hydrolysis and eIF2/GDP release from the initiation complex. We show that this function is encoded in their independently folded IRES domain II and that it depends both on its characteristic bent conformation and two conserved RNA motifs, an apical hairpin loop and a loop E. Our data suggest a general mode of subunit joining in HCV and HCV-like IRESes.     

A central fragment of ribosomal protein S26 containing the eukaryote-specific motif YxxPKxYxK is a key component of the ribosomal binding site of mRNA region 5' of the E site codon

The eukaryotic ribosomal protein S26e (rpS26e) lacking eubacterial counterparts is a key component of the ribosomal binding site of mRNA region 5' of the codon positioned at the exit site. Here, we determined the rpS26e oligopeptide neighboring mRNA on the human 80S ribosome using mRNA analogues bearing perfluorophenyl azide-derivatized nucleotides at designed locations. The protein was cross-linked to mRNA analogues in specific ribosomal complexes, in which the derivatized nucleotide was located at positions -3 to -9. Digestion of cross-linked rpS26e with various specific proteolytic agents followed by identification of the resulting modified oligopeptides made it possible to map the cross-links to fragment 60-71. This fragment contains the motif YxxPKxYxK conserved in eukaryotic but not in archaeal rpS26e. Analysis of X-ray structure of the Tetrahymena thermophila 40S subunit showed that this motif is not implicated in the intraribosomal interactions, implying its involvement in translation process in a eukaryote-specific manner. Comparison of the results obtained with data on positioning of ribosomal ligands on the 40S subunit lead us to suggest that this motif is involved in interaction with both the 5'-untranslated region of mRNA and the initiation factor eIF3 specific for eukaryotes, providing new insights into molecular mechanisms of translation in eukaryotes.     

Crystal structure of human eIF3k, the first structure of eIF3 subunits

eIF3k, the smallest subunit of eukaryotic initiation factor 3 (eIF3), interacts with several other subunits of eIF3 and the 40 S ribosomal subunit. eIF3k is conserved among high eukaryotes, including mammals, insects, and plants, and it is ubiquitously expressed in human tissues. Interestingly, eIF3k does not exist in some species of yeast. Thus, eIF3k may play a unique regulatory role in higher organisms. Here we report the crystal structure of human eIF3k, the first high-resolution structure of an eIF3 component. This novel structure contains two distinct domains, a HEAT (named for Huntington, elongation factor 3, A subunit of protein phosphatase 2A, target of rapamycin) repeat-like HAM (HEAT analogous motif) domain and a winged-helix-like WH domain. Through structural comparison and sequence conservation analysis, we show that eIF3k has three putative protein-binding surfaces and has potential RNA binding activity. The structure provides key information for understanding the structure and function of the eIF3 complex.     

The archaeal eIF2 homologue: functional properties of an ancient translation initiation factor

The eukaryotic translation initiation factor 2 (eIF2) is pivotal for delivery of the initiator tRNA (tRNAi) to the ribosome. Here, we report the functional characterization of the archaeal homologue, a/eIF2. We have cloned the genes encoding the three subunits of a/eIF2 from the thermophilic archaeon Sulfolobus solfataricus, and have assayed the activities of the purified recombinant proteins in vitro. We demonstrate that the trimeric factor reconstituted from the recombinant polypeptides has properties similar to those of its eukaryal homologue: it interacts with GTP and Met-tRNAi, and stimulates binding of the latter to the small ribosomal subunit. However, the archaeal protein differs in some functional aspects from its eukaryal counterpart. In contrast to eIF2, a/eIF2 has similar affinities for GDP and GTP, and the β-subunit does not contribute to tRNAi binding. The detailed analysis of the complete trimer and of its isolated subunits is discussed in light of the evolutionary history of the eIF2-like proteins.     

Translation driven by an eIF4G core domain in vivo.

Most eukaryotic mRNAs possess a 5' cap structure (m(7)GpppN) and a 3' poly(A) tail which promote translation initiation by binding the eukaryotic translation initiation factor (eIF)4E and the poly(A) binding protein (PABP), respectively. eIF4G can bridge between eIF4E and PABP, and-through eIF3-is thought to establish a link to the small ribosomal subunit. We fused the C-terminal region of human eIF4GI lacking both the eIF4E- and PABP-binding sites, to the IRE binding protein IRP-1. This chimeric protein suffices to direct the translation of the downstream cistron of bicistronic mRNAs bearing IREs in their intercistronic space in vivo. This function is preserved even when translation via the 5' end is inhibited. Deletion analysis defined the conserved central domain (amino acids 642-1091) of eIF4G as an autonomous 'ribosome recruitment core' and implicated eIF4A as a critical binding partner. Our data reveal the sufficiency of the conserved eIF4G ribosome recruitment core to drive productive mRNA translation in living cells. The C-terminal third of eIF4G is dispensable, and may serve as a regulatory domain.     

Role of aIF5B in archaeal translation initiation

In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. In eukaryotes, the role of eIF5B in ribosomal subunit joining is established and structural data showing eIF5B bound to the full ribosome were obtained. To achieve its function, eIF5B collaborates with eIF1A. However, structural data illustrating how these two factors interact on the small ribosomal subunit have long been awaited. The role of the archaeal counterparts, aIF5B and aIF1A, remains to be extensively addressed. Here, we study the late steps of Pyrococcus abyssi translation initiation. Using in vitro reconstituted initiation complexes and light scattering, we show that aIF5B bound to GTP accelerates subunit joining without the need for GTP hydrolysis. We report the crystallographic structures of aIF5B bound to GDP and GTP and analyze domain movements associated to these two nucleotide states. Finally, we present the cryo-EM structure of an initiation complex containing 30S bound to mRNA, Met-tRNA_i^Met, aIF5B and aIF1A at 2.7 Å resolution. Structural data shows how archaeal 5B and 1A factors cooperate to induce a conformation of the initiator tRNA favorable to subunit joining. Archaeal and eukaryotic features of late steps of translation initiation are discussed.     

Viral stress-inducible protein p56 inhibits translation by blocking the interaction of eIF3 with the ternary complex eIF2.GTP.Met-tRNAi

Viral stress-inducible protein p56 is produced in response to viral stress-inducing agents such as double-stranded RNA and interferon, as well as other poorly understood mechanisms of viral infection. It has been shown previously that p56 is able to bind the eukaryotic initiation factor 3e(eIF3e) (p48/Int-6) subunit of the eukaryotic translation initiation factor eIF3 and function as an inhibitor of translation in vitro and in vivo. The exact mechanism by which p56 is able to interfere with protein synthesis is not understood. Based on the known roles of eIF3 in the initiation pathway, we employed assays designed to individually look at specific functions of eIF3 and the effect of p56 on these eIF3-mediated functions. These assays examined the effect of p56 on ribosome dissociation, the eIF3.eIF4F interaction, and enhancement of the ternary complex eIF2.GTP.Met-tRNAi formation. Here we report that p56 is able to inhibit translation initiation specifically at the level of eIF3.ternary complex formation. The effect of p56-mediated inhibition was also examined in two different contexts, cap-mediated and encephalomyocarditis virus internal ribosomal entry site-mediated translation. Whereas cap-dependent initiation was severely inhibited by p56, internal ribosomal entry site-mediated translation appeared to be insensitive to p56.     

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

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.     

Structural dynamics of the yeast Shwachman-Diamond syndrome protein (Sdo1) on the ribosome and its implication in the 60S subunit maturation

The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast. Our data show that Sdo1p interacts tightly with the mature 60S subunit in vitro through its domain I and II, and is capable of bridging two 60S subunits to form a stable 2:2 dimer. Structural analysis indicates that Sdo1p bind to the ribosomal P-site, in the proximity of uL16 and uL5, and with direct contact to H69 and H38. The dynamic nature of Sdo1p on the 60S subunit, together with its strategic binding position, suggests a surveillance role of Sdo1p in monitoring the conformational maturation of the ribosomal P-site. Altogether, our data support a conformational signal-relay cascade during late-stage 60S maturation, involving uL16, Sdo1p, and Efl1p, which interrogates the functional P-site to control the departure of the anti-association factor eIF6.     

rRNA Suppressor of a Eukaryotic Translation Initiation Factor 5B/Initiation Factor 2 Mutant Reveals a Binding Site for Translational GTPases on the Small Ribosomal Subunit

The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.     

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.     

Cloning and characterization of the p42 subunit of mammalian translation initiation factor 3 (eIF3): demonstration that eIF3 interacts with eIF5 in mammalian cells.

Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit protein complex that plays an essential role in the binding of the initiator methionyl-tRNA and mRNA to the 40S ribosomal subunit to form the 40S initiation complex. cDNAs encoding all the subunits of mammalian eIF3 except the p42 subunit have been cloned in several laboratories. Here we report the cloning and characterization of a human cDNA encoding the p42 subunit of mammalian eIF3. The open reading frame of the cDNA, which encodes a protein of 320 amino acids (calculated Mr35 614) has been expressed in Escherichia coli and the recombinant protein has been purified to homogeneity. The purified protein binds RNA in agreement with the presence of a putative RNA binding motif in the deduced amino acid sequence. The protein shows 33% identity and 53% similarity with the Tif35p subunit (YDR 429C) of yeast eIF3. Transfection experiments demonstrated that polyhistidine-tagged p42 protein, transiently expressed in human U20S cells, was incorporated into endogenous eIF3. Furthermore, eIF3 isolated from transfected cell lysates contains bound eIF5 indicating that a specific physical interaction between eIF5 and eIF3 may play an important role in the function of eIF5 during translation initiation in eukaryotic cells.     

Function and ribosomal localization of aIF6, a translational regulator shared by archaea and eukarya

The translation factor IF6 is shared by the Archaea and the Eukarya, but is not found in Bacteria. The properties of eukaryal IF6 (eIF6) have been extensively studied, but remain somewhat elusive. eIF6 behaves as a ribosome-anti-association factor and is involved in miRNA-mediated gene silencing; however, it also seems to participate in ribosome synthesis and export. Here we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homologue (aIF6). We find that aIF6 binds specifically to the 50S ribosomal subunits, hindering the formation of 70S ribosomes and strongly inhibiting translation. aIF6 is uniformly expressed along the cell cycle, but it is upregulated following both cold- and heat shock. The aIF6 ribosomal binding site lies in the middle of the 30-S interacting surface of the 50S subunit, including a number of critical RNA and protein determinants involved in subunit association. The data suggest that the IF6 protein evolved in the archaeal–eukaryal lineage to modulate translational efficiency under unfavourable environmental conditions, perhaps acquiring additional functions during eukaryotic evolution.     

Control of Paip1-Eukayrotic Translation Initiation Factor 3 Interaction by Amino Acids through S6 Kinase

The simultaneous interaction of poly(A)-binding protein (PABP) with eukaryotic translation initiation factor 4G (eIF4G) and the mRNA 3′ poly(A) tail promotes translation initiation. We previously showed that the interaction of PABP-interacting protein 1 (Paip1) with PABP and eukaryotic translation initiation factor 3 (eIF3; via the eIF3g subunit) further stimulates translation. Here, we demonstrate that the interaction of eIF3 with Paip1 is regulated by amino acids through the mTORC1 signaling pathway. The Paip1-eIF3 interaction is impaired by the mTORC1 inhibitors, rapamycin and PP242. We show that ribosomal protein S6 kinases 1 and 2 (S6K1/2) promote the interaction of eIF3 with Paip1. The enhancement of Paip1-eIF3 interaction by amino acids is abrogated by an S6K inhibitor or shRNA against S6K1/2. S6K1 interacts with eIF3f and, in vitro, phosphorylates eIF3. Finally, we show that S6K inhibition leads to a reduction in translation by Paip1. We propose that S6K1/2 phosphorylate eIF3 to stimulate Paip1-eIF3 interaction and consequent translation initiation. Taken together, these data demonstrate that eIF3 is a new translation target of the mTOR/S6K pathway.