The phosphorylation state of poly(A)-binding protein specifies its binding to poly(A) RNA and its interaction with eukaryotic initiation factor (eIF) 4F, eIFiso4F, and eIF4B

The poly(A)-binding protein (PABP) interacts with the eukaryotic initiation factor (eIF) 4G (or eIFiso4G), the large subunit of eIF4F (or eIFiso4F) to promote translation initiation. In plants, PABP also interacts with eIF4B, a factor that assists eIF4F function. PABP is a phosphoprotein, although the function of its phosphorylation has not been previously investigated. In this study, we have purified the phosphorylated and hypophosphorylated isoforms of PABP from wheat to examine whether its phosphorylation state affects its binding to poly(A) RNA and its interaction with eIF4G, eIFiso4G, or eIF4B. Phosphorylated PABP exhibited cooperative binding to poly(A) RNA even under non-stoichiometric binding conditions, whereas multiple molecules of hypophosphorylated PABP bound to poly(A) RNA only after free poly(A) RNA was no longer available. Together, phosphorylated and hypophosphorylated PABP exhibited synergistic binding. eIF4B interacted with PABP in a phosphorylation state-specific manner; native eIF4B increased the RNA binding activity specifically of phosphorylated PABP and was greater than 14-fold more effective than was recombinant eIF4B, whereas eIF4F promoted the cooperative binding of hypophosphorylated PABP. These data suggest that the phosphorylation state of PABP specifies the type of binding to poly(A) RNA and its interaction with its partner proteins.     

Wheat eukaryotic initiation factor 4B organizes assembly of RNA and eIFiso4G, eIF4A, and poly(A)-binding protein

The eukaryotic translation initiation factor (eIF) 4B promotes the RNA-dependent ATP hydrolysis activity and ATP-dependent RNA helicase activity of eIF4A and eIF4F during translation initiation. Although this function is conserved among plants, animals, and yeast, eIF4B is one of the least conserved of initiation factors at the sequence level. To gain insight into its functional conservation, the organization of the functional domains of eIF4B from wheat has been investigated. Plant eIF4B contains three RNA binding domains, one more than reported for mammalian or yeast eIF4B, and each domain exhibits a preference for purine-rich RNA. In addition to a conserved RNA recognition motif and a C-terminal RNA binding domain, wheat eIF4B contains a novel N-terminal RNA binding domain that requires a short, lysine-rich containing sequence. Both the lysine-rich motif and an adjacent, C-proximal motif are conserved with an N-proximal sequence in human and yeast eIF4B. The C-proximal motif within the N-terminal RNA binding domain in wheat eIF4B is required for interaction with eIFiso4G, an interaction not reported for other eIF4B proteins. Moreover, each RNA binding domain requires dimerization for binding activity. Two binding sites for the poly(A)-binding protein were mapped to a region within each of two conserved 41-amino acid repeat domains on either side of the C-terminal RNA binding domain. eIF4A bound to an adjacent region within each repeat, supporting a central role for these conserved eIF4B domains in facilitating interaction with other components of the translational machinery. These results support the notion that eIF4B functions by organizing multiple components of the translation initiation machinery and RNA.     

Disruption of the interaction of mammalian protein synthesis eukaryotic initiation factor 4B with the poly(A)-binding protein by caspase- and viral protease-mediated cleavages

Eukaryotic initiation factor (eIF) 4B interacts with several components of the initiation pathway and is targeted for cleavage during apoptosis. In a cell-free system, cleavage of eIF4B by caspase-3 coincides with a general inhibition of protein synthetic activity. Affinity chromatography demonstrates that mammalian eIF4B interacts with the poly(A)-binding protein and that a region consisting of the N-terminal 80 amino acids of eIF4B is both necessary and sufficient for such binding. This interaction is lost when eIF4B is cleaved by caspase-3, which removes the N-terminal 45 amino acids. Similarly, the association of eIF4B with the poly(A)-binding protein in vivo is reduced when cells are induced to undergo apoptosis. Cleavage of the poly(A)-binding protein itself, using human rhinovirus 3C protease, also eliminates the interaction with eIF4B. Thus, disruption of the association between mammalian eIF4B and the poly(A)-binding protein can occur during both apoptosis and picornaviral infection and is likely to contribute to the inhibition of translation observed under these conditions.     

Poly(A)-binding protein interaction with elF4G stimulates picornavirus IRES-dependent translation.

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated, at least in part, by elF4G, which bridges the mRNA termini by simultaneous binding the poly(A)-binding protein (PABP) and the cap-binding protein, elF4E. The poly(A) tail also stimulates translation from the internal ribosome binding sites (IRES) of a number of picornaviruses. elF4G is likely to mediate this translational stimulation through its direct interaction with the IRES. Here, we support this hypothesis by cleaving elF4G to separate the PABP-binding site from the portion that promotes internal initiation. elF4G cleavage abrogates the stimulatory effect of poly(A) tail on translation. In addition, translation in extracts in which elF4G is cleaved is resistant to inhibition by the PABP-binding protein 2 (Paip2). The elF4G cleavage-induced loss of the stimulatory effect of poly(A) on translation was mimicked by the addition of the C-terminal portion of elF4G. Thus, PABP stimulates picornavirus translation through its interaction with elF4G.     

Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.     

Wheat germ poly(A)-binding protein increases the ATPase and the RNA helicase activity of translation initiation factors eIF4A, eIF4B, and eIF-iso4F

Recent studies demonstrated that wheat germ poly(A)-binding protein (PABP) interacted with translation eukaryotic initiation factor (eIF)-iso4G and eIF4B, and these interactions increased the poly(A) binding activity of PABP (Le, H., Tanguay, R. L., Balasta, M. L., Wei, C. C., Browning, K. S., Metz, A. M., Goss, D. J., and Gallie, D. R. (1997) J. Biol. Chem. 272, 16247-16255) and the cap binding activity of eIF-iso4F (Wei, C. C., Balasta, M. L., Ren, J., and Goss, D. J. (1998) Biochemistry 37, 1910-1916). We report here that the interaction between PABP and eIF-iso4G has a substantial effect on the ATPase activity and RNA helicase activity of (eIF4A + eIF4B + eIF-iso4F) complex. ATPase kinetic assays show, in the presence of poly(U), PABP can increase the parameter (k(cat)/K(m)) by 3.5-fold with a 2-fold decrease of K(m) for the (eIF4A + eIF-iso4F) complex. In the presence of globin messenger RNA, the ATPase activity of the complex (eIF4A + eIF-iso4F) was increased 2-fold by the presence of PABP. RNA helicase assays demonstrated that the presence of PABP enhanced the RNA duplex unwinding activity of the initiation factor complex. These results suggest that, in terms of the scanning model of translation initiation, PABP may enhance the mRNA scanning rate of the complex formed by eIF4A, eIF4B, and eIF4F or eIF-(iso)4F and increase the rate of translation.     

The RNA binding domains of the nuclear poly(A)-binding protein

The nuclear poly(A)-binding protein (PABPN1) is involved in the synthesis of the mRNA poly(A) tails in most eukaryotes. We report that the protein contains two RNA binding domains, a ribonucleoprotein-type RNA binding domain (RNP domain) located approximately in the middle of the protein sequence and an arginine-rich C-terminal domain. The C-terminal domain also promotes self-association of PABPN1 and moderately cooperative binding to RNA. Whereas the isolated RNP domain binds specifically to poly(A), the isolated C-terminal domain binds non-specifically to RNA and other polyanions. Despite this nonspecific RNA binding by the C-terminal domain, selection experiments show that adenosine residues throughout the entire minimal binding site of approximately 11 nucleotides are recognized specifically. UV-induced cross-links with oligo(A) carrying photoactivatable nucleotides at different positions all map to the RNP domain, suggesting that most or all of the base-specific contacts are made by the RNP domain, whereas the C-terminal domain may contribute nonspecific contacts, conceivably to the same nucleotides. Asymmetric dimethylation of 13 arginine residues in the C-terminal domain has no detectable influence on the interaction of the protein with RNA. The N-terminal domain of PABPN1 is not required for RNA binding but is essential for the stimulation of poly(A) polymerase.     

Poly(A) RNA and Paip2 act as allosteric regulators of poly(A)-binding protein

When bound to the 3′ poly(A) tail of mRNA, poly(A)-binding protein (PABP) modulates mRNA translation and stability through its association with various proteins. By visualizing individual PABP molecules in real time, we found that PABP, containing four RNA recognition motifs (RRMs), adopts a conformation on poly(A) binding in which RRM1 is in proximity to RRM4. This conformational change is due to the bending of the region between RRM2 and RRM3. PABP-interacting protein 2 actively disrupts the bent structure of PABP to the extended structure, resulting in the inhibition of PABP-poly(A) binding. These results suggest that the changes in the configuration of PABP induced by interactions with various effector molecules, such as poly(A) and PABP-interacting protein 2, play pivotal roles in its function.     

Effects of poly(A)-binding protein on the interactions of translation initiation factor eIF4F and eIF4F.4B with internal ribosome entry site (IRES) of tobacco etch virus RNA

In wheat germ, the interaction between poly(A)-binding protein and eukaryotic initiation factor eIF 4G increases the affinity of eIF4E for the cap by 20-40-fold. Recent findings that wheat germ eIF4G is required for interaction with the IRES, pseudoknot 1 (PK1), of tobacco etch virus to promote cap-independent translation led us to investigate the effects of PABP on the interaction of eIF4F with PK1. The fluorescence anisotropy data showed addition of PABP to eIF4F increased the binding affinity approximately 2.0-fold for PK1 RNA as compared with eIF4F alone. Addition of both PABP and eIF4B to eIF4F enhance binding affinity to PK1 about 4-fold, showing an additive effect rather than the large increase in affinity shown for cap binding. The van't Hoff analyses showed that PK1 RNA binding to eIF4F, eIF4F.PABP, eIF4F.4B and eIF4F.4B.PABP is enthalpy-driven and entropy-favorable. PABP and eIF4B decreased the entropic contribution 65% for binding of PK1 RNA to eIF4F. The lowering of entropy for the formation of eIF4F.4B.PABP-PK1 complex suggested reduced hydrophobic interactions for complex formation. Overall, these results demonstrate the first direct effect of PABP on the interaction of eIF4F and eIF4F.4B with PK1 RNA.     

Poly(A)-binding protein facilitates translation of an uncapped/nonpolyadenylated viral RNA by binding to the 3' untranslated region

Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3'CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3'CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3'CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3'CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3'CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3'CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA.     

Poly(A)-binding protein-interacting protein 2, a strong regulator of vascular endothelial growth factor mRNA

Expression of vascular endothelial growth factor (VEGF) is tightly regulated, particularly at the level of its mRNA stability, which is essentially mediated through the 3'-untranslated region (3'-UTR) of VEGF mRNA. To identify new protein partners regulating VEGF mRNA stability, we screened a cDNA expression library with an RNA probe corresponding to the entire VEGF mRNA 3'-UTR. We identified the "poly(A)-binding protein-interacting protein 2" (PAIP2) as a new VEGF mRNA 3'-UTR interacting protein. By RNA electromobility shift assays, we showed that PAIP2 binds to two distinct regions of a domain encompassing base 1 to 1280 of the VEGF 3'-UTR. Such in vitro interaction was confirmed using cell extracts in which PAIP2 expression is induced by tetracycline (Tet-on cells). Moreover, we demonstrated by RNA affinity purification as well as by ribonucleoprotein complexes immunoprecipitation, that PAIP2 interacts with VEGF mRNA in vivo. Using an in vitro RNA degradation assay, the half-life of VEGF 3'-UTR was found to be increased by overexpressing PAIP2. PAIP2 stabilizes endogenous VEGF mRNA in Tet-on cells, leading to increased VEGF secretion. Moreover, RNAi-mediated knock-down of PAIP2 significantly reduces the steady-state levels of endogenous VEGF mRNA. We also showed, by in vitro protein-protein interactions and co-immunoprecipitation experiments, that PAIP2 interacts with HuR, an already known VEGF mRNA-binding protein, suggesting cooperation of both proteins for VEGF mRNA stabilization. Hence, PAIP2 appears to be a crucial regulator of VEGF mRNA and as a consequence, any variation in its expression could modulate angiogenesis.     

A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3.     

Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G.

Although the cap structure and the poly(A) tail are on opposite ends of the mRNA molecule, previous work has suggested that they interact to enhance translation and inhibit mRNA degradation. Here we present biochemical data that show that the proteins bound to the mRNA cap (eIF-4F) and poly(A) tail (Pab1p) are physically associated in extracts from the yeast Saccharomyces cerevisiae. Specifically, we find that Pab1p co-purifies and co-immunoprecipitates with the eIF-4G subunit of eIF-4F. The Pab1p binding site on the recombinant yeast eIF-4G protein Tif4632p was mapped to a 114-amino-acid region just proximal to its eIF-4E binding site. Pab1p only bound to this region when complexed to poly(A). These data support the model that the Pablp-poly(A) tail complex on mRNA can interact with the cap structure via eIF-4G.     

Poly(A)-binding protein and eRF3 are associated in vivo in human and Xenopus cells

An interaction between human poly(A)-binding protein (PABP) et human eRF3 has been demonstrated using a double-hybrid approach and in vitro assays. Here, we show that the binding of both proteins is conserved through evolution. We also demonstrate that the last 39 C-terminal amino acids of PABP contain the interface that interacts with eRF3. This region includes helix 5, identified by RMN, which is conserved in all known PABPs. Lastly, we demonstrate that eRF3 et PABP molecules interact in vivo.     

Translational control by neuroguidin, a eukaryotic initiation factor 4E and CPEB binding protein

CPEB-mediated translation is important in early development and neuronal synaptic plasticity. Here, we describe a new eukaryotic initiation factor 4E (eIF4E) binding protein, Neuroguidin (Ngd), and its interaction with CPEB. In the mammalian nervous system, Ngd is detected as puncta in axons and dendrites and in growth cones and filopodia. Ngd contains three motifs that resemble those present in eIF4G, 4EBP, Cup, and Maskin, all of which are eIF4E binding proteins. Ngd binds eIF4E directly, and all three motifs must be deleted to abrogate the interaction between these two proteins. In injected Xenopus oocytes, Ngd binds CPEB and, most importantly, represses translation in a cytoplasmic polyadenylation element (CPE)-dependent manner. In Xenopus embryos, Ngd is found in both neural tube and neural crest cells. The injection of morpholino-containing antisense oligonucleotides directed against ngd mRNA disrupts neural tube closure and neural crest migration; however, the wild-type phenotype is restored by the injection of a rescuing ngd mRNA. These data suggest that Ngd guides neural development by regulating the translation of CPE-containing mRNAs.     

Phosphorylation dynamics of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) is discordant with its potential to interact with eukaryotic initiation factor 4E (eIF4E)

Mammalian target of rapamycin (mTOR) controls cellular growth and proliferation by virtue of its ability to regulate protein translation. Eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) — a key mTOR substrate, binds and sequesters eIF4E to impede translation initiation that is supposedly overcome upon 4E-BP1 phosphorylation by mTOR. Ambiguity surrounding the precise identity of mTOR regulated sites in 4E-BP1 and their invariable resistance to mTOR inactivation raises concerns about phospho-regulated model proposed for 4E:4E-BP1 interaction. Our attempt to mimic dephosphorylation associated with rapamycin response by introducing phospho deficient mutants for sites implicated in regulating 4E:4E-BP1 interaction individually or globally highlighted no obvious difference in the quantum of their association with CAP bound 4E when compared with their phosphomimicked counterparts or the wild type 4E-BP1. TOS or RAIP motif deletion variants compromised for raptor binding and resultant phosphodeficiency did little to influence their association with CAP bound 4E. Interestingly ectopic expression of ribosomal protein S6 kinase 1 (S6K1) that restored 4E-BP1 sensitivity to rapamycin/Torin reflected by instant loss of 4E-BP1 phosphorylation, failed to bring about any obvious change in 4E:4E-BP1 stoichiometry. Our data clearly demonstrate a potential disconnect between rapamycin response of 4E-BP1 and its association with CAP bound 4E.