Influence of the Length of the Phosphate Chain in mRNA 5′ Cap Analogues on Their Interaction with Eukaryotic Initiation Factor 4E

Abstract

The recognition of the 5′mRNA cap structure m⁷G(5′)ppp(5′)N by one of the components of the initiation translation machinery, the eIF4E factor, plays a pivotal role in regulation of the protein synthesis. In the present study we have shown two opposing roles of the cap phosphate chain in the specific eIF4E-cap interaction. The extension of the phosphate chain enhances the binding of the cap to the unphosphorylated eIF4E but destabilises the eIF4E-cap complex in case of the phosphorylated protein.     

The eukaryotic mRNA decapping protein Dcp1 interacts physically and functionally with the eIF4F translation initiation complex

Dcp1 plays a key role in the mRNA decay process in Saccharomyces cerevisiae, cleaving off the 5' cap to leave an end susceptible to exonucleolytic degradation. The eukaryotic initiation factor complex eIF4F, which in yeast contains the core components eIF4E and eIF4G, uses the cap as a binding site, serving as an initial point of assembly for the translation apparatus, and also binds the poly(A) binding protein Pab1. We show that Dcp1 binds to eIF4G and Pab1 as free proteins, as well as to the complex eIF4E-eIF4G-Pab1. Dcp1 interacts with the N-terminal region of eIF4G but does not compete significantly with eIF4E or Pab1 for binding to eIF4G. Most importantly, eIF4G acts as a function-enhancing recruitment factor for Dcp1. However, eIF4E blocks this effect as a component of the high affinity cap-binding complex eIF4E-eIF4G. Indeed, cooperative enhancement of the eIF4E-cap interaction stabilizes yeast mRNAs in vivo. These data on interactions at the interface between translation and mRNA decay suggest how events at the 5' cap and 3' poly(A) tail might be coupled.     

Influence of electric charge variation at residues 209 and 159 on the interaction of eIF4E with the mRNA 5' terminus

Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient protein synthesis in cap-dependent translation. The protein specifically binds the cap structure at the mRNA 5' terminus and facilitates the assembly of the mRNA with other initiation factors and the 40S ribosomal subunit. Phosphorylation of eIF4E is implicated in the regulation of the initiation step of translation. However, the molecular mechanism of this regulation still remains unclear. To address this problem, we have determined the binding affinities of eIF4E specifically mutated at position 209 or 159 for a series of novel mono- and dinucleotide cap analogues by a fluorometric time-synchronized titration method. A 1.5-3-fold reduction in the affinity of cap for the S209E mutant and a 1-2-fold increase in the affinity of cap for the S209K mutant, depending on the negative charge of phosphate chains, indicate that phosphorylation at Ser209 creates electrostatic repulsion between the protein and the negatively charged cap structure. The inhibition of the ability to bind cap analogues by the K159A mutant and its phosphorylated counterpart shows significant participation of Lys159 in the binding of the capped mRNA. Both structural modifications, phosphorylation and the replacement of lysine with alanine, result in an increase in the negative Gibbs free energy of association that is proportional to the length of the cap phosphate chain and additive, i.e., equal to the sum of the individual destabilizing changes of DeltaG degrees. The possible implication of these results for the mechanism of control of eIF4E by phosphorylation, especially for the "clamping model", is discussed.     

Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins

mRNA 5'-cap recognition by the eukaryotic translation initiation factor eIF4E has been exhaustively characterized with the aid of a novel fluorometric, time-synchronized titration method, and X-ray crystallography. The association constant values of recombinant eIF4E for 20 different cap analogues cover six orders of magnitude; with the highest affinity observed for m(7)GTP (approximately 1.1 x 10(8) M(-1)). The affinity of the cap analogues for eIF4E correlates with their ability to inhibit in vitro translation. The association constants yield contributions of non-covalent interactions involving single structural elements of the cap to the free energy of binding, giving a reliable starting point to rational drug design. The free energy of 7-methylguanine stacking and hydrogen bonding (-4.9 kcal/mol) is separate from the energies of phosphate chain interactions (-3.0, -1.9, -0.9 kcal/mol for alpha, beta, gamma phosphates, respectively), supporting two-step mechanism of the binding. The negatively charged phosphate groups of the cap act as a molecular anchor, enabling further formation of the intermolecular contacts within the cap-binding slot. Stabilization of the stacked Trp102/m(7)G/Trp56 configuration is a precondition to form three hydrogen bonds with Glu103 and Trp102. Electrostatically steered eIF4E-cap association is accompanied by additional hydration of the complex by approximately 65 water molecules, and by ionic equilibria shift. Temperature dependence reveals the enthalpy-driven and entropy-opposed character of the m(7)GTP-eIF4E binding, which results from dominant charge-related interactions (DeltaH degrees =-17.8 kcal/mol, DeltaS degrees= -23.6 cal/mol K). For recruitment of synthetic eIF4GI, eIF4GII, and 4E-BP1 peptides to eIF4E, all the association constants were approximately 10(7) M(-1), in decreasing order: eIF4GI>4E-BP1>eIF4GII approximately 4E-BP1(P-Ser65) approximately 4E-BP1(P-Ser65/Thr70). Phosphorylation of 4E-BP1 at Ser65 and Thr70 is insufficient to prevent binding to eIF4E. Enhancement of the eIF4E affinity for cap occurs after binding to eIF4G peptides.     

Specific domains in yeast translation initiation factor eIF4G strongly bias RNA unwinding activity of the eIF4F complex toward duplexes with 5'-overhangs

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex is recruited to the 5'-end of an mRNA through its interaction with the 7-methylguanosine cap, and it subsequently scans along the mRNA to locate the start codon. Both mRNA recruitment and scanning require the removal of secondary structure within the mRNA. Eukaryotic translation initiation factor 4A is an essential component of the translational machinery thought to participate in the clearing of secondary structural elements in the 5'-untranslated regions of mRNAs. eIF4A is part of the 5'-7-methylguanosine cap-binding complex, eIF4F, along with eIF4E, the cap-binding protein, and the scaffolding protein eIF4G. Here, we show that Saccharomyces cerevisiae eIF4F has a strong preference for unwinding an RNA duplex with a single-stranded 5'-overhang versus the same duplex with a 3'-overhang or without an overhang. In contrast, eIF4A on its own has little RNA substrate specificity. Using a series of deletion constructs of eIF4G, we demonstrate that its three previously elucidated RNA binding domains work together to provide eIF4F with its 5'-end specificity, both by promoting unwinding of substrates with 5'-overhangs and inhibiting unwinding of substrates with 3'-overhangs. Our data suggest that the RNA binding domains of eIF4G provide the S. cerevisiae eIF4F complex with a second mechanism, in addition to the eIF4E-cap interaction, for directing the binding of pre-initiation complexes to the 5'-ends of mRNAs and for biasing scanning in the 5' to 3' direction.     

Phosphorylation of eIF4E attenuates its interaction with mRNA 5' cap analogs by electrostatic repulsion: intein-mediated protein ligation strategy to obtain phosphorylated protein

Phosphorylation of the eukaryotic initiation factor eIF4E in response to mitogenic stimuli and cytokines is implicated in the regulation of the initiation step of translation. It still remains unclear how the phosphorylation of eIF4E regulates the translation. To address this problem, we applied a unique technique in protein engineering, intein-mediated protein ligation, to synthesize eIF4E, which is selectively phosphorylated at Ser 209. Using selectively chosen synthetic cap analogs, we compared quantitatively the cap affinity for phosphorylated and unphosphorylated eIF4E by a fluorometric time-synchronized titration method. A 1.5- to 4.5-fold reduction of the cap affinity for phosphorylated eIF4E was observed, depending on the negative charge of the 5'-to-5' phosphate chains as well as the presence of a longer tetraribonucleotide strand. Possible implications for understanding the regulation of eIF4E functioning, cap complex formation, and stability, are discussed.     

Structural changes of eIF4E upon binding to the mRNA 5' monomethylguanosine and trimethylguanosine Cap

Recognition of the 5' cap by the eukaryotic initiation factor 4E (eIF4E) is the rate-limiting step in the ribosome recruitment to mRNAs. The regular cap consists of 7-monomethylguanosine (MMG) linked by a 5'-5' triphosphate bridge to the first transcribed nucleoside, while some primitive eukaryotes possess a N (2), N (2),7-trimethylguanosine (TMG) cap structure as a result of trans splicing. Mammalian eIF4E is highly specific to the MMG form of the cap in terms of association constants and thermodynamic driving force. We have investigated conformational changes of eIF4E induced by interaction with two cap analogues, 7-methyl-GTP and N (2), N (2),7-trimethyl-GTP. Hydrogen-deuterium exchange and electrospray mass spectrometry were applied to probe local dynamics of murine eIF4E in the apo and cap-bound forms. The data show that the cap binding induces long-range conformational changes in the protein, not only in the cap-binding pocket but also in a distant region of the 4E-BP/eIF4G binding site. Formation of the complex with 7-methyl-GTP makes the eIF4E structure more compact, while binding of N (2), N (2),7-trimethyl-GTP leads to higher solvent accessibility of the protein backbone in comparison with the apo form. The results suggest that the additional double methylation at the N (2)-amino group of the cap causes sterical effects upon binding to mammalian eIF4E which influence the overall solution dynamics of the protein, thus precluding formation of a tight complex.     

Folding transitions during assembly of the eukaryotic mRNA cap-binding complex

The cap-binding protein eIF4E is the first in a chain of translation initiation factors that recruit 40S ribosomal subunits to the 5' end of eukaryotic mRNA. During cap-dependent translation, this protein binds to the 5'-terminal m(7)Gppp cap of the mRNA, as well as to the adaptor protein eIF4G. The latter then interacts with small ribosomal subunit-bound proteins, thereby promoting the mRNA recruitment process. Here, we show apo-eIF4E to be a protein that contains extensive unstructured regions, which are induced to fold upon recognition of the cap structure. Binding of eIF4G to apo-eIF4E likewise induces folding of the protein into a state that is similar to, but not identical with, that of cap-bound eIF4E. At the same time, binding of each of the binding partners of eIF4E modulates the kinetics with which it interacts with the other partner. We present structural, kinetic and mutagenesis data that allow us to deduce some of the detailed folding transitions that take place during the eIF4E interactions.     

Potyvirus terminal protein VPg, effector of host eukaryotic initiation factor eIF4E

Potyvirus RNA contains at the 5' end a covalently linked virus-encoded protein VPg, which is required for virus infectivity. This role has been attributed to VPg interaction with the eukaryotic translation initiation factor eIF4E, a cap-binding protein. We characterized the dissociation constants for the interaction of the potato virus Y VPg with different plant eIF4Es and its isoforms and mapped the eIF(iso)4E attachment region on VPg. VPg/eIF4E interaction results in the inhibition of cell-free protein synthesis, and we show that it stems from the liberation of the cap moiety from the complex with eIF4E. Since VPg does not attach the cap, it appears that VPg induces changes in the eIF4E structure, diminishing its affinity to the cap. We show here that the initiation complex scaffold protein eIF(iso)4G increases VPg interaction with eIF(iso)4E. These data together suggest similar cap and VPg interactions with eIF4E and characterize VPg as a novel eIF4E-binding protein, which inhibits host protein synthesis at a very early stage of the initiation complex formation through the inhibition of cap attachment to the initiation factor eIF4E.     

Translational control by the poly(A) binding protein: a check for mRNA integrity

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the capbinding protein eIF4E and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this "closed loop" mRNP among other effects enhance the affinity of eIF4E for the 5' cap by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picomavirus' internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to initiation complex formation. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP.     

Suppression of translation during in vitro maturation of pig oocytes despite enhanced formation of cap-binding protein complex eIF4F and 4E-BP1 hyperphosphorylation

In this study, we document that the overall rate of protein synthesis decreases during in vitro maturation (IVM) of pig oocytes despite enhanced formation of the 5' cap structure eIF4F. Within somatic/interphase cells, formation of the eIF4F protein complex correlates very well with overall rates of protein translation, and the formation of this complex is controlled primarily by the availability of the 5' cap binding protein eIF4E. We show that the eIF4E inhibitory protein, 4E-BP1, becomes phosphorylated during IVM, which results in gradual release of eIF4E from 4E-BP1, as documented by immunoprecipitation analyses. Isoelectric focusing and Western blotting experiments show conclusively that eIF4E becomes gradually phosphorylated with a maximum at metaphase II (M II). The activity of eIF4E and its ability to bind mRNA also increases during oocyte maturation as documented in experiments with m7-methyl GTP-Sepharose, which mimics the cap structure of mRNA. Complementary analysis of flow-through fraction for 4E-BP1, and eIF4G proteins additionally provides evidence for enhanced formation of cap-binding protein complex eIF4F. Altogether, our results bring new insights to the regulation of translation initiation during meiotic division, and more specifically clarify that 4E-BP1 hyper-phosphorylation is not the cause of the observed suppression of overall translation rates.     

Weak binding affinity of human 4EHP for mRNA cap analogs

Ribosome recruitment to the majority of eukaryotic mRNAs is facilitated by the interaction of the cap binding protein, eIF4E, with the mRNA 5' cap structure. eIF4E stimulates translation through its interaction with a scaffolding protein, eIF4G, which helps to recruit the ribosome. Metazoans also contain a homolog of eIF4E, termed 4EHP, which binds the cap structure, but not eIF4G, and thus cannot stimulate translation, but it instead inhibits the translation of only one known, and possibly subset mRNAs. To understand why 4EHP does not inhibit general translation, we studied the binding affinity of 4EHP for cap analogs using two methods: fluorescence titration and stopped-flow measurements. We show that 4EHP binds cap analogs m(7)GpppG and m(7)GTP with 30 and 100 lower affinity than eIF4E. Thus, 4EHP cannot compete with eIF4E for binding to the cap structure of most mRNAs.     

Phosphorylation of eukaryotic translation initiation factor 4E is critical for growth

Eukaryotic translation initiation factor 4E (eIF4E) binds to the cap structure at the 5' end of mRNAs and is a critical target for the control of protein synthesis. eIF4E is phosphorylated in many systems in response to extracellular stimuli, but biochemical evidence to date has been equivocal as to the biological significance of this modification. Here we use a genetic approach to this problem. We show that, in Drosophila melanogaster, homozygous eIF4E mutants arrest growth during larval development. In Drosophila eIF4EI, Ser251 corresponds to Ser209 of mammalian eIF4E, which is phosphorylated in response to extracellular signals. We find that, in vivo, eIF4EI Ser251 mutants cannot incorporate labeled phosphate. Furthermore, transgenic Drosophila organisms expressing eIF4E(Ser251Ala) in an eIF4E mutant background have reduced viability. Escapers develop more slowly than control siblings and are smaller. These genetic data provide evidence that eIF4E phosphorylation is biologically significant and is essential for normal growth and development.     

The emerging roles of translation factor eIF4E in the nucleus

The emerging field of nuclear eIF research has yielded many surprises and led to the dissolution of some dogmatic/ideological viewpoints of the place of translation in the regulation of gene expression. Eukaryotic initiation factors (eIFs) are classically defined by their cytoplasmic location and ability to regulate the initiation phase of protein synthesis. For instance, in the cytoplasm, the m7G cap-binding protein eIF4E plays a distinct role in cap-dependent translation initiation. Disruption of eIF4E's regulatory function drastically effects cell growth and may lead to oncogenic transformation. A growing number of studies indicate that many eIFs, including a substantial fraction of eIF4E, are found in the nucleus. Indeed, nuclear eIF4E participates in a variety of important RNA-processing events including the nucleocytoplasmic transport of specific, growth regulatory mRNAs. Although unexpected, it is possible that some eIFs regulate protein synthesis within the nucleus. This review will focus on the novel, nuclear functions of eIF4E and how they contribute to eIF4E's growth-activating and oncogenic properties. Both the cytoplasmic and nuclear functions of eIF4E appear to be dependent on its intrinsic ability to bind to the 5' m7G cap of mRNA. For example, Promyelocytic Leukemia Protein (PML) potentially acts as a negative regulator of nuclear eIF4E function by decreasing eIF4E's affinity for the m7G cap. Therefore, eIF4E protein is flexible enough to utilize a common biochemical activity, such as m7G cap binding, to participate in divergent processes in different cellular compartments.     

Structures of the human eIF4E homologous protein, h4EHP, in its m7GTP-bound and unliganded forms

All eukaryotic cellular mRNAs contain a 5' m(7)GpppN cap. In addition to conferring stability to the mRNA, the cap is required for pre-mRNA splicing, nuclear export and translation by providing an anchor point for protein binding. In translation, the interaction between the cap and the eukaryotic initiation factor 4E (eIF4E) is important in the recruitment of the mRNAs to the ribosome. Human 4EHP (h4EHP) is a homologue of eIF4E. Like eIF4E it is able to bind the cap but it appears to play a different cellular role, possibly being involved in the fine-tuning of protein expression levels. Here we use X-ray crystallography and isothermal titration calorimetry (ITC) to investigate further the binding of cap analogues and peptides to h4EHP. m(7)GTP binds to 4EHP 200-fold more weakly than it does to eIF4E with the guanine base sandwiched by a tyrosine and a tryptophan instead of two tryptophan residues as seen in eIF4E. The tyrosine resides on a loop that is longer in h4EHP than in eIF4E. The consequent conformational difference between the proteins allows the tyrosine to mimic the six-membered ring of the tryptophan in eIF4E and adopt an orientation that is similar to that seen for equivalent residues in other non-homologous cap-binding proteins. In the absence of ligand the binding site is incompletely formed with one of the aromatic residues being disordered and the side-chain of the other adopting a novel conformation. A peptide derived from the eIF4E inhibitory protein, 4E-BP1 binds h4EHP 100-fold less strongly than eIF4E but in a similar manner. Overall the data, combined with sequence analyses of 4EHP from evolutionary diverse species, strongly support the hypothesis that 4EHP plays a physiological role utilizing both cap-binding and protein-binding functions but which is distinct from eIF4E.     

Stopped-flow kinetic analysis of eIF4E and phosphorylated eIF4E binding to cap analogs and capped oligoribonucleotides: evidence for a one-step binding mechanism

Recruitment of eukaryotic mRNA to the 48 S initiation complex is rate-limiting for protein synthesis under normal conditions. Binding of the 5' -terminal cap structure of mRNA to eIF4E is a critical event during this process. Mammalian eIF4E is phosphorylated at Ser-209 by Mnk1 and Mnk2 kinases. We investigated the interaction of both eIF4E and phosphorylated eIF4E (eIF4E(P)) with cap analogs and capped oligoribonucleotides by stopped-flow kinetics. For m(7)GpppG, the rate constant of association, k(on), was dependent on ionic strength, decreasing progressively up to 350 mm KCl, but the rate constant of dissociation, k(off), was independent of ionic strength. Phosphorylation of eIF4E decreased k(on) by 2.1-2.3-fold at 50-100 mm KCl but had progressively less effect at higher ionic strengths, being negligible at 350 mm. Contrary to published evidence, eIF4E phosphorylation had no effect on k(off). Several observations supported a simple one-step binding mechanism, in contrast to published reports of a two-step mechanism. The kinetic function that best fit the data changed from single- to double-exponential as the eIF4E concentration was increased. However, measuring k(off) for dissociation of a pre-formed eIF4E.m(7)GpppG complex suggested that the double-exponential kinetics were caused by dissociation of eIF4E dimers, not a two-step mechanism. Addition of a 12-nucleotide chain to the cap structure increased affinity at high ionic strength for both eIF4E (24-fold) and eIF4E(P) (7-fold), primarily due to a decrease in k(off). This suggests that additional stabilizing interactions between capped oligoribonucleotides and eIF4E, which do not occur with cap analogs alone, act to slow dissociation.