Interaction of three Caenorhabditis elegans isoforms of translation initiation factor eIF4E with mono- and trimethylated mRNA 5' cap analogues

Translation initiation factor eIF4E binds the m(7)G cap of eukaryotic mRNAs and mediates recruitment of mRNA to the ribosome during cap-dependent translation initiation. This event is the rate-limiting step of translation and a major target for translational control. In the nematode Caenorhabditis elegans, about 70% of genes express mRNAs with an unusual cap structure containing m(3)(2,2,7)G, which is poorly recognized by mammalian eIF4E. C. elegans expresses five isoforms of eIF4E (IFE-1, IFE-2, etc.). Three of these (IFE-3, IFE-4 and IFE-5) were investigated by means of spectroscopy and structural modelling based on mouse eIF4E bound to m(7)GDP. Intrinsic fluorescence quenching of Trp residues in the IFEs by iodide ions indicated structural differences between the apo and m(7)G cap bound proteins. Fluorescence quenching by selected cap analogues showed that only IFE-5 forms specific complexes with both m(7)G- and m(3)(2,2,7)G-containing caps (K(as) 2 x 10(6) M(-1) to 7 x 10(6) M(-1)) whereas IFE-3 and IFE-4 discriminated strongly in favor of m(7)G-containing caps. These spectroscopic results quantitatively confirm earlier qualitative data derived from affinity chromatography. The dependence of K(as) on pH indicated optimal cap binding of IFE-3, IFE-4 and IFE-5 at pH 7.2, lower by 0.4 pH units than that of eIF4E from human erythrocytes. These results provide insight into the molecular mechanism of recognition of structurally different caps by the highly homologous IFEs.     

Functional characterization of five eIF4E isoforms in Caenorhabditis elegans

Recognition of the 5'-cap structure of mRNA by eIF4E is a critical step in the recruitment of most mRNAs to the ribosome. In Caenorhabditis elegans, approximately 70% of mRNAs contain an unusual 2,2,7-trimethylguanosine cap structure as a result of trans-splicing onto the 5' end of the pre-mRNA. The characterization of three eIF4E isoforms in C. elegans (IFE-1, IFE-2, and IFE-3) was reported previously. The present study describes two more eIF4E isoforms expressed in C. elegans, IFE-4 and IFE-5. We analyzed the requirement of each isoform for viability by RNA interference. IFE-3, the most closely related to mammalian eIF4E-1, binds only 7-methylguanosine caps and is essential for viability. In contrast, three closely related isoforms (IFE-1, IFE-2, and IFE-5) bind 2,2, 7-trimethylguanosine caps and are partially redundant, but at least one functional isoform is required for viability. IFE-4, which binds only 7-methylguanosine caps, is most closely related to an unusual eIF4E isoform found in plants (nCBP) and mammals (4E-HP) and is not essential for viability in any combination of IFE knockout. ife-2, ife-3, ife-4, and ife-5 mRNAs are themselves trans-spliced to SL1 spliced leaders. ife-1 mRNA is trans-spliced to an SL2 leader, indicating that its gene resides in a downstream position of an operon.     

Identification in the ancient protist Giardia lamblia of two eukaryotic translation initiation factor 4E homologues with distinctive functions

Eukaryotic translation initiation factor 4E (eIF4E) binds to the m(7)GTP of capped mRNAs and is an essential component of the translational machinery that recruits the 40S small ribosomal subunit. We describe here the identification and characterization of two eIF4E homologues in an ancient protist, Giardia lamblia. Using m(7)GTP-Sepharose affinity column chromatography, a specific binding protein was isolated and identified as Giardia eIF4E2. The other homologue, Giardia eIF4E1, bound only to the m(2,2,7)GpppN structure. Although neither homologue can rescue the function of yeast eIF4E, a knockdown of eIF4E2 mRNA in Giardia by a virus-based antisense ribozyme decreased translation, which was shown to use m(7)GpppN-capped mRNA as a template. Thus, eIF4E2 is likely the cap-binding protein in a translation initiation complex. The same knockdown approach indicated that eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of both homologues in the cytoplasm. But eIF4E1 was also found concentrated and colocalized with the m(2,2,7)GpppN cap, 16S-like rRNA, and fibrillarin in the nucleolus-like structure in the nucleus. eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein was bound to Giardia small nuclear RNAs D and H known to have an m(2,2,7)GpppN cap, thus suggesting a novel function not yet observed among other eIF4Es in eukaryotes.     

Translation of a small subset of Caenorhabditis elegans mRNAs is dependent on a specific eukaryotic translation initiation factor 4E isoform

The mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) participates in protein synthesis initiation, translational repression of specific mRNAs, and nucleocytoplasmic shuttling. Multiple isoforms of eIF4E are expressed in a variety of organisms, but their specific roles are poorly understood. We investigated one Caenorhabditis elegans isoform, IFE-4, which has homologues in plants and mammals. IFE-4::green fluorescent protein (GFP) was expressed in pharyngeal and tail neurons, body wall muscle, spermatheca, and vulva. Knockout of ife-4 by RNA interference (RNAi) or a null mutation produced a pleiotropic phenotype that included egg-laying defects. Sedimentation analysis demonstrated that IFE-4, but not IFE-1, was present in 48S initiation complexes, indicating that it participates in protein synthesis initiation. mRNAs affected by ife-4 knockout were determined by DNA microarray analysis of polysomal distribution. Polysome shifts, in the absence of total mRNA changes, were observed for only 33 of the 18,967 C. elegans mRNAs tested, of which a disproportionate number were related to egg laying and were expressed in neurons and/or muscle. Translational regulation was confirmed by reduced levels of DAF-12, EGL-15, and KIN-29. The functions of these proteins can explain some phenotypes observed in ife-4 knockout mutants. These results indicate that translation of a limited subset of mRNAs is dependent on a specific isoform of eIF4E.     

An isoform of eIF4E is a component of germ granules and is required for spermatogenesis in C. elegans

Control of gene expression at the translational level is crucial for many developmental processes. The mRNA cap-binding protein, eIF4E, is a key player in regulation of translation initiation; appropriate levels of eIF4E are essential for normal cell-cycle regulation and tissue differentiation. The observation that eIF4E levels are elevated during gametogenesis in several organisms suggests that eIF4E might have a specific role in gamete formation as well. We show that one of the five isoforms of C. elegans eIF4E, IFE-1, is enriched in the germline and is a component of germ granules (P granules). The association of IFE-1 with P granules requires the P-granule protein PGL-1. In vitro PGL-1 interacts directly with IFE-1, but not with the other four isoforms of eIF4E. Analysis of animals depleted of IFE-1 by RNAi shows that IFE-1 is required for spermatogenesis, specifically for efficient progression through the meiotic divisions and for the production of functional sperm, in both hermaphrodites and males. The requirement for IFE-1 is highly sensitive to temperature. IFE-1 is not required for oogenesis, as ife-1(RNAi) hermaphrodites produce viable progeny when normal sperm are supplied. Consistent with a primary role in spermatogenesis, ife-1 mRNA levels are highest in regions of the gonad undergoing spermatogenesis. Our results suggest that C. elegans spermatogenesis requires either this specific isoform of eIF4E or an elevated level of eIF4E.     

Cap-dependent deadenylation of mRNA

Poly(A) tail removal is often the initial and rate-limiting step in mRNA decay and is also responsible for translational silencing of maternal mRNAs during oocyte maturation and early development. Here we report that deadenylation in HeLa cell extracts and by a purified mammalian poly(A)-specific exoribonuclease, PARN (previously designated deadenylating nuclease, DAN), is stimulated by the presence of an m(7)-guanosine cap on substrate RNAs. Known cap-binding proteins, such as eIF4E and the nuclear cap-binding complex, are not detectable in the enzyme preparation, and PARN itself binds to m(7)GTP-Sepharose and is eluted specifically with the cap analog m(7)GTP. Xenopus PARN is known to catalyze mRNA deadenylation during oocyte maturation. The enzyme is depleted from oocyte extract with m(7)GTP-Sepharose, can be photocross-linked to the m(7)GpppG cap and deadenylates m(7)GpppG-capped RNAs more efficiently than ApppG-capped RNAs both in vitro and in vivo. These data provide additional evidence that PARN is responsible for deadenylation during oocyte maturation and suggest that interactions between 5' cap and 3' poly(A) tail may integrate translational efficiency with mRNA stability.     

Evolutionary changes in the Leishmania eIF4F complex involve variations in the eIF4E–eIF4G interactions

Translation initiation in eukaryotes is mediated by assembly of the eIF4F complex over the m⁷GTP cap structure at the 5′-end of mRNAs. This requires an interaction between eIF4E and eIF4G, two eIF4F subunits. The Leishmania orthologs of eIF4E are structurally diverged from their higher eukaryote counterparts, since they have evolved to bind the unique trypanosomatid cap-4 structure. Here, we characterize a key eIF4G candidate from Leishmania parasites (LeishIF4G-3) that contains a conserved MIF4G domain. LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m⁷GTP-Sepharose column and to bind directly to LeishIF4E. In higher eukaryotes the eIF4E-eIF4G interaction is based on a conserved peptide signature [Y(X₄)Lϕ], where X is any amino acid and Φ is a hydrophobic residue. A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27). However, the binding motif varies extensively: in addition to Y20 and L25, binding strictly requires the presence of F23, whereas the hydrophobic amino acid (Φ) is dispensable. The LeishIF4E–LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies. In view of these diversities, the characterization of the parasite eIF4E–eIF4G interaction may not only serve as a novel target for inhibiting Leishmaniasis but also provide important insight for future drug discovery.     

Nematode m7GpppG and m3(2,2,7)GpppG decapping: activities in Ascaris embryos and characterization of C. elegans scavenger DcpS

A spliced leader contributes the mature 5'ends of many mRNAs in trans-splicing organisms. Trans-spliced metazoan mRNAs acquire an m3(2,2,7)GpppN cap from the added spliced leader exon. The presence of these caps, along with the typical m7GpppN cap on non-trans-spliced mRNAs, requires that cellular mRNA cap-binding proteins and mRNA metabolism deal with different cap structures. We have developed and used an in vitro system to examine mRNA degradation and decapping activities in nematode embryo extracts. The predominant pathway of mRNA decay is a 3' to 5' pathway with exoribonuclease degradation of the RNA followed by hydrolysis of resulting mRNA cap by a scavenger (DcpS-like) decapping activity. Direct decapping of mRNA by a Dcp1/Dcp2-like activity does occur, but is approximately 15-fold less active than the 3' to 5' pathway. The DcpS-like activity in nematode embryo extracts hydrolyzes both m7GpppG and m3(2,2,7)GpppG dinucleoside triphosphates. The Dcp1/Dcp2-like activity in extracts also hydrolyzes these two cap structures at the 5' ends of RNAs. Interestingly, recombinant nematode DcpS differs from its human ortholog in its substrate length requirement and in its capacity to hydrolyze m3(2,2,7)GpppG.     

Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania

The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.     

Cap-binding activity of an eIF4E homolog from Leishmania

All eukaryotic mRNAs possess a 5'-cap (m(7)GpppN) that is recognized by a family of cap-binding proteins. These participate in various processes, such as RNA transport and stabilization, as well as in assembly of the translation initiation complex. The 5'-cap of trypanosomatids is complex; in addition to 7-methyl guanosine, it includes unique modifications on the first four transcribed nucleotides, and is thus denoted cap-4. Here we analyze a cap-binding protein of Leishmania, in an attempt to understand the structural features that promote its binding to this unusual cap. LeishIF4E-1, a homolog of eIF4E, contains the conserved cap-binding pocket, similar to its mouse counterpart. The mouse eIF4E has a higher K(as) for all cap analogs tested, as compared with LeishIF4E-1. However, whereas the mouse eIF4E shows a fivefold higher affinity for m(7)GTP than for a chemically synthesized cap-4 structure, LeishIF4E-1 shows similar affinities for both ligands. A sequence alignment shows that LeishIF4E-1 lacks the region that parallels the C terminus in the murine eIF4E. Truncation of this region in the mouse protein reduces the difference that is observed between its binding to m(7)GTP and cap-4, prior to this deletion. We hypothesize that variations in the structure of LeishIF4E-1, possibly also the absence of a region that is homologous to the C terminus of the mouse protein, promote its ability to interact with the cap-4 structure. LeishIF4E-1 is distributed in the cytoplasm, but its function is not clear yet, because it cannot substitute the mammalian eIF4E in a rabbit reticulocyte in vitro translation system.     

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.     

Structural basis for nematode eIF4E binding an m(2,2,7)G-Cap and its implications for translation initiation

Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m^2,2,7G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an m^2,2,7G-cap is unknown. Here, we describe the first structure of an eIF4E with an m^2,2,7G-cap and compare it to the cognate m⁷G-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4E binds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m^2,2,7G-cap. Nematode and mammalian eIF4E both have a low affinity for m^2,2,7G-cap compared with the m⁷G-cap. Nematode eIF4E binding to the m⁷G-cap, m^2,2,7G-cap and the m^2,2,7G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m^2,2,7G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m^2,2,7G-cap is present. These data have implications for the contribution of 5′-UTRs in mRNA translation and the function of different eIF4E isoforms.     

Multiple roles for the C-terminal domain of eIF5 in translation initiation complex assembly and GTPase activation

eIF5 stimulates the GTPase activity of eIF2 bound to Met-tRNA(i)(Met), and its C-terminal domain (eIF5-CTD) bridges interaction between eIF2 and eIF3/eIF1 in a multifactor complex containing Met-tRNA(i)(Met). The tif5-7A mutation in eIF5-CTD, which destabilizes the multifactor complex in vivo, reduced the binding of Met-tRNA(i)(Met) and mRNA to 40S subunits in vitro. Interestingly, eIF5-CTD bound simultaneously to the eIF4G subunit of the cap-binding complex and the NIP1 subunit of eIF3. These interactions may enhance association of eIF4G with eIF3 to promote mRNA binding to the ribosome. In vivo, tif5-7A eliminated eIF5 as a stable component of the pre-initiation complex and led to accumulation of 48S complexes containing eIF2; thus, conversion of 48S to 80S complexes is the rate-limiting defect in this mutant. We propose that eIF5-CTD stimulates binding of Met-tRNA(i)(Met) and mRNA to 40S subunits through interactions with eIF2, eIF3 and eIF4G; however, its most important function is to anchor eIF5 to other components of the 48S complex in a manner required to couple GTP hydrolysis to AUG recognition during the scanning phase of initiation.     

Structural Insights into Parasite eIF4E Binding Specificity for m7G and m2,2,7G mRNA Caps

The eukaryotic translation initiation factor eIF4E recognizes the mRNA cap, a key step in translation initiation. Here we have characterized eIF4E from the human parasite Schistosoma mansoni. Schistosome mRNAs have either the typical monomethylguanosine (m⁷G) or a trimethylguanosine (m^2,2,7G) cap derived from spliced leader trans-splicing. Quantitative fluorescence titration analyses demonstrated that schistosome eIF4E has similar binding specificity for both caps. We present the first crystal structure of an eIF4E with similar binding specificity for m⁷G and m^2,2,7G caps. The eIF4E·m⁷GpppG structure demonstrates that the schistosome protein binds monomethyl cap in a manner similar to that of single specificity eIF4Es and exhibits a structure similar to other known eIF4Es. The structure suggests an alternate orientation of a conserved, key Glu-90 in the cap-binding pocket that may contribute to dual binding specificity and a position for mRNA bound to eIF4E consistent with biochemical data. Comparison of NMR chemical shift perturbations in schistosome eIF4E on binding m⁷GpppG and m^2,2,7GpppG identified key differences between the two complexes. Isothermal titration calorimetry demonstrated significant thermodynamics differences for the binding process with the two caps (m⁷G versus m^2,2,7G). Overall the NMR and isothermal titration calorimetry data suggest the importance of intrinsic conformational flexibility in the schistosome eIF4E that enables binding to m^2,2,7G cap.     

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, {"type":"entrez-protein","attrs":{"text":"CGP57380","term_id":"877393391","term_text":"CGP57380"}}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 m⁷GTP-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.     

The structure of eukaryotic translation initiation factor-4E from wheat reveals a novel disulfide bond

Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m(7) guanosine nucleotide at the 5' end of eukaryotic messenger RNAs; this protein-RNA interaction is an essential step in the initiation of protein synthesis. The structure of eIF4E from wheat (Triticum aestivum) was investigated using a combination of x-ray crystallography and nuclear magnetic resonance (NMR) methods. The overall fold of the crystallized protein was similar to eIF4E from other species, with eight beta-strands, three alpha-helices, and three extended loops. Surprisingly, the wild-type protein did not crystallize with m(7)GTP in its binding site, despite the ligand being present in solution; conformational changes in the cap-binding loops created a large cavity at the usual cap-binding site. The eIF4E crystallized in a dimeric form with one of the cap-binding loops of one monomer inserted into the cavity of the other. The protein also contained an intramolecular disulfide bridge between two cysteines (Cys) that are conserved only in plants. A Cys-to-serine mutant of wheat eIF4E, which lacked the ability to form the disulfide, crystallized with m(7)GDP in its binding pocket, with a structure similar to that of the eIF4E-cap complex of other species. NMR spectroscopy was used to show that the Cys that form the disulfide in the crystal are reduced in solution but can be induced to form the disulfide under oxidizing conditions. The observation that the disulfide-forming Cys are conserved in plants raises the possibility that their oxidation state may have a role in regulating protein function. NMR provided evidence that in oxidized eIF4E, the loop that is open in the ligand-free crystal dimer is relatively flexible in solution. An NMR-based binding assay showed that the reduced wheat eIF4E, the oxidized form with the disulfide, and the Cys-to-serine mutant protein each bind m(7)GTP in a similar and labile manner, with dissociation rates in the range of 20 to 100 s(-1).     

Mutations in the S4-H2 loop of eIF4E which increase the affinity for m7GTP

Eukaryotic initiation factor 4E (eIF4E) binds the 5'-cap of eukaryotic mRNAs and overexpression of eIF4E in epithelial cell cancers correlates with the metastases/tissue invasion phenotype. Photolabeling of eIF4E with [gamma-32P]8-azidoguanosine 5'-triphosphate (8-N3GTP) demonstrated cross-linking at Lys-119 in the S4-H2 loop which is distant from the m7GTP binding site [Marcotrigiano et al. (1997) Cell 89, 951-961; Friedland et al. (1997) Protein Sci. 6, 125-131]. Modeling studies indicate that 8-N3GTP cross-linked with Lys-119 because it binds a site that is occupied by the second nucleotide of a bound mRNA. Mutagenesis of the S4-H2 loop produced proteins with a 5-10-fold higher affinity for m7GTP than wild-type eIF4E. These mutants of eIF4E may have uses in selectively purifying mRNAs with intact 5'-ends or in determining how the promyelocytic leukemia protein decreases the affinity of eIF4E for mRNA caps.     

Dynamical insight into <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> eIF4E recognition specificity for mono-and trimethylated structures of mRNA 5′ cap

Specific recognition and binding of the ribonucleic acid 5′ termini (mRNA 5′ cap) by the eukaryotic translation initiation factor 4E (eIF4E) is a key, rate limiting step in translation initiation. Contrary to mammalian and yeast eIF4Es that discriminate in favor of 7-methylguanosine cap, three out of five eIF4E isoforms from the nematode Caenorhabditis elegans as well as eIF4Es from the parasites Schistosome mansoni and Ascaris suum, exhibit dual binding specificity for both 7-methylguanosine-and N²,N²,7-trimethylguanosine cap. To address the problem of the differences in the mechanism of the cap recognition by those highly homologic proteins, we carried out molecular dynamics simulations in water of three factors, IFE-3 and IFE-5 isoforms from C. elegans and murine eIF4E, in the apo form as well as in the complexes with 7-methyl-GDP and N²,N²,7-trimethyl-GDP. The results clearly pointed to a dynamical mechanism of discrimination between each type of the cap, viz. differences in mobility of the loops located at the entrance into the protein binding pockets during the cap association and dissociation. Additionally, our data showed that the hydrogen bond involving the N²-amino group of 7-methylguanosine and the carboxylate of glutamic acid was not stable. The dynamic mechanism proposed here differs from a typical, static one in that the differences in the protein-ligand binding specificity cannot be ascribed to formation and/or disruption of well defined stabilizing contacts.