A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation

Most eukaryotic mRNAs possess a 5' cap and a 3' poly(A) tail, both of which are required for efficient translation. In yeast and plants, binding of eIF4G to poly(A)-binding protein (PABP) was implicated in poly(A)-dependent translation. In mammals, however, there has been no evidence that eIF4G binds PABP. Using 5' rapid amplification of cDNA, we have extended the known human eIF4GI open reading frame from the N-terminus by 156 amino acids. Co-immunoprecipitation experiments showed that the extended eIF4GI binds PABP, while the N-terminally truncated original eIF4GI cannot. Deletion analysis identified a 29 amino acid sequence in the new N-terminal region as the PABP-binding site. The 29 amino acid stretch is almost identical in eIF4GI and eIF4GII, and the full-length eIF4GII also binds PABP. As previously shown for yeast, human eIF4G binds to a fragment composed of RRM1 and RRM2 of PABP. In an in vitro translation system, an N-terminal fragment which includes the PABP-binding site inhibits poly(A)-dependent translation, but has no effect on translation of a deadenylated mRNA. These results indicate that, in addition to a recently identified mammalian PABP-binding protein, PAIP-1, eIF4G binds PABP and probably functions in poly(A)-dependent translation in mammalian cells.     

Interaction of polyadenylate-binding protein with the eIF4G homologue PAIP enhances translation

In the initiation of translation in eukaryotes, binding of the small ribosomal subunit to the messenger RNA results from recognition of the 5' cap structure (m7GpppX) of the mRNA by the cap-binding complex eIF4F. eIF4F is itself a three-subunit complex comprising the cap-binding protein eIF4E, eIF4A, an ATP-dependent RNA helicase, and eIF4G, which interacts with both eIF4A and eIF4E and enhances cap binding by eIF4E. The mRNA 3' polyadenylate tail and the associated poly(A)-binding protein (PABP) also regulate translational initiation, probably by interacting with the 5' end of the mRNA. In yeast and plants, PABP interacts with eIF4G but no such interaction has been reported in mammalian cells. Here, we describe a new human PABP-interacting protein, PAIP-I, whose sequence is similar to the central portion of eIF4G and which interacts with eIF4A. Overexpression of PAIP-1 in COS-7 cells stimulates translation, perhaps by providing a physical link between the mRNA termini.     

Vaccinia virus protein synthesis has a low requirement for the intact translation initiation factor eIF4F, the cap-binding complex, within infected cells

The role of the cap-binding complex, eIF4F, in the translation of vaccinia virus mRNAs has been analyzed within infected cells. Plasmid DNAs, which express dicistronic mRNAs containing a picornavirus internal ribosome entry site, produced within vaccinia virus-infected cells both beta-glucuronidase and a cell surface-targeted single-chain antibody (sFv). Cells expressing sFv were selected from nonexpressing cells, enabling analysis of protein synthesis specifically within the transfected cells. Coexpression of poliovirus 2A or foot-and-mouth disease virus Lb proteases, which cleaved translation initiation factor eIF4G, greatly inhibited cap-dependent protein (beta-glucuronidase) synthesis. Under these conditions, internal ribosome entry site-directed expression of sFv continued and cell selection was maintained. Furthermore, vaccinia virus protein synthesis persisted in the selected cells containing cleaved eIF4G. Thus, late vaccinia virus protein synthesis has a low requirement for the intact cap-binding complex eIF4F. This may be attributed to the short unstructured 5' noncoding regions of the vaccinia virus mRNAs, possibly aided by the presence of poly(A) at both 5' and 3' termini.     

Cleavage of Poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo: another mechanism for host protein synthesis shutoff?

Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and enterovirus groups results in the shutoff of host protein synthesis but allows viral protein synthesis to proceed. Although considerable evidence suggests that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease in the case of coxsackievirus), several experimental observations are at variance with this view. Thus, the cleavage of other cellular proteins could contribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70-kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in translation initiation. We have now found that PABP is also proteolytically cleaved during coxsackievirus infection of HeLa cells. The cleavage of PABP correlated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experiments with purified rabbit PABP and recombinant human PABP as well as in vivo experiments with Xenopus oocytes and recombinant Xenopus PABP demonstrate that the cleavage is catalyzed by 2A protease directly. N- and C-terminal sequencing indicates that cleavage occurs uniquely in human PABP at 482VANTSTQTM downward arrowGPRPAAAAAA500, separating the four N-terminal RNA recognition motifs (80%) from the C-terminal homodimerization domain (20%). The N-terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate translation system. These results suggest that the cleavage of PABP may be another mechanism by which picornaviruses alter the rate and spectrum of protein synthesis.     

Cleavage of poly(A)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro

Many enteroviruses, members of the family Picornaviridae, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3' end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5'-end-3'-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in higher eukaryotes.     

Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E

Human eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA cap structure and interacts with eIF4G, which serves as a scaffold protein for the assembly of eIF4E and eIF4A to form the eIF4F complex. eIF4E is an important modulator of cell growth and proliferation. It is the least abundant component of the translation initiation machinery and its activity is modulated by phosphorylation and interaction with eIF4E-binding proteins (4E-BPs). One strong candidate for the eIF4E kinase is the recently cloned MAPK-activated protein kinase, Mnk1, which phosphorylates eIF4E on its physiological site Ser209 in vitro. Here we report that Mnk1 is associated with the eIF4F complex via its interaction with the C-terminal region of eIF4G. Moreover, the phosphorylation of an eIF4E mutant lacking eIF4G-binding capability is severely impaired in cells. We propose a model whereby, in addition to its role in eIF4F assembly, eIF4G provides a docking site for Mnk1 to phosphorylate eIF4E. We also show that Mnk1 interacts with the C-terminal region of the translational inhibitor p97, an eIF4G-related protein that does not bind eIF4E, raising the possibility that p97 can block phosphorylation of eIF4E by sequestering Mnk1.     

Malignant transformation by overproduction of translation initiation factor eIF4G

N4G3, a cell line that overexpresses translation initiation factor eIF4G, one of the components of eIF4F, was made by stable transfection of the human eIF4G cDNA into NIH3T3 cells. The cells expressed 80-100 times greater levels of eIF4G mRNA than did NIH3T3 cells. N4G3 cells formed transformed foci on a monolayer of cells, showed anchorage-independent growth, and formed tumors in nude mice. These results indicate that overexpression of eIF4G caused malignant transformation of NIH3T3 cells. It is also known that overexpression of eIF4E, another component of eIF4F, causes transformation of NIH3T3 cells. However, there was no difference in the amount of eIF4E protein between N4G3 and NIH3T3 cells, indicating that cell transformation does not involve a change in eIF4E levels. The results may be due to an effect of eIF4G on translational control of protein synthesis directed by mRNAs having long 5'-untranslated region.     

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.     

The association of initiation factor 4F with poly(A)-binding protein is enhanced in serum-stimulated Xenopus kidney cells

Serum stimulation of cultured Xenopus kidney cells results in enhanced phosphorylation of the translational initiation factor (eIF) 4E and promotes a 2.8-fold increase in the binding of the adapter protein eIF4G to eIF4E, to form the functional initiation factor complex eIF4F. Here we demonstrate the serum-stimulated co-isolation of the poly(A)-binding protein (PABP) with the eIF4F complex. This apparent interaction of PABP with eIF4F suggests that a mechanism shown to be important in the control of translation in the yeast Saccharomyces cerevisiae also operates in vertebrate cells. We also present evidence that the signaling pathways modulating eIF4E phosphorylation and function in Xenopus kidney cells differ from those in several mammalian cell types studied previously. Experiments with the immunosuppressant rapamycin suggest that the mTOR signaling pathway is involved in serum-promoted eIF4E phosphorylation and association with eIF4G. Moreover, we could find little evidence for regulation of eIF4E function via interaction with the specific binding proteins 4E-BP1 or 4E-BP2 in these cells. Although rapamycin abrogated serum-enhanced rates of protein synthesis and the interaction of eIF4G with eIF4E, it did not prevent the increase in association of eIF4G with PABP. This suggests that serum stimulates the interaction between eIF4G and PABP by a distinct mechanism that is independent of both the mTOR pathway and the enhanced association of eIF4G with eIF4E.     

Implication of eIF2B rather than eIF4E in the regulation of global protein synthesis by amino acids in L6 myoblasts

The present study was designed to investigate the mechanism through which leucine and histidine regulate translation initiation in L6 myoblasts. The results show that both amino acids stimulate initiation and coordinately regulate the activity of eukaryotic initiation factor eIF2B. The changes in eIF2B activity could be explained in part by modulation of the phosphorylation state of the alpha-subunit of eIF2. The activity changes might also be a result of modulation of the phosphorylation state of the eIF2B epsilon-subunit, because deprivation of either amino acid caused a decrease in eIF2Bepsilon kinase activity. Leucine, but not histidine, additionally caused a redistribution of eIF4E from the inactive eIF4E.4E-BP1 complex to the active eIF4E.eIF4G complex. The redistribution was a result of increased phosphorylation of 4E-BP1. The changes in 4E-BP1 phosphorylation and eIF4E redistribution associated with leucine deprivation were not observed in the presence of insulin. However, the leucine- and histidine-induced alterations in global protein synthesis and eIF2B activity were maintained in the presence of the hormone. Overall, the results suggest that both leucine and histidine regulate global protein synthesis through modulation of eIF2B activity. Furthermore, under the conditions employed herein, alterations in eIF4E availability are not rate-controlling for global protein synthesis but might be necessary for regulation of translation of specific mRNAs.     

A novel functional human eukaryotic translation initiation factor 4G

Mammalian eukaryotic translation initiation factor 4F (eIF4F) is a cap-binding protein complex consisting of three subunits: eIF4E, eIF4A, and eIF4G. In yeast and plants, two related eIF4G species are encoded by two different genes. To date, however, only one functional eIF4G polypeptide, referred to here as eIF4GI, has been identified in mammals. Here we describe the discovery and functional characterization of a closely related homolog, referred to as eIF4GII. eIF4GI and eIF4GII share 46% identity at the amino acid level and possess an overall similarity of 56%. The homology is particularly high in certain regions of the central and carboxy portions, while the amino-terminal regions are more divergent. Far-Western analysis and coimmunoprecipitation experiments were used to demonstrate that eIF4GII directly interacts with eIF4E, eIF4A, and eIF3. eIF4GII, like eIF4GI, is also cleaved upon picornavirus infection. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A to cleave endogenous eIF4G. Finally, eIF4GII exists as a complex with eIF4E in HeLa cells, because eIF4GII and eIF4E can be purified together by cap affinity chromatography. Taken together, our findings indicate that eIF4GII is a functional homolog of eIF4GI. These results may have important implications for the understanding of the mechanism of shutoff of host protein synthesis following picornavirus infection.     

A poly(A) binding protein functions in the chloroplast as a message-specific translation factor

High-affinity binding of a set of proteins with specificity for the 5' untranslated region (UTR) of the Chlamydomonas reinhardtii chloroplast psbA mRNA correlates with light-regulated translational activation of this message. We have isolated a cDNA encoding the main psbA RNA binding protein, RB47, and identified this protein as a member of the poly(A) binding protein family. Poly(A) binding proteins are a family of eukaryotic, cytoplasmic proteins thought to bind poly(A) tails of mRNAs and play a role in translational regulation. In vitro translation of RNA transcribed from the RB47 cDNA produces a precursor protein that is efficiently transported into the chloroplast and processed to the mature 47-kDa protein. RB47 expressed and purified from Escherichia coli binds to the psbA 5' UTR with similar specificity and affinity as RB47 isolated from C. reinhardtii chloroplasts. The identification of a normally cytoplasmic translation factor in the chloroplast suggests that the prokaryotic-like chloroplast translation machinery utilizes a eukaryotic-like initiation factor to regulate the translation of a key chloroplast mRNA. These data also suggest that poly(A) binding proteins may play a wider role in translation regulation than previously appreciated.     

Effects of premilking and postmilking teat disinfectants on teat skin condition

Most eukaryotic mRNAs contain a 5' cap (m7GppX) and a 3' poly(A) tail to increase synergistically the translational efficiency. Recently, the poly(A) binding protein (PABP) and cap-binding protein, eIF-4F, were found to interact [Le et al. (1997) J. Biol. Chem. 272, 16247-16255; Tarun and Sachs (1996) EMBO J. 15, 7168-7177]. These data suggest that PABP may exert its effect on translational efficiency either by increasing the formation of initiation factor-mRNA complex or by enhancing ribosome recycling. To investigate the functional consequences of these interactions, the fluorescent cap analogue, ant-m7GTP, which is an environmentally sensitive fluorescent probe [Ren and Goss (1996) Nucleic Acids Res. 24, 3629-3634] was used to investigate the cap-binding affinity. Our data show that the binding of eIF-(iso)4F or eIF-4F to cap analogue enhanced their binding affinity toward PABP approximately 40-fold. Similarly, the eIF-4F/PABP or eIF-(iso)4F/PABP complexes show a 40-fold enhancement of cap analogue binding as compared to eIF-4F or eIF-(iso)4F alone. At least part of the enhancement of the translational initiation by PABP can be accounted for by direct changes in cap-binding affinity. The interactions of these components also suggest a mechanism whereby the poly(A) tail is brought into close proximity with m7G cap. This effect was examined by fluorescence energy transfer, and it was determined that the PABP/eIF-4F complex could bind both poly(A) and 5' cap simultaneously.     

The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast Saccharomyces cerevisiae

Initiation factor eIF4G is an essential protein required for initiation of mRNA translation via the 5' cap-dependent pathway. It interacts with eIF4E (the mRNA 5' cap-binding protein) and serves as an anchor for the assembly of further initiation factors. With treatment of Saccharomyces cerevisiae with rapamycin or with entry of cells into the diauxic phase, eIF4G is rapidly degraded, whereas initiation factors eIF4E and eIF4A remain stable. We propose that nutritional deprivation or interruption of the TOR signal transduction pathway induces eIF4G degradation.     

Identification and characterization of a novel cap-binding protein from Arabidopsis thaliana

Cap-binding proteins specifically bind to the 7-methyl guanosine (m7G) functional group at the 5' end of eukaryotic mRNAs. A novel Arabidopsis thaliana protein has been identified that has sequence similarity to cap-binding proteins but is clearly a different form of the protein. The most obvious primary sequence difference is the substitution of two of the eight conserved tryptophan residues with other aromatic amino acids in the novel protein. Analogous forms of this novel protein appear to be present in other higher eukaryotes but not in yeast. Analysis of the native and recombinant forms of the novel protein by retention on m7GTP-Sepharose indicate that it is a functional cap-binding protein. Measurements of the dissociation constant for this protein indicate that it binds m7GTP 5-20-fold tighter than eukaryotic initiation factor (eIF)(iso)4E. The novel protein also supports the initiation of translation of capped mRNA in vitro. Biochemical analysis and yeast two-hybrid data indicate that it interacts with eIF(iso)4G to form a complex. Based on these observations, this protein appears to be able to function as a cap-binding protein and is given the designation of novel cap-binding protein (nCBP).     

Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein

eIF4E, the mRNA cap binding protein, is a master switch that controls eukaryotic translation. To be active, it must bind eIF4G and form the eIF4F complex, which also contains eIF4A. Translation is downregulated by association of eIF4E with 4E-BP, which occupies the eIF4G binding site. Signalling events acting on 4E-BP cause it to dissociate from eIF4E, and eIF4E is then free to bind eIF4G to form the active eIF4F complex. We have solved the structure of the yeast eIF4E/m7Gpp complex in a CHAPS micelle. We determined the position of the second nucleotide in a complex with m7GpppA, and identified the 4E-BP binding site. eIF4E has a curved eight-stranded antiparallel beta-sheet, decorated with three helices on the convex face and three smaller helices inserted in connecting loops. The m7G of the cap is intercalated into a stack of tryptophans in the concave face. The 4E-BP binding site is located in a region encompassing one edge of the beta-sheet, the adjacent helix a2 and several regions of non-regular secondary structure. It is adjacent to, but does not overlap the cap-binding site.     

Translation eukaryotic initiation factor 4G recognizes a specific structural element within the internal ribosome entry site of encephalomyocarditis virus RNA

A complex of eukaryotic initiation factors (eIFs) 4A, 4E, and 4G (collectively termed eIF4F) plays a key role in recruiting mRNAs to ribosomes during translation initiation. The site of ribosomal entry onto most mRNAs is determined by interaction of the 5'-terminal cap with eIF4E; eIFs 4A and 4G may facilitate ribosomal entry by modifying mRNA structure near the cap and by interacting with ribosome-associated factors. eIF4G recruits uncapped encephalomyocarditis virus (EMCV) mRNA to ribosomes without the involvement of eIF4E by binding directly to the approximately 450-nucleotide long EMCV internal ribosome entry site (IRES). We have used chemical and enzymatic probing to map the eIF4G binding site to a structural element within the J-K domain of the EMCV IRES that consists of an oligo(A) loop at the junction of three helices. The oligo(A) loop itself is not sufficient to form stable complexes with eIF4G since alteration of its structural context abolished its interaction with eIF4G. Addition of wild type or trans-dominant mutant forms of eIF4A to binary IRES.eIF4G complexes did not further alter the pattern of chemical/enzymatic modification of the IRES.     

HSP101 functions as a specific translational regulatory protein whose activity is regulated by nutrient status

The 5' leader (Omega) of tobacco mosaic viral RNA functions as a translational enhancer. Sequence analysis of a 102-kD protein, identified previously as a specific Omega RNA-binding protein, revealed homology to the HSP101/HSP104/ClpB family of heat shock proteins and its expression in yeast complemented a thermotolerance defect caused by a deletion of the HSP104 gene. Up to a 50-fold increase in the translation of Omega-luc, but not luc mRNA was observed in yeast expressing the tobacco HSP101 whereas Omega failed to enhance translation in the absence of HSP101. Therefore, HSP101 and Omega comprise a two-component translational regulatory mechanism that can be recapitulated in yeast. Analysis of HSP101 function in yeast translation mutants suggested that the initiation factor (eIF) 3 and specifically one (TIF4632) of the two eIF4G proteins were required for the HSP101-mediated enhancement. The RNA-binding and translational regulatory activities of HSP101 were inactive in respiring cells or in cells subject to nutrient limitation, but its thermotolerance function remained unaffected. This is the first identification of a protein required for specific translational enhancement of capped mRNAs, the first report of a translational regulatory function for any heat-shock protein, and the first functional distinction between the two eIF4G proteins present in eukaryotes.