Eukaryotic Translation Initiation Factor 4G Research Articles

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.

Eukaryotic Initiation Factor eIFiso4G1 and eIFiso4G2 Are Isoforms Exhibiting Distinct Functional Differences in Supporting Translation in Arabidopsis

The eukaryotic translation initiation factor (eIF) 4G is required during protein synthesis to promote the assembly of several factors involved in the recruitment of a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, the eIF4G isoforms in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization but both can interact with eIF4A, eIF4B, eIF4E isoforms, and the poly(A)-binding protein. Nevertheless, eIF4G and eIFiso4G from wheat exhibit preferences in the mRNAs they translate optimally. For example, mRNA containing the 5'-leader (called Ω) of tobacco mosaic virus preferentially uses eIF4G in wheat germ lysate. In this study, the eIF4G isoform specificity of Ω was used to examine functional differences of the eIF4G isoforms in Arabidopsis. As in wheat, Ω-mediated translation was reduced in an eif4g null mutant. Loss of the eIFiso4G1 isoform, which is similar in sequence to wheat eIFiso4G, did not substantially affect Ω-mediated translation. However, loss of the eIFiso4G2 isoform substantially reduced Ω-mediated translation. eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. Loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during Ω-mediated translation.

Cleavage of DAP5 by coxsackievirus B3 2A protease facilitates viral replication and enhances apoptosis by altering translation of IRES-containing genes.

Cleavage of eukaryotic translation initiation factor 4G (eIF4G) by enterovirus proteases during infection leads to the shutoff of cellular cap-dependent translation, but does not affect the initiation of cap-independent translation of mRNAs containing an internal ribosome entry site (IRES). Death-associated protein 5 (DAP5), a structural homolog of eIF4G, is a translation initiation factor specific for IRES-containing mRNAs. Coxsackievirus B3 (CVB3) is a positive single-stranded RNA virus and a primary causal agent of human myocarditis. Its RNA genome harbors an IRES within the 5'-untranslated region and is translated by a cap-independent, IRES-driven mechanism. Previously, we have shown that DAP5 is cleaved during CVB3 infection. However, the protease responsible for cleavage, cleavage site and effects on the translation of target genes during CVB3 infection have not been investigated. In the present study, we demonstrated that viral protease 2A but not 3C is responsible for DAP5 cleavage, generating 45- and 52-kDa N- (DAP5-N) and C-terminal (DAP5-C) fragments, respectively. By site-directed mutagenesis, we found that DAP5 is cleaved at amino acid G434. Upon cleavage, DAP5-N largely translocated to the nucleus at the later time points of infection, whereas the DAP5-C largely remained in the cytoplasm. Overexpression of these DAP5 truncates demonstrated that DAP5-N retained the capability of initiating IRES-driven translation of apoptosis-associated p53, but not the prosurvival Bcl-2 (B-cell lymphoma 2) when compared with the full-length DAP5. Similarly, DAP5-N expression promoted CVB3 replication and progeny release; on the other hand, DAP5-C exerted a dominant-negative effect on cap-dependent translation. Taken together, viral protease 2A-mediated cleavage of DAP5 results in the production of two truncates that exert differential effects on protein translation of the IRES-containing genes, leading to enhanced host cell death.

Host MicroRNA miR-197 Plays a Negative Regulatory Role in the Enterovirus 71 Infectious Cycle by Targeting the RAN Protein.

Enterovirus 71 (EV71), a member of Picornaviridae, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a time-dependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3' untranslated region (UTR) reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication. Enterovirus 71 (EV71) is a picornavirus with a positive-sense single-stranded RNA that globally inhibits the cellular translational system, mainly by cleaving cellular eukaryotic translation initiation factor 4G (eIF4G) and poly(A)-binding protein (PABP), which inhibits the association of the ribosome with the host capped mRNA. We used a microRNA (miRNA) microarray chip to identify the host miRNA 197 (miR-197) that was downregulated by EV71. We also used quantitative mass spectrometry and a target site prediction tool to identify the miR-197 target genes. During viral infection, the expression of the target protein RAN was upregulated considerably, and there was a parallel downregulation of miR-197. The nuclear transport of viral 3D/3CD protein and of the host proteins involved in viral replication proceeded in an RAN-dependent manner. We have identified a new mechanism in picornavirus through which EV71-induced cellular miRNA downregulation can regulate host protein levels to facilitate viral replication.

Requirement of the eukaryotic translation initiation factor 4F complex in hepatitis E virus replication

Hepatitis E virus (HEV) infection, one of the foremost causes of acute hepatitis, is becoming a health problem of increasing magnitude. As other viruses, HEV exploits elements from host cell biochemistry, but we understand little as to which components of the human hepatocellular machinery are perverted for HEV multiplication. It is, however, known that the eukaryotic translation initiation factors 4F (eIF4F) complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, serves as an important component for the translation and replication of many viruses. Here we aim to investigate the role of three subunits of the eIF4F complex: eukaryotic translation initiation factor 4A (eIF4A), eukaryotic translation initiation factor 4G (eIF4G) and eukaryotic translation initiation factor 4E (eIF4E) in HEV replication. We found that efficient replication of HEV requires eIF4A, eIF4G and eIF4E. Consistently, the negative regulatory factors of this complex: programmed cell death 4 (PDCD4) and eIF4E-binding protein 1 (4E-BP1) exert anti-HEV activities, which further illustrates the requirement for eIF4A and eIF4E in supporting HEV replication. Notably, phosphorylation of eIF4E induced by MNK1/2 activation is not involved in HEV replication. Although ribavirin and interferon-α (IFN-α), the most often-used off-label drugs for treating hepatitis E, interact with this complex, their antiviral activities are independent of eIF4E. In contrast, eIF4E silencing provokes enhanced anti-HEV activity of these compounds. Thus, HEV replication requires eIF4F complex and targeting essential elements of this complex provides important clues for the development of novel antiviral therapy against HEV.

Phylogenetic analysis reveals dynamic evolution of the poly(A)-binding protein gene family in plants.

BackgroundThe poly(A)-binding protein (PABP) binds the poly(A) tail of eukaryotic mRNAs and functions to maintain the integrity of the mRNA while promoting protein synthesis through its interaction with eukaryotic translation initiation factor (eIF) 4G and eIF4B. PABP is encoded by a single gene in yeast and marine algae but during plant evolution the PABP gene family expanded substantially, underwent sequence divergence into three subclasses, and acquired tissue-specificity in gene family member expression. Although such changes suggest functional specialization, the size of the family and its sequence divergence have complicated an understanding of which gene family members may be foundational and which may represent more recent expansions of the family to meet the specific needs of speciation. Here, we examine the evolution of the plant PABP gene family to provide insight into these aspects of the family that may yield clues into the function of individual family members.ResultsThe PABP gene family had expanded to two members by the appearance of fresh water algae and four members in non-vascular plants. In lycophytes, the first sequence divergence yielding a specific class member occurs. The earliest members of the gene family share greatest similarity to those modern members whose expression is confined to reproductive tissues, suggesting that supporting reproductive-associated gene expression is the most conserved function of this family. A family member sharing similarity to modern vegetative-associated members first appears in gymnosperms. Further elaboration of the reproductive-associated and vegetative-associated members occurred during the evolution of flowering plants.ConclusionsExpansion of the plant PABP gene family began prior to the colonization of land. By the evolution of lycophytes, the first class member whose expression is confined to reproductive tissues in higher plants had appeared. A second class member whose expression is vegetative-associated appeared in gymnosperms and all three modern classes had fully evolved by the appearance of the first known basal angiosperm. The size of each PABP class underwent further expansion during subsequent evolution, especially in the Brassicaceae, suggesting that the family is undergoing dynamic evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-014-0238-4) contains supplementary material, which is available to authorized users.

Phosphoproteomics Reveals the Effect of Ethylene in Soybean Root under Flooding Stress

Flooding has severe negative effects on soybean growth. To explore the flooding-responsive mechanisms in early-stage soybean, a phosphoproteomic approach was used. Two-day-old soybean plants were treated without or with flooding for 3, 6, 12, and 24 h, and root tip proteins were then extracted and analyzed at each time point. After 3 h of flooding exposure, the fresh weight of soybeans increased, whereas the ATP content of soybean root tips decreased. Using a gel-free proteomic technique, a total of 114 phosphoproteins were identified in the root tip samples, and 34 of the phosphoproteins were significantly changed with respect to phosphorylation status after 3 h of flooding stress. Among these phosphoproteins, eukaryotic translation initiation factors were dephosphorylated, whereas several protein synthesis-related proteins were phosphorylated. The mRNA expression levels of sucrose phosphate synthase 1F and eukaryotic translation initiation factor 4 G were down-regulated, whereas UDP-glucose 6-dehydrogenase mRNA expression was up-regulated during growth but down-regulated under flooding stress. Furthermore, bioinformatic protein interaction analysis of flooding-responsive proteins based on temporal phosphorylation patterns indicated that eukaryotic translation initiation factor 4 G was located in the center of the network during flooding. Soybean eukaryotic translation initiation factor 4 G has homology to programmed cell death 4 protein and is implicated in ethylene signaling. The weight of soybeans was increased with treatment by an ethylene-releasing agent under flooding condition, but it was decreased when plants were exposed to an ethylene receptor antagonist. These results suggest that the ethylene signaling pathway plays an important role, via the protein phosphorylation, in mechanisms of plant tolerance to the initial stages of flooding stress in soybean root tips.

Norovirus Translation Requires an Interaction between the C Terminus of the Genome-linked Viral Protein VPg and Eukaryotic Translation Initiation Factor 4G

Viruses have evolved a variety of mechanisms to usurp the host cell translation machinery to enable translation of the viral genome in the presence of high levels of cellular mRNAs. Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5' end of the viral RNA. To further characterize this novel mechanism of translation initiation, we have used proteomics to identify the components of the norovirus translation initiation factor complex. This approach revealed that VPg binds directly to the eIF4F complex, with a high affinity interaction occurring between VPg and eIF4G. Mutational analyses indicated that the C-terminal region of VPg is important for the VPg-eIF4G interaction; viruses with mutations that alter or disrupt this interaction are debilitated or non-viable. Our results shed new light on the unusual mechanisms of protein-directed translation initiation.

Eukaryotic Translation Initiation Factor eIFiso4G Is Required to Regulate Violaxanthin De-epoxidase Expression in Arabidopsis

The eukaryotic translation initiation factor (eIF) 4G is a scaffold protein that organizes the assembly of those initiation factors needed to recruit the 40 S ribosomal subunit to an mRNA. Plants, like many eukaryotes, express two eIF4G isoforms. eIFiso4G, one of the isoforms specific to plants, is unique among eukaryotic eIF4G proteins in that it is highly divergent and unusually small in size, raising the possibility of functional specialization. In this study, the role of eIFiso4G in plant growth was investigated using null mutants for the eIF4G isoforms in Arabidopsis. eIFiso4G loss of function mutants exhibited smaller cell, leaf, plant size, and biomass accumulation that correlated with its reduced photosynthetic activity, phenotypes not observed with the eIF4G loss of function mutant. Although no change in photorespiration or dark respiration was observed in the eIFiso4G loss of function mutant, a reduction in chlorophyll levels and an increase in the level of nonphotochemical quenching were observed. An increase in xanthophyll cycle activity and the generation of reactive oxygen species contributed to the qE and qI components of nonphotochemical quenching, respectively. An increase in the transcript and protein levels of violaxanthin de-epoxidase in the eIFiso4G loss of function mutant and an increase in its xanthophyll de-epoxidation state correlated with the higher qE associated with loss of eIFiso4G expression. These observations indicate that eIFiso4G expression is required to regulate violaxanthin de-epoxidase expression and to support photosynthetic activity.

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.

EIF4G1 R1205H and VPS35 D620N mutations are rare in Parkinson's disease from South Africa

The R1205H mutation in the eukaryotic translation initiation factor 4G1 (EIF4G1) gene and the D620N mutation in the vacuolar protein sorting 35 (VPS35) gene were recently found in patients with autosomal dominant or sporadic forms of Parkinson's disease (PD). In the present study, 418 South African PD patients and 528 control subjects of diverse ethnicities were screened using the KASP (Kompetitive Allele Specific PCR) genotyping assay. The mutations were not found in our study, suggesting that they are not a common cause of PD in South African patients. Further studies are needed on the frequency of these 2 mutations in other sub-Saharan African populations.

The unique evolution of the programmed cell death 4 protein in plants

BackgroundThe programmed cell death 4 (PDCD4) protein is induced in animals during apoptosis and functions to inhibit translation and tumor promoter-induced neoplastic transformation. PDCD4 is composed of two MA3 domains that share similarity with the single MA3 domain present in the eukaryotic translation initiation factor (eIF) 4G, which serves as a scaffold protein to assemble several initiation factors needed for the recruitment of the 40S ribosomal subunit to an mRNA. Although eIF4A is an ATP-dependent RNA helicase that binds the MA3 domain of eIF4G to promote translation initiation, binding of eIF4A to the MA3 domains of PDCD4 inhibits protein synthesis. Genes encoding PDCD4 are present in many lower eukaryotes and in plants, but PDCD4 in higher plants is unique in that it contains four MA3 domains and has been implicated in ethylene signaling and abiotic stress responses. Here, we examine the evolution of PDCD4 in plants.ResultsIn older algal lineages, PDCD4 contains two MA3 domains similar to the homolog in animals. By the appearance of early land plants, however, PDCD4 is composed of four MA3 domains which likely is the result of a duplication of the two MA3 domain form of the protein. Evidence from fresh water algae, from which land plants evolved, suggests that the duplication event occurred prior to the colonization of land. PDCD4 in more recently evolved chlorophytes also contains four MA3 domains but this may have resulted from an independent duplication event. Expansion and divergence of the PDCD4 gene family occurred during land plant evolution with the appearance of a distinct gene member following the evolution of basal angiosperms.ConclusionsThe appearance of a unique form of PDCD4 in plants correlates with the appearance of components of the ethylene signaling pathway, suggesting that it may represent the adaptation of an existing protein involved in programmed cell death to one that functions in abiotic stress responses through hormone signaling.

An open conformation determined by a structural switch for 2A protease from coxsackievirus A16

Coxsackievirus A16 belongs to the family Picornaviridae, and is a major agent of hand-foot-and-mouth disease that infects mostly children, and to date no vaccines or antiviral therapies are available. 2A protease of enterovirus is a nonstructural protein and possesses both self-cleavage activity and the ability to cleave the eukaryotic translation initiation factor 4G. Here we present the crystal structure of coxsackievirus A16 2A protease, which interestingly forms hexamers in crystal as well as in solution. This structure shows an open conformation, with its active site accessible, ready for substrate binding and cleavage activity. In conjunction with a previously reported "closed" state structure of human rhinovirus 2, we were able to develop a detailed hypothesis for the conformational conversion triggered by two "switcher" residues Glu88 and Tyr89 located within the bll2-cII loop. Substrate recognition assays revealed that amino acid residues P1', P2 and P4 are essential for substrate specificity, which was verified by our substrate binding model. In addition, we compared the in vitro cleavage efficiency of 2A proteases from coxsackievirus A16 and enterovirus 71 upon the same substrates by fluorescence resonance energy transfer (FRET), and observed higher protease activity of enterovirus 71 compared to that of coxsackievirus A16. In conclusion, our study shows an open conformation of coxsackievirus A16 2A protease and the underlying mechanisms for conformational conversion and substrate specificity. These new insights should facilitate the future rational design of efficient 2A protease inhibitors.

Control of Translation and miRNA-Dependent Repression by a Novel Poly(A) Binding Protein, hnRNP-Q

Author SummaryThe regulation of mRNA translation and stability is of paramount importance for almost every cellular function. In eukaryotes, the poly(A) binding protein (PABP) is a central regulator of both global and mRNA-specific translation. PABP simultaneously interacts with the 3′ poly(A) tail of the mRNA and the eukaryotic translation initiation factor 4G (eIF4G). These interactions circularize the mRNA and stimulate translation. PABP also regulates specific mRNAs by promoting miRNA-dependent deadenylation and translational repression. A key step in understanding PABP's functions is to identify factors that affect its association with the poly(A) tail. Here we show that the cytoplasmic isoform of the mouse heterogeneous nuclear ribonucleoprotein Q (hnRNP-Q2/SYNCRIP), which exhibits binding preference to poly(A), interacts with the poly(A) tail by default when PABP binding is inhibited. In addition, hnRNP-Q2 competes with PABP for binding to the poly(A) tail. Depleting hnRNP-Q2 stimulates translation in cell-free extracts and in cultured cells, in agreement with its function as translational repressor. In addition, hnRNP-Q2 impeded miRNA-mediated deadenylation and repression of target mRNAs, which requires PABP. Thus, competition from hnRNP-Q2 provides a novel mechanism by which multiple functions of PABP are regulated. This regulation could play important roles in various biological processes, such as development, viral infection, and human disease.

Eukaryotic initiation factor 4B and the poly(A)-binding protein bind eIF4G competitively.

The eukaryotic translation initiation factor (eIF) 4G functions as a scaffold protein that assembles components of the translation initiation complex required to recruit the 40S ribosomal subunit to an mRNA. Although many eukaryotes express two highly similar eIF4G isoforms, those in plants are highly divergent in size and sequence from one another and are referred to as eIF4G and eIFiso4G. Although the domain organization of eIFiso4G differs substantially from eIF4G orthologs in other species, the domain organization of plant eIF4G is largely unknown despite the fact that it is more similar in size and sequence to eIF4G of other eukaryotes. In this study, we show that eIF4G differs from eIFiso4G in that it contains two distinct interaction domains for the poly(A) binding protein (PABP) and eIF4B but is similar to eIFiso4G in having two eIF4A interaction domains. PABP and eIF4B bind the same N-terminal region of eIF4G as they do to a region C-proximal to the HEAT-1 domain in the middle domain of eIF4G, resulting in competitive binding between eIF4B and PABP to each site. eIF4G also differs from eIFiso4G in that no competitive binding was observed between PABP and eIF4A or between eIF4B and eIF4A to its HEAT-1-containing region. These results demonstrate that despite substantial differences in size, sequence, and domain organization, PABP and eIF4B bind to eIF4G and eIFiso4G competitively.

Dynamic Regulation of the Translation Initiation Helicase Complex by Mitogenic Signal Transduction to Eukaryotic Translation Initiation Factor 4G

Eukaryotic translation initiation factor 4F (eIF4F), comprising the cap-binding protein eIF4E, the helicase eIF4A, and the central scaffold eIF4G, is a convergence node for a complex signaling network that controls protein synthesis. Together with eIF3 and eIF4A/4B, eIF4G recruits ribosomal subunits to mRNAs and facilitates 5' untranslated region unwinding. Mammalian eIF4G contains three HEAT domains and unstructured regions involved in protein-protein interactions. Despite detailed eIF4G structure data, the mechanisms controlling initiation scaffold formation remain obscure. We found a new, highly regulated eIF4B/-3 binding site within the HEAT-1/-2 interdomain linker, harboring two phosphorylation sites that we identified as substrates for Erk1/2 and casein kinase 2. Phorbol ester-induced sequential phosphorylation of both sites detached HEAT-2 from the complex with eIF4A/-4B/-3 and stimulated the association of HEAT-3 with the mitogen-activated protein kinase signal integrating kinase Mnk1. Our results provide a mechanistic link between intracellular signal transduction and dynamic initiation complex formation coordinated by flexible eIF4G structure.

L‐Serine deficiency caused by genetic Phgdh deletion leads to robust induction of 4E‐BP1 and subsequent repression of translation initiation in the developing central nervous system

Targeted disruption in mice of the gene encoding D-3-phosphoglycerate dehydrogenase (Phgdh) results in embryonic lethality associated with a striking reduction in free L-serine and growth retardation including severe brain malformation. We previously observed a severe impairment in neurogenesis of the central nervous system of Phgdh knockout (KO) embryos and a reduction in the protein content of their brains. Although these findings suggest that L-serine deficiency links attenuation of mRNA translation to severe developmental malformation of the central nervous system, the underlying key molecular event remains unexplored. Here we demonstrate that mRNA of Eif4ebp1 encoding eukaryotic initiation factor 4 binding protein 1 and its protein, 4E-BP1, are markedly induced in the central nervous system of Phgdh KO embryos, whereas a modest induction is observed in the liver. The increase in 4E-BP1 was associated with a decrease in the cap initiation complex in the brain, as shown by lower levels of eukaryotic translation initiation factor 4G bound to eukaryotic translation initiation factor 4E (eIF4E) and increased eIF4E interaction with 4E-BP1 based on 7-methyl-GTP chromatography. eIF4E protein and polysomes were also diminished in Phgdh KO embryos. Induction of Eif4ebp1 mRNA and of 4E-BP1 was reproduced in mouse embryonic fibroblasts established from Phgdh KO embryos under the condition of L-serine deprivation. Induction of Eif4ebp1 mRNA was suppressed only when L-serine was supplemented in the culture medium, indicating that reduced L-serine availability regulates the induction of Eif4ebp1/4E-BP1. These data suggest that elevated levels of 4E-BP1 may be involved in a mechanism to arrest brain development in Phgdh KO embryos.

Backbone resonance assignment of the HEAT1-domain of the human eukaryotic translation initiation factor 4GI

Controlling translation during protein synthesis is crucial for cell proliferation and differentiation. Protein translation is orchestrated by an assembly of various protein components at the ribosomal subunits. The eukaryotic translation initiation factor 4G (eIF4G) plays an important role in the formation of the translation initiation complex eIF4F consisting of eIF4G, the ATP dependent RNA helicase eIF4A and the cap binding protein eIF4E. One of the functions of eIF4G is the enhancement of the activity of eIF4A facilitated mainly through binding to the HEAT1 domain of eIF4G. In order to understand the interaction of HEAT1 with eIF4A and other components during translation initiation backbone assignment is essential. Here we report the (1)H, (13)C and (15)N backbone assignment for the HEAT1 domain of human eIF4G isoform I (eIF4GI-HEAT1), the first of three HEAT domains of eIF4G (29 kDa) as a basis for the elucidation of its structure and interactions with its binding partners, necessary for understanding the mechanism of its biological function.

Yeast Eukaryotic Initiation Factor 4B (eIF4B) Enhances Complex Assembly between eIF4A and eIF4G in Vivo

Translation initiation factor eIF4F (eukaryotic initiation factor 4F), composed of eIF4E, eIF4G, and eIF4A, binds to the m(7)G cap structure of mRNA and stimulates recruitment of the 43S preinitiation complex and subsequent scanning to the initiation codon. The HEAT domain of eIF4G stabilizes the active conformation of eIF4A required for its RNA helicase activity. Mammalian eIF4B also stimulates eIF4A activity, but this function appears to be lacking in yeast, making it unclear how yeast eIF4B (yeIF4B/Tif3) stimulates translation. We identified Ts(-) mutations in the HEAT domains of yeast eIF4G1 and eIF4G2 that are suppressed by overexpressing either yeIF4B or eIF4A, whereas others are suppressed only by eIF4A overexpression. Importantly, suppression of HEAT domain substitutions by yeIF4B overexpression was correlated with the restoration of native eIF4A·eIF4G complexes in vivo, and the rescue of specific mutant eIF4A·eIF4G complexes by yeIF4B was reconstituted in vitro. Association of eIF4A with WT eIF4G in vivo also was enhanced by yeIF4B overexpression and was impaired in cells lacking yeIF4B. Furthermore, we detected native complexes containing eIF4G and yeIF4B but lacking eIF4A. These and other findings lead us to propose that yeIF4B acts in vivo to promote eIF4F assembly by enhancing a conformation of the HEAT domain of yeast eIF4G conducive for stable binding to eIF4A.

Sequential Eukaryotic Translation Initiation Factor 5 (eIF5) Binding to the Charged Disordered Segments of eIF4G and eIF2β Stabilizes the 48S Preinitiation Complex and Promotes Its Shift to the Initiation Mode

During translation initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eukaryotic translation initiation factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2β bind three common partners, eIF5, eIF1, and mRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2β-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner reversed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2β-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2β mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involving the eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2β and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNA(i)(Met):mRNA binding to the P site.