The helicase protein DHX29 promotes translation initiation,cell proliferation,and tumorigenesis

Translational control plays an important role in cell growth and tumorigenesis. Cap-dependent translation initiation of mammalian mRNAs with structured 5′UTRs requires the DExH-box protein, DHX29, in vitro. Here we show that DHX29 is important for translation in vivo. Down-regulation of DHX29 leads to impaired translation, resulting in disassembly of polysomes and accumulation of mRNA-free 80S monomers. DHX29 depletion also impedes cancer cell growth in culture and in xenografts. Thus, DHX29 is a bona fide translation initiation factor that potentially can be exploited as a target to inhibit cancer cell growth.Initiation is a tightly regulated rate-limiting step in the translation of eukaryotic mRNAs. Ribosome recruitment to the mRNA commences with binding of translation initiation factor 4F (eIF4F) to the 7-methyl guanosine cap structure, which is present at the 5′ end of all nuclear-encoded eukaryotic mRNAs (1). eIF4F (comprising the cap-binding protein eIF4E, the DEAD-box RNA helicase eIF4A and eIF4G, a scaffold for binding eIF4E and eIF4A) binds to the cap, unwinds (with the aid of eIF4A) the cap-proximal region of the mRNA, and, through interaction with the ribosome-bound eIF3, recruits the 40S ribosomal subunit to the mRNA (2–4). The 40S subunit then scans the 5′ UTR in a 5′ to 3′ direction until it encounters an initiation codon. A subsequent joining of the 60S ribosomal subunit and release of eIFs result in formation of an elongation-competent 80S ribosome.Secondary structures in 5′UTRs of mRNAs are thought to become unwound to allow ribosomal complexes to move along the mRNA in search of the initiation codon. Thus, in addition to its role in the initial attachment of ribosomal complexes to mRNA, eIF4A is believed to assist ribosomal complexes during scanning (5). Recent observations suggest that the process of eukaryotic initiation requires additional members of the DEAD/DExH-box protein family; for instance, a DEAD-box protein, yeast Ded1, and its mammalian homologue, DDX3, are biochemically and genetically implicated in translation initiation on long structured 5′UTRs (6), and another DExH-box protein, DHX29, strongly stimulates cap-dependent initiation on mRNAs with structured 5′UTRs in vitro (7). Here we studied the importance of DHX29 for translation in vivo and characterized it as a novel factor required for cell proliferation.     

Molecular mechanisms of translation initiation in eukaryotes

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5' end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.     

Mapping the binding interface between human eukaryotic initiation factors 1A and 5B: a new interaction between old partners

The translation initiation factors (IFs) IF1/eIF1A and IF2e/IF5B have been conserved throughout all kingdoms. Although the central roles of the bacterial factors IF1 and IF2 were established long ago, the importance of their eukaryotic homologs, eukaryotic IFs (eIFs) eIF1A and eIF5B, has only recently become evident. The translation machinery in eukaryotes is more complex and accordingly, eIF1A and eIF5B seem to have acquired a number of new functions while also retaining many of the roles of bacterial IF1 and IF2. IF1 and IF2 have been shown to interact on the ribosome but no binding has been detected for the free factors. In contrast, yeast eIF1A and eIF5B have been reported to interact in the absence of ribosomes. Here, we have identified the binding interface between human eIF1A and the C-terminal domain of eIF5B by using solution NMR. That interaction interface involves the C termini of the two proteins, which are not present in bacterial IF1 and IF2. The interaction is, therefore, unique to eukaryotes. A structural model for the interaction of eIF1A and eIF5B in the context of the ribosome is presented. We propose that eIF1A and eIF5B simultaneously interact at two sites that are >50 A apart: through their C termini as reported here, and through an interface previously identified in bacterial IF1 and IF2. The binding between the C termini of eIF1A and eIF5B has implications for eukaryote-specific mechanisms of recruitment and release of translation IFs from the ribosome.     

Initiation factor 2 crystal structure reveals a different domain organization from eukaryotic initiation factor 5B and mechanism among translational GTPases

The initiation of protein synthesis uses initiation factor 2 (IF2) in prokaryotes and a related protein named eukaryotic initiation factor 5B (eIF5B) in eukaryotes. IF2 is a GTPase that positions the initiator tRNA on the 30S ribosomal initiation complex and stimulates its assembly to the 50S ribosomal subunit to make the 70S ribosome. The 3.1-Å resolution X-ray crystal structures of the full-length Thermus thermophilus apo IF2 and its complex with GDP presented here exhibit two different conformations (all of its domains except C2 domain are visible). Unlike all other translational GTPases, IF2 does not have an effecter domain that stably contacts the switch II region of the GTPase domain. The domain organization of IF2 is inconsistent with the “articulated lever” mechanism of communication between the GTPase and initiator tRNA binding domains that has been proposed for eIF5B. Previous cryo-electron microscopy reconstructions, NMR experiments, and this structure show that IF2 transitions from being flexible in solution to an extended conformation when interacting with ribosomal complexes.The synthesis of proteins in prokaryotes is divided into three distinct processes: initiation, elongation, and termination. The initiation of translation in prokaryotes is directed by three initiation factors (IF1, IF2, and IF3) that govern the binding and positioning of the mRNA, as well as the initiator tRNA, and the joining of ribosomal subunits to form a 70S complex that is ready for the elongation stage of protein synthesis.IF2 is a GTPase that functions to position the initiator tRNA within the 30S ribosomal initiation complex (30S IC) and promotes its joining with the 50S ribosomal subunit to form a 70S ribosome. IF2 is encoded by a single copy of the infB gene and is completely conserved in bacteria (1). The flexible structure of the N terminus has the largest sequence variability among different species (2). Variability also exists within a species; for instance, Escherichia coli IF2 has three isoforms, which vary in the length of their N-terminal domain due to three distinct start sites for its translation initiation (1). The C-terminal part of IF2 (G, II, C1, and C2 domains) contains the highly conserved GTPase domain (G domain) and C2 domain, which interact with the initiator tRNA.The C2 domain recognizes and protects the formylated Met of the initiator tRNA from hydrolysis (3, 4). The formylation of Met results in a fivefold increase in the binding affinity of the tRNA and is made in a G-nucleotide–independent fashion (3, 5). This interaction permits IF2 to assist in positioning the initiator tRNA within a 30S initiation complex and guide the formation of a functional 30S IC on the establishment of the P-site codon–anticodon interaction (2, 6, 7). A functional 30S IC is competent for the 50S ribosomal subunit to join, which is mediated by the formation of the intersubunit salt bridges via an interaction between IF2 and L12 (8). Once the 50S ribosomal subunit joins, IF2 comes into contact with the GTPase activation center, GTP is hydrolyzed by IF2, and IF2 dissociates from the ribosome (2, 9).IF2 is a GTPase homologous to other translational GTPases such as EF-Tu, EF-G, LepA, and RF3 (1). All translational GTPases use a conserved mechanism for GTP hydrolysis in which switch I, switch II and the P-loop are stimulated through interaction with II and VI domains of the 23S rRNA in addition to L11 and L7/L12 of the large ribosomal subunit (1). GTP hydrolysis by IF2 causes it to dissociate from the 70S IC and organizes the 70S ribosome for peptide elongation by a rotation of the ribosomal subunits relative to each other (9–11). Cryo-EM studies of the 30S and 70S particles have shown that the final function of IF2 before it leaves the ribosome is to position the CCA end of the initiator tRNA near the peptidyl transferase center (6, 7, 9, 10).Low-resolution cryo-electron microscopy (cryo-EM) reconstructions have provided structural information about the interactions that govern the initiation process of 70S ribosome assembly in prokaryotes for protein synthesis. No structure from full-length IF2 at atomic resolution has previously been determined. Until recently, the only structural information at atomic resolution on IF2 has been NMR models of four separate domains of IF2 (N, G, C1, and C2) (12–15).The crystal structures of eukaryotic initiation factor 5B (eIF5B) from Methanobacterium thermoautotrophicum have been used to construct a homology model of IF2 to interpret the electron density from cryo-EM reconstructions of the 30S and 70S ICs with IF2 (6, 7, 9, 10). Small angle X-ray scattering (SAXS) studies of E. coli IF2 domains IV–VI (domains G, II, C1, and C2) indicate that its domains are organized differently compared with eIF5B (16). Allen et al. (10) found that the structure of the E. coli IF2 in their cryo-EM reconstruction of a 70S IC differed significantly with that of the crystal structure of M. thermoautotrophicum guanosine 5′-[β,γ-imido]triphosphateeIF5B.Structural information is important for understanding, first how IF2 influences the position of the initiator tRNA in the 30S and 70S ICs, and second how IF2 facilitates joining of the ribosomal subunits to form a 70S ribosome ready for peptide elongation. We address these aspects here and in a companion paper (17).We determined the X-ray crystal structures of Thermus thermophilus IF2 from a full-length protein both with and without GDP bound at 3.09-Å resolution, which improves the model available for interpreting the cryo-EM reconstructions of the 30S and 70S ICs. These two structures exhibit two different conformations of IF2 (in apo and GDP-bound forms), and all of its domains except C2 domain are visible. The initial molecular replacement solution was determined by using a homology model based on an NMR model of the G domain from Geobacillus stearothermophilus (PDB ID code 2LKD) and aligned with the G2 and G3 domains (IF2 G and II domains) of LepA from Aquifex aeolicus (PDB ID codes 2YWE, 2YWF, 2YWG, and 2YWH). While this work was being completed, a crystal structure of the first 363 residues of the T. thermophilus IF2 was determined and that model was then used as an improved search model for molecular replacement (18).This structure of IF2 determined of residues 3–467 exhibits a different 3D organization compared with the homologous domains of eIF5B. We conclude that the organization of the IF2 domains indicates that the communication between the GTPase domain and initiator tRNA binding domain cannot be explained by the “articulated lever” mechanism proposed previously for eIF5B. This difference in domain organization appears to reflect different functions of the two proteins in translation initiation between prokaryotes and eukaryotes. Our crystal structure of isolated IF2 shows that IF2 is unique among crystal structures of isolated translational GTPases because the effecter domains do not directly contact or form a stable interaction with the switch II region of the GTPase domain.     

Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA

Protein synthesis in all cells begins with recruitment of the small ribosomal subunit to the initiation codon in a messenger RNA. In some eukaryotic viruses, RNA upstream of the coding region forms an internal ribosome entry site (IRES) that directly binds to the 40S ribosomal subunit and enables translation initiation in the absence of many canonical translation initiation factors. The hepatitis C virus (HCV) IRES RNA requires just two initiation factors, eukaryotic initiation factor (eIF) 2 and eIF3, to form preinitiation 48S ribosomal complexes that subsequently assemble into translation-competent ribosomes. Using an RNA-based affinity purification approach, we show here that HCV IRES RNA facilitates eIF2 function through its interactions with eIF3 and the 40S ribosomal subunit. Although the wild-type IRES assembles normally into 48S and 80S ribosomal complexes in human cell extract, mutant IRES RNAs become trapped at the 48S assembly stage. Trapped 48S complexes formed by IRES mutants with reduced eIF3 binding affinity nonetheless contain eIF3, consistent with inherent eIF3-40S subunit affinity. Intriguingly, however, one of these IRES mutants prevents stable association of both eIF3 and eIF2, preventing initiator tRNA deposition and explaining the block in 80S assembly. In contrast, an IRES mutant unable to induce a conformational change in the 40S subunit, as observed previously by single-particle cryoelectron microscopy, blocks 80S formation at a later stage in assembly. These data suggest that the IRES RNA coordinates interactions of eIF3 and eIF2 on the ribosome required to position the initiator tRNA on the mRNA in the ribosomal peptidyl-tRNA site (P site).     

Translation of hepatitis C virus RNA

The 341-nucleotide 5' non-translated region is the most conserved part of the hepatitis C virus (HCV) genome. It contains a highly structured internal ribosomal entry site (IRES) that mediates cap-independent initiation of translation of the viral polyprotein by a mechanism that is unprecedented in eukaryotes. The first step in translation initiation is assembly of eukaryotic initiation factor (eIF) 3, eIF2, GTP, initiator tRNA and a 40S ribosomal subunit into a 43S preinitiation complex. The HCV IRES recruits this complex and directs its precise attachment at the initiation codon to form a 48S complex in a process that does not involve eIFs 4A, 4B or 4F. The IRES contains sites that bind independently with the eIF3 and 40S subunit components of 43S complexes, and structural determinants that ensure the correct spatial orientation of these binding sites so that the 48S complex assembles precisely at the initiation codon.     

Translation of hepatitis C virus RNA

The 341-nucleotide 5' non-translated region is the most conserved part of the hepatitis C virus (HCV) genome. It contains a highly structured internal ribosomal entry site (IRES) that mediates cap-independent initiation of translation of the viral polyprotein by a mechanism that is unprecedented in eukaryotes. The first step in translation initiation is assembly of eukaryotic initiation factor (eIF) 3, eIF2, GTP, initiator tRNA and a 40S ribosomal subunit into a 43S preinitiation complex. The HCV IRES recruits this complex and directs its precise attachment at the initiation codon to form a 48S complex in a process that does not involve eIFs 4A, 4B or 4F. The IRES contains sites that bind independently with the eIF3 and 40S subunit components of 43S complexes, and structural determinants that ensure the correct spatial orientation of these binding sites so that the 48S complex assembles precisely at the initiation codon.     

Initiation factor 2, tRNA, and 50S subunits cooperatively stabilize mRNAs on the ribosome during initiation

Initiation factor 2 (IF2) is a key factor in initiation of bacterial protein synthesis. It recruits initiator tRNA to the small ribosomal subunit and facilitates joining of the large ribosomal subunit. Using reconstituted translation system of Escherichia coli and optical tweezers, we directly measure the rupture force between single ribosomal complexes and mRNAs for initiation complexes in the presence and the absence of IF2. We demonstrate that IF2 together with codon recognition by initiator tRNA increases the force required to dislocate mRNA from the ribosome complexes; mRNA stabilization by IF2 required the presence of a joined 50S subunit, and was independent of bound guanine nucleotide. IF2 thus helps lock the 70S ribosome over the start codon during initiation, thus maintaining reading frame. Our results show how mRNA is progressively stabilized on the ribosome through distinct steps of initiation.     

Initiation context modulates autoregulation of eukaryotic translation initiation factor 1 (eIF1)

The central feature of standard eukaryotic translation initiation is small ribosome subunit loading at the 5' cap followed by its 5' to 3' scanning for a start codon. The preferred start is an AUG codon in an optimal context. Elaborate cellular machinery exists to ensure the fidelity of start codon selection. Eukaryotic initiation factor 1 (eIF1) plays a central role in this process. Here we show that the translation of eIF1 homologs in eukaryotes from diverse taxa involves initiation from an AUG codon in a poor context. Using human eIF1 as a model, we show that this poor context is necessary for an autoregulatory negative feedback loop in which a high level of eIF1 inhibits its own translation, establishing that variability in the stringency of start codon selection is used for gene regulation in eukaryotes. We show that the stringency of start codon selection (preferential utilization of optimal start sites) is increased to a surprising degree by overexpressing eIF1. The capacity for the cellular level of eIF1 to impact initiation through the variable stringency of initiation codon selection likely has significant consequences for the proteome in eukaryotes.     

Defective ribosome assembly in Shwachman-Diamond syndrome

Shwachman-Diamond syndrome (SDS), a recessive leukemia predisposition disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, skeletal abnormalities and poor growth, is caused by mutations in the highly conserved SBDS gene. Here, we test the hypothesis that defective ribosome biogenesis underlies the pathogenesis of SDS. We create conditional mutants in the essential SBDS ortholog of the ancient eukaryote Dictyostelium discoideum using temperature-sensitive, self-splicing inteins, showing that mutant cells fail to grow at the restrictive temperature because ribosomal subunit joining is markedly impaired. Remarkably, wild type human SBDS complements the growth and ribosome assembly defects in mutant Dictyostelium cells, but disease-associated human SBDS variants are defective. SBDS directly interacts with the GTPase elongation factor-like 1 (EFL1) on nascent 60S subunits in vivo and together they catalyze eviction of the ribosome antiassociation factor eukaryotic initiation factor 6 (eIF6), a prerequisite for the translational activation of ribosomes. Importantly, lymphoblasts from SDS patients harbor a striking defect in ribosomal subunit joining whose magnitude is inversely proportional to the level of SBDS protein. These findings in Dictyostelium and SDS patient cells provide compelling support for the hypothesis that SDS is a ribosomopathy caused by corruption of an essential cytoplasmic step in 60S subunit maturation.     

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression

Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser/Thr protein kinase that plays an important role in cell cycle progression. Although its function in this process remains unclear, it is known to be required for the G(1) and G(2)/M phase transitions in yeast. Here, we show that CK2 activity changes notably during cell cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, eukaryotic translation initiation factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are major sites of phosphorylation by CK2. This is confirmed using eIF5 mutants that lack CK2 sites; the phosphorylation levels of mutant eIF5 proteins are significantly reduced, relative to WT eIF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous eIF5, and that they perturb synchronous progression of cells through S to M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature eIF5/eIF2/eIF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy. These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation.     

Molecular cloning and expression of cDNA for mammalian translation initiation factor 5.

Eukaryotic translation initiation factor 5 (eIF-5) catalyzes the hydrolysis of GTP bound to the 40S ribosomal initiation complex (40S.AUG.Met-tRNAf-eIF-2.GTP) with the subsequent joining of a 60S ribosomal subunit resulting in the formation of a functional 80S initiation complex. A rat cDNA that encodes eIF-5 has been isolated and expressed in Escherichia coli to yield a catalytically active eIF-5 protein. The 3.55-kb cDNA encodes a protein of 429 amino acids (calculated M(r) 48,926) with properties that are similar to eIF-5 isolated from rabbit reticulocyte lysates. The deduced amino acid sequence of eIF-5 contains sequence motifs characteristic of proteins of the GTPase superfamily.     

Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation

RNA helicases are the largest group of enzymes in eukaryotic RNA metabolism. The DEXD/H-box putative RNA helicases form the helicase superfamily II, whose members are defined by seven highly conserved amino acid motifs, making specific targeting of selected members a challenging pharmacological problem. The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase that works in conjunction with eIF4B and eIF4H and as a subunit of eIF4F to prepare the mRNA template for ribosome binding, possibly by unwinding the secondary structure proximal to the 5' m7GpppN cap structure. We report the identification and characterization of a small molecule inhibitor of eukaryotic translation initiation that acts in an unusual manner by stimulating eIF4A-associated activities. Our results suggest that proper control of eIF4A helicase activity is necessary for efficient ribosome binding and demonstrate the feasibility of selectively targeting DEAD-box RNA helicases with small molecules.     

Universal conservation in translation initiation revealed by human and archaeal homologs of bacterial translation initiation factor IF2

Binding of initiator methionyl-tRNA to ribosomes is catalyzed in prokaryotes by initiation factor (IF) IF2 and in eukaryotes by eIF2. The discovery of both IF2 and eIF2 homologs in yeast and archaea suggested that these microbes possess an evolutionarily intermediate protein synthesis apparatus. We describe the identification of a human IF2 homolog, and we demonstrate by using in vivo and in vitro assays that human IF2 functions as a translation factor. In addition, we show that archaea IF2 can substitute for its yeast homolog both in vivo and in vitro. We propose a universally conserved function for IF2 in facilitating the proper binding of initiator methionyl-tRNA to the ribosomal P site.     

Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3)

Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli, revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.     

Modulation of protein translation by Nck-1

In mammals, Nck represented by two genes, is a 47-kDa SH2/SH3 domain-containing protein lacking intrinsic enzymatic function. Here, we reported that the first and the third SH3 domains of Nck-1 interact with the C-terminal region of the beta subunit of the eukaryotic initiation factor 2 (eIF2 beta). Binding of eIF2 beta was specific to the SH3 domains of Nck-1, and in vivo, the interaction Nck/eIF2 beta was demonstrated by reciprocal coimmunoprecipitations. In addition, Nck was detected in a molecular complex with eIF2 beta in an enriched ribosomal fraction, whereas no other SH2/SH3 domain-containing adapters were found. Cell fractionation studies demonstrated that the presence of Nck in purified ribosomal fractions was enhanced after insulin stimulation, suggesting that growth factors dynamically regulate translocation of Nck to ribosomes. In HEK293 cells, we observed that transient overexpression of Nck-1 significantly enhanced Cap-dependent and -independent protein translation. This effect of Nck-1 required the integrity of its first and third SH3 domains originally found to interact with eIF2 beta. Finally, in vitro, Nck-1 also increased protein translation, revealing a direct role for Nck-1 in this process. Our study demonstrates that in addition to mediate receptor tyrosine kinase signaling, Nck-1 modulates protein translation potentially through its direct interaction with an intrinsic component of the protein translation machinery.     

Upregulation of eIF5B controls cell-cycle arrest and specific developmental stages

Proliferation arrest and distinct developmental stages alter and decrease general translation yet maintain ongoing translation. The factors that support translation in these conditions remain to be characterized. We investigated an altered translation factor in three cell states considered to have reduced general translation: immature Xenopus laevis oocytes, mouse ES cells, and the transition state of proliferating mammalian cells to quiescence (G0) upon growth-factor deprivation. Our data reveal a transient increase of eukaryotic translation initiation factor 5B (eIF5B), the eukaryotic ortholog of bacterial initiation factor IF2, in these conditions. eIF5B promotes 60S ribosome subunit joining and pre-40S subunit proofreading. eIF5B has also been shown to promote the translation of viral and stress-related mRNAs and can contribute indirectly to supporting or stabilizing initiator methionyl tRNA (tRNA-Met_i) association with the ribosome. We find that eIF5B is a limiting factor for translation in these three conditions. The increased eIF5B levels lead to increased eIF5B complexes with tRNA-Met_i upon serum starvation of THP1 mammalian cells. In addition, increased phosphorylation of eukaryotic initiation factor 2α, the translation factor that recruits initiator tRNA-Met_i for general translation, is observed in these conditions. Importantly, we find that eIF5B is an antagonist of G0 and G0-like states, as eIF5B depletion reduces maturation of G0-like, immature oocytes and hastens early G0 arrest in serum-starved THP1 cells. Consistently, eIF5B overexpression promotes maturation of G0-like immature oocytes and causes cell death, an alternative to G0, in serum-starved THP1 cells. These data reveal a critical role for a translation factor that regulates specific cell-cycle transition and developmental stages.Specific cell states and transitions, including distinct developmental stages and cell-cycle arrest, alter and decrease general translation (1, 2) yet exhibit ongoing translation (3). In immature Xenopus laevis oocytes, translation of mRNAs is regulated and active after maturation (3, 4); however, mRNAs are translated during immature stages preceding maturation (5). Similarly, canonical translation is altered in mouse ES cells until differentiation (6), but translation ensues in ES cells (7), indicating that uncharacterized factors operate to support general translation in these cell states.The transition from immature to mature oocytes shows some features similar to the entry into mammalian G1/cell cycle from quiescence (G0) (8), an assortment of reversible, cell-cycle–arrested states that can withstand unfavorable environments (9). Serum deprivation of proliferating mammalian cells induces an early stage of transient stress that alters gene expression; cells subjected to such stress either adapt to these nonproliferative conditions and proceed further into G0 or alternatively undergo cell death (9). Early (1 day) serum starvation represents a transient and heterogeneous state and is distinct from prolonged serum starvation in cells that have entered G0 and late G0 (9). The reprogramming of gene expression during this transition involves a decrease in overall translation, yet ongoing translation is observed (10), indicating that undiscovered factors support translation in these conditions.The mechanism of translation involves several steps, including recruitment of the initiator methionyl tRNA (tRNA-Met_i) to the small 40S ribosome subunit by eukaryotic initiation factor 2 (eIF2) and joining of the large 60S ribosome subunit by eukaryotic initiation factor 5B (eIF5B) (1, 2, 11). eIF5B is the mammalian ortholog of bacterial IF2 that is important in the formation of the 30S initiation complex, stimulates 50S association to form 70S complexes, and can contribute to stabilizing tRNA-Met_i association with the ribosome (2, 11–13). eIF5B functions in 60S ribosome subunit joining during canonical translation (2, 13, 14) and is involved in pre-40S ribosome subunit proofing (15, 16). eIF5B is also required for the translation of a few viral and specialized mRNAs (12, 17–20) and can contribute to supporting or stabilizing tRNA-Met_i association, including in specific conditions where phosphorylation of the eIF2 subunit, eIF2α, is also observed (12, 17–19). In some cases, phosphorylation of eIF2α is sufficiently increased relative to the levels of its guanine nucleotide exchange factor (eIF2B), which leads to decreased release of phosphorylated eIF2α from eIF2B. This decrease prevents the recycling of inactive eIF2α-GDP to the active eIF2α-GTP form and can cause altered translation (2, 21, 22).Here we investigated an altered translation factor in cell states considered to have reduced general translation: in late immature oocytes, in ES cells, and during the transient stress induced in the early stages of growth-factor deprivation of proliferating mammalian cells (3, 4, 6, 7, 10). We find that eIF5B is transiently increased in these three conditions. The role of eIF5B in 60S subunit joining and other functions (2, 13–16) may be important for translation in these conditions and may indirectly enable tRNA-Met_i association. Accordingly, depletion of eIF5B reduces translation, indicating that eIF5B is limiting, in part, for translation in these three conditions. In serum-starved cells, the increased levels of eIF5B lead to increased formation of eIF5B complexes with tRNA-Met_i, consistent with its previously described role with viral and stress mRNAs (12, 17–19). Additionally, increased phosphorylation of eIF2α is observed in these three conditions. Importantly, we find that eIF5B overexpression promotes the transition away from G0 or G0-like states (immature oocytes; ref. 8), enhancing the maturation of late G0-like immature oocytes and causing cell death, the alternative to G0, upon serum deprivation of proliferating mammalian cells. These data suggest that eIF5B promotes translation in these specific conditions and is an antagonist of G0 and G0-like states; consistently, eIF5B depletion decreases translation, promotes the immature state of oocytes, and enables earlier G0 arrest upon serum starvation of proliferating mammalian cells. These studies reveal a critical role for a translation factor that is important for general translation under specific conditions and regulates distinct cell and developmental stages.     

Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3

The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome.     

Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity

Elevated eukaryotic initiation factor 4E (eIF4E) levels frequently occur in a variety of human cancers. Overexpression of eIF4E promotes cellular transformation by selectively increasing the translation of proliferative and prosurvival mRNAs. These mRNAs possess highly structured 5′-UTRs that impede ribosome recruitment and scanning, yet the mechanism for how eIF4E abundance elevates their translation is not easily explained by its cap-binding activity. Here, we show that eIF4E possesses an unexpected second function in translation initiation by strongly stimulating eukaryotic initiation factor 4A (eIF4A) helicase activity. Importantly, we demonstrate that this activity promotes mRNA restructuring in a manner that is independent of its cap-binding function. To explain these findings, we show that the eIF4E-binding site in eukaryotic initiation factor 4G (eIF4G) functions as an autoinhibitory domain to modulate its ability to stimulate eIF4A helicase activity. Binding of eIF4E counteracts this autoinhibition, enabling eIF4G to stimulate eIF4A helicase activity. Finally, we have successfully separated the two functions of eIF4E to show that its helicase promoting activity increases the rate of translation by a mechanism that is distinct from its cap-binding function. Based on our results, we propose that maintaining a connection between eIF4E and eIF4G throughout scanning provides a plausible mechanism to explain how eIF4E abundance selectively stimulates the translation of highly structured proliferation and tumor-promoting mRNAs.