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.     

Drosophila Me31B is a Dual eIF4E-Interacting Protein

Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). In this study, we show that Drosophila eIF4E-1 and eIF4E-3 occur in PBs along the DEAD-box RNA helicase Me31B. We show that Me31B interacts with eIF4E-1 and eIF4E-3 by means of yeast two-hybrid system, FRET in D. melanogaster S2 cells and coimmunoprecipitation in testis. Truncation and point mutations of Me31B proteins show two eIF4E-binding sites located in different protein domains. Residues Y401-L407 (at the carboxy-terminus) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminus) are critical for interaction with eIF4E-3. The residue W117 in eIF4E-1 and the homolog position F103 in eIF4E-3 are necessary for Me31B-eIF4E interaction suggesting that the change of tryptophan to phenylalanine provides specificity. Me31B represents a novel type of eIF4E-interacting protein with dual and specific interaction domains that might be recognized by different eIF4E isoforms in different tissues, adding complexity to the control of gene expression in eukaryotes.     

Eukaryotic initiation factor 4E-3 is essential for meiotic chromosome segregation, cytokinesis and male fertility in Drosophila

Gene expression is translationally regulated during many cellular and developmental processes. Translation can be modulated by affecting the recruitment of mRNAs to the ribosome, which involves recognition of the 5' cap structure by the cap-binding protein eIF4E. Drosophila has several genes encoding eIF4E-related proteins, but the biological role of most of them remains unknown. Here, we report that Drosophila eIF4E-3 is required specifically during spermatogenesis. Males lacking eIF4E-3 are sterile, showing defects in meiotic chromosome segregation, cytokinesis, nuclear shaping and individualization. We show that eIF4E-3 physically interacts with both eIF4G and eIF4G-2, the latter being a factor crucial for spermatocyte meiosis. In eIF4E-3 mutant testes, many proteins are present at different levels than in wild type, suggesting widespread effects on translation. Our results imply that eIF4E-3 forms specific eIF4F complexes that are essential for spermatogenesis.     

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.     

Binding preference of eIF4E for 4E-binding protein isoform and function of eIF4E N-terminal flexible region for interaction, studied by SPR analysis

To investigate the binding preference of eIF4E for the three eIF4E-binding isoforms (4E-BP1-3) and the function of N-terminal flexible region of eIF4E for their interactions, the binding parameters of recombinant full-length and N-terminal residues-deleted eIF4Es with 4E-BP1-3 were investigated by the surface plasmon resonance (SPR) analysis. Consequently, it was clarified that 4E-BP2 exhibits the highest binding affinity for both m7GTP-bound and -unbound full-length eIF4Es when compared with 4E-BP1 and 4E-BP3. This is primarily due to the difference among their dissociation rates, because their association rates are almost the same. Interestingly, the deletion of the 33 N-terminal residues of eIF4E increased its binding affinities for 4E-BP1 and 4E-BP2 markedly, whereas such a change was not observed by at least the N-terminal deletion up to 26 residues. In contrast, the binding parameters of 4E-BP3 were hardly influenced by N-terminal deletion up to 33 residues. From the comparison of the amino acid sequences of 4E-BP1-3, the present result indicates the importance of N-terminal flexible region of eIF4E for the suppressive binding with 4E-BP1 and 2, together with the possible contribution of N-terminal sequence of 4E-BP isoform to the regulative binding to eIF4E.     

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.     

Selective involvement of members of the eukaryotic initiation factor 4E family in the infection of Arabidopsis thaliana by potyviruses

Arabidopsis thaliana plants with mutations in the genes encoding eukaryotic initiation factor (eIF4E) or isoform of eIF4E (eIF(iso)4E) were tested for susceptibility to Clover yellow vein virus (ClYVV), a member of the genus Potyvirus. ClYVV accumulated in both inoculated and upper uninoculated leaves of mutant plants lacking eIF(iso)4E, but not in mutant plants lacking eIF4E. In contrast, Turnip mosaic virus (TuMV), another member of the genus Potyvirus, multiplied in mutant plants lacking eIF4E but not in mutant plants lacking eIF(iso)4E. These results suggest the selective involvement of members of the eIF4E family in infection by potyviruses.     

Two zebrafish eIF4E family members are differentially expressed and functionally divergent

Eukaryotic translation initiation factor 4E (eIF4E) is an essential component of the translational machinery that binds m(7)GTP and mediates the recruitment of capped mRNAs by the small ribosomal subunit. Recently, a number of proteins with homology to eIF4E have been reported in plants, invertebrates, and mammals. Together with the prototypical translation factor, these constitute a new family of structurally related proteins. To distinguish the prototypical translation factor eIF4E from other family members, it has been termed eIF4E-1 (Keiper, B. D., Lamphear, B. J., Deshpande, A. M., Jankowska-Anyszka, M., Aamodt, E. J., Blumenthal, T., and Rhoads, R. E. (2000) J. Biol. Chem. 275, 10590-10596). We describe the characterization of two eIF4E family members in the zebrafish Danio rerio. Based on their relative identities with human eIF4E-1, these zebrafish proteins are termed eIF4E-1A (82%) and eIF4E-1B (66%). eIF4E-1B, originally termed eIF4E(L), has been reported previously as the zebrafish eIF4E-1 counterpart (Fahrenkrug, S. C., Dahlquist, M. O., Clark, K., and Hackett, P. B. (1999) Differentiation 65, 191-201; Fahrenkrug, S. C., Joshi, B., Hackett, P. B., and Jagus, R. (2000) Differentiation 66, 15-22). Sequence comparisons suggest that the two genes probably evolved from a duplication event that occurred during vertebrate evolution. eIF4E-1A is expressed ubiquitously in zebrafish, whereas expression of eIF4E-1B is restricted to early embryonic development and to gonads and muscle of the tissues investigated. The ability of these two zebrafish proteins to bind m(7)GTP, eIF4G, and 4E-BP, as well as to complement yeast conditionally deficient in functional eIF4E, show that eIF4E-1A is a functional equivalent of human eIF4E-1. Surprisingly, although eIF4E-1B possesses all known residues thought to be required for interaction with the cap structure, eIF4G, and 4E-BPs, it fails to interact with any of these components, suggesting that this protein serves a role other than that assigned to eIF4E.     

Structural basis for mRNA Cap-Binding regulation of eukaryotic initiation factor 4E by 4E-binding protein, studied by spectroscopic, X-ray crystal structural, and molecular dynamics simulation methods

Taking advantage of the Trp73 residue located close to the 4E-BP binding site of eIF4E, the interaction between the 4E-BP isoform and eIF4E was investigated by the Trp fluorescence titration method. Although no significant difference was observed among the association constants of three 4E-BP isoforms, the binding preference of 4E-BP2 over 4E-BP1 and -BP3 was shown, probably due to the effect of a 4E-BP2-specific LDRR (60-63) sequence for the binding with eIF4E. By contrast, surface plasmon resonance (SPR) analyses showed the binding preference of 4E-BP1, although the difference among the isoforms was also not significant. This inconsistency with fluorescence analysis likely resulted from the different observation points of the interaction, i.e., local and overall interactions observed by the fluorescence and SPR methods, respectively. To clarify the structural basis for these spectroscopic results, the crystal structure of the ternary complex of m7GpppA-eIF4E-4E-BP1 fragment (Thr36-Thr70) was analyzed by the X-ray diffraction method. Crystal structure analysis at 2.1 A resolution revealed that the 4E-BP1 fragment, assigned to the Pro47-Pro66 peptide moiety, adopted a reverse L-shaped conformation involving the beta sheet and alpha-helical structures and was located at the root of the handle of the temple-bell-shaped eIF4E through hydrophilic and hydrophobic interactions. Based on the observed binding mode, possible interactions with the three 4E-BP isoforms have been discussed. On the other hand, since the crystal structural comparison with the previously determined m7GpppA-eIF4E-4E binary complex showed that the docking of the 4E-BP1 fragment does not significantly affect the overall tertiary structure and cap-binding scaffold of eIF4E, the dynamic regulation of the cap-binding of eIF4E by 4E-BP1 was investigated by molecular dynamics (MD) simulations. Consequently, the simulation suggested that (i) the helical region of the 4E-BP1 peptide is important for the binding with eIF4E, (ii) the existence of a cap structure stabilizes the binding of eIF4E with 4E-BP, (iii) the binding of 4E-BP stabilizes the cap-binding pocket of eIF4E, and (iv) the phosphorylation of Ser67 alone does not induce the separation of 4E-BP from eIF4E, but increases the structural rigidity of 4E-BP. These results provide the structural basis for the mRNA cap-binding regulation of eIF4E by 4E-BP.     

Translation initiation factors eIF4E and eIFiso4E are required for polysome formation and regulate plant growth in tobacco

Eukaryotic initiation factor eIF4E plays a pivotal role in translation initiation. As a component of the ternary eIF4F complex, eIF4E interacts with the mRNA cap structure to facilitate recruitment of the 40S ribosomal subunit onto mRNA. Plants contain two distinct cap-binding proteins, eIF4E and eIFiso4E, that assemble into different eIF4F complexes. To study the functional roles of eIF4E and eIFiso4E in tobacco, we isolated two corresponding cDNAs, NteIF4E1 and NteIFiso4E1, and used these to deplete cap-binding protein levels in planta by antisense downregulation. Antibodies raised against recombinant NteIF4E1 detected three distinct cap-binding proteins in tobacco leaf extracts; NteIF4E and two isoforms of NteIFiso4E. The three cap-binding proteins were immuno-detected in all tissues analysed and were coordinately regulated, with peak expression in anthers and pollen. Transgenic tobacco plants showing significant depletion of either NteIF4E or the two NteIFiso4E isoforms displayed normal vegetative development and were fully fertile. Interestingly, NteIFiso4E depletion resulted in a compensatory increase in NteIF4E levels, whereas the down-regulation of NteIF4E did not trigger a reciprocal increase in NteIFiso4E levels. The antisense depletion of both NteIF4E and NteIFiso4E resulted in plants with a semi-dwarf phenotype and an overall reduction in polyribosome loading, demonstrating that both eIF4E and eIFiso4E support translation initiation in planta, which suggests their potential role in the regulation of plant growth.     

Tethered-function analysis reveals that elF4E can recruit ribosomes independent of its binding to the cap structure

The cap-binding complex elF4F is involved in ribosome recruitment during the initiation phase of translation and is composed of three subunits: elF4E, -4G, and -4A. The m7GpppN cap-binding subunit eIF4E binds the N-terminal region of eIF4G, which in turn contacts eIF4A through its central and C-terminal regions. We have previously shown, through a tethered-function approach in transfected HeLa cells, that the binding of eIF4G to an mRNA is sufficient to drive productive translation (De Gregorio et al., EMBO J, 1999, 18:4865-4874). Here we exploit this approach to assess which of the other subunits of elF4F can exert this function. eIF4AI or mutant forms of eIF4E were fused to the RNA-binding domain of the lambda phage antiterminator protein N to generate the chimeric proteins lambda4A, lambda4E-102 (abolished cap binding), and lambda4E-73-102 (impaired binding to both, the cap and eIF4G). The fusion proteins were directed to a bicistronic reporter mRNA by means of interaction with a specific lambda-N binding site (boxB) in the intercistronic space. We show that lambda4E-102, but neither the double mutant lambda4E-73-102 nor lambda4A, suffices to promote translation of the downstream gene in this assay. Coimmunoprecipitation analyses confirmed that all lambda-fusion proteins are capable of interacting with the appropriate endogenous eIF4F subunits. These results reveal that eIF4E, as well as eIF4G, can drive ribosome recruitment independent of a physical link to the cap structure. In spite of its interaction with endogenous eIF4G, lambda4A does not display this property. eIF4A thus appears to supply an essential auxiliary function to eIF4F that may require its ability to cycle into and out of this complex.     

Characterization of mammalian eIF4E-family members.

The translational factor eukaryotic initiation factor 4E (eIF4E) is a central component in the initiation and regulation of translation in eukaryotic cells. Through its interaction with the 5' cap structure of mRNA, eIF4E functions to recruit mRNAs to the ribosome. The accumulation of expressed sequence tag sequences has allowed the identification of three different eIF4E-family members in mammals termed eIF4E-1, eIF4E-2 (4EHP, 4E-LP) and eIF4E-3, which differ in their structural signatures, functional characteristics and expression patterns. Unlike eIF4E-1, which is found in all eukaryotes, orthologues for eIF4E-2 appear to be restricted to metazoans, while those for eIF4E-3 have been found only in chordates. Like prototypical eIF4E-1, eIF4E-2 was found to be ubiquitously expressed, with the highest levels in the testis. Expression of eIF4E-3 was detected only in heart, skeletal muscle, lung and spleen. Similarly to eIF4E-1, both eIF4E-2 and eIF4E-3 can bind to the mRNA cap-structure. However, in contrast to eIF4E-1 which interacts with both the scaffold protein, eIF4G and the translational repressor proteins, the eIF4E-binding proteins (4E-BPs), eIF4E-2 and eIF4E-3 each possesses a range of partial activities. eIF4E-2 does not interact with eIF4G, but does interact with 4E-BPs. Conversely, eIF4E-3 interacts with eIF4G, but not with 4E-BPs. Neither eIF4E-2 nor eIF4E-3 is able to rescue the lethality of eIF4E gene deletion in yeast. It is hypothesized that each eIF4E-family member fills a specialized niche in the recruitment of mRNAs by the ribosome through differences in their abilities to bind cap and/or to interact with eIF4G and the 4E-BPs.     

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.     

CPEB interacts with an ovary-specific eIF4E and 4E-T in early Xenopus oocytes

CPEB (cytoplasmic polyadenylation element-binding protein) is an important regulator of translation in oocytes and neurons. Although previous studies of CPEB in late Xenopus oocytes involve the eIF4E-binding protein maskin as the key factor for the repression of maternal mRNA, a second mechanism must exist, since maskin is absent earlier in oogenesis. Using co-immunoprecipitation and gel filtration assays, we show that CPEB specifically interacts, via protein/protein interactions, with the RNA helicase Xp54, the RNA-binding proteins P100(Pat1) and RAP55, the eIF4E-binding protein 4E-T, and an eIF4E protein. Remarkably, these CPEB complex proteins have been characterized, in one or more organism, as P-body, maternal, or neuronal granule components. We do not detect interactions with eIF4E1a, the canonical cap-binding factor, eIF4G, or eIF4A or with proteins expressed late in oogenesis, including maskin, PARN, and 4E-BP1. The eIF4E protein was identified as eIF4E1b, a close homolog of eIF4E1a, whose expression is restricted to oocytes and early embryos. Although eIF4E1b possesses all residues required for cap and eIF4G binding, it binds m(7)GTP weakly, and in pull-down assays, rather than binding eIF4G, it binds 4E-T, in a manner independent of the consensus eIF4E-binding site, YSKEELL. Wild type and Y-A mutant 4E-T (which binds eIF4E1b but not eIF4E1a), when tethered to a reporter mRNA, represses its translation in a cap-dependent manner, and injection of eIF4E1b antibody accelerates meiotic maturation. Altogether, our data suggest that CPEB, partnered with several highly conserved RNA-binding partners, inhibits protein synthesis in oocytes using a novel pairing of 4E-T and eIF4E1b.     

Cap binding-independent recruitment of eIF4E to cytoplasmic foci

Eukaryotic translation initiation factor 4E (eIF4E) is required for cap-dependent initiation. In addition, eIF4E occurs in cytoplasmic foci such as processing bodies (PB) and stress granules (SG). We examined the role of key functional amino acid residues of eIF4E in the recruitment of this protein to cytoplasmic foci. We demonstrate that tryptophan residues required for mRNA cap recognition are not required for the recruitment of eIF4E to SG or PB. We show that a tryptophan residue required for protein-protein interactions is essential for the accumulation of eIF4E in granules. Moreover, we show, by the analysis of two Drosophila eIF4E isoforms, that the tryptophan residue is the common feature for eIF4E for the transfer of active mRNA from polysomes to other ribonucleoprotein particles in the cytoplasm. This residue resides in a putative interaction domain different than the eIF4E-BP domain. We conclude that protein-protein interactions rather than interactions with the mRNA are essential for the recruitment of eIF4E and for a putative nucleation function.     

Discrimination between mono- and trimethylated cap structures by two isoforms of Caenorhabditis elegans eIF4E

Primitive eukaryotes like Caenorhabditis elegans produce mRNAs capped with either m(7)GTP or m(3)(2,2,7)GTP. Caenorhabditis elegans also expresses five isoforms of the cap-binding protein eIF4E. Some isoforms (e.g. IFE-3) bind to m(7)GTP-Sepharose exclusively, whereas others (e.g. IFE-5) bind to both m(7)GTP- and m(3)(2,2,7)GTP-Sepharose. To examine specificity differences, we devised molecular models of the tertiary structures of IFE-3 and IFE-5, based on the known structure of mouse eIF4E-1. We then substituted amino acid sequences of IFE-5 with homologous sequences from IFE-3. As few as two changes (N64Y/V65L) converted the cap specificity of IFE-5 to essentially that of IFE-3. Molecular dynamics simulations suggested that the width and depth of the cap-binding cavity were larger in IFE-5 than in IFE-3 or the N64Y/V65L variant, supporting a model in which IFE-3 discriminates against m(3)(2,2,7)GTP by steric hindrance. Furthermore, the affinity of IFE-5 (but not IFE-3) for m(3)(2,2,7)GTP was reversibly increased when thiol reagents were removed. This was correlated with the formation of a disulfide bond between Cys-122 and Cys-126. Thus, translation of m(3)(2,2,7)GTP-capped mRNAs may be regulated by intracellular redox state.     

Two Arabidopsis Loci Encode Novel Eukaryotic Initiation Factor 4E Isoforms That Are Functionally Distinct from the Conserved Plant Eukaryotic Initiation Factor 4E

Canonical translation initiation in eukaryotes begins with the Eukaryotic Initiation Factor 4F (eIF4F) complex, made up of eIF4E, which recognizes the 7-methylguanosine cap of messenger RNA, and eIF4G, which serves as a scaffold to recruit other translation initiation factors that ultimately assemble the 80S ribosome. Many eukaryotes have secondary EIF4E genes with divergent properties. The model plant Arabidopsis (Arabidopsis thaliana) encodes two such genes in tandem loci on chromosome 1, EIF4E1B (At1g29550) and EIF4E1C (At1g29590). This work identifies EIF4E1B/EIF4E1C-type genes as a Brassicaceae-specific diverged form of EIF4E. There is little evidence for EIF4E1C gene expression; however, the EIF4E1B gene appears to be expressed at low levels in most tissues, though microarray and RNA Sequencing data support enrichment in reproductive tissue. Purified recombinant eIF4E1b and eIF4E1c proteins retain cap-binding ability and form functional complexes in vitro with eIF4G. The eIF4E1b/eIF4E1c-type proteins support translation in yeast (Saccharomyces cerevisiae) but promote translation initiation in vitro at a lower rate compared with eIF4E. Findings from surface plasmon resonance studies indicate that eIF4E1b and eIF4E1c are unlikely to bind eIF4G in vivo when in competition with eIF4E. This study concludes that eIF4E1b/eIF4E1c-type proteins, although bona fide cap-binding proteins, have divergent properties and, based on apparent limited tissue distribution in Arabidopsis, should be considered functionally distinct from the canonical plant eIF4E involved in translation initiation.Cap-dependent translation in eukaryotes begins with recognition of the 7-methylguanosine cap at the 5′ end of an mRNA by the translation initiation factor eIF4E, which forms the eIF4F complex with the scaffolding protein eIF4G. The binding of the RNA helicase eIF4A along with eIF4B promotes unwinding of mRNA secondary structure (Aitken and Lorsch, 2012). The eIF4F complex then serves to circularize mRNA by interaction of eIF4G with poly(A) binding protein and recruit the preinitiation complex through binding of eIF4G to eIF3 and eIF5, ultimately leading to the assembly of the 80S ribosome (Aitken and Lorsch, 2012). eIF4E is an attractive target for global regulation of translational activity through its position at the earliest step, mRNA cap recognition. In many organisms, eIF4E availability is regulated by 4E-binding proteins as well as phosphorylation and sumoylation (Jackson et al., 2010; Xu et al., 2010). However, plants appear to lack 4E-binding proteins, and the role of phosphorylation of eIF4E in translational control is less clear (Pierrat et al., 2007).The eIF4E proteins generally thought to be involved in translation initiation are Class I eIF4E proteins (Joshi et al., 2005), of which two exist in flowering plants: eIF4E, which pairs with eIF4G to form the eIF4F complex, and the plant-specific isoform eIFiso4E, which pairs with eIFiso4G to form eIFiso4F (Mayberry et al., 2011; Patrick and Browning, 2012). Class I eIF4E family members have conserved Trp residues at positions equivalent to Trp-43 and Trp-56 of Homo sapiens eIF4E (Joshi et al., 2005), and the canonical members of this class, such as plant eIF4E and eIFiso4E, have the ability to promote translation through binding of mRNA cap structure and eIF4G (or eIFiso4G).In some organisms, however, secondary Class I isoforms exist with expression patterns and functions divergent from the conserved eIF4E (Rhoads, 2009). Caenorhabditis elegans has four isoforms involved in differentiation between mono- and trimethylated mRNA caps (Keiper et al., 2000) and have specialized roles for regulation of certain sets of mRNAs, particularly in the germline (Amiri et al., 2001; Song et al., 2010). Trypanosoma brucei has four isoforms with varying ability to bind cap analog and eIF4G isoforms (Freire et al., 2011). Schizosaccharomyces pombe has a second eIF4E isoform, eIF4E2, which is nonessential under normal growth conditions, but accumulates in response to high temperatures (Ptushkina et al., 2001). It cannot, however, complement deletion of EIF4E1, and while it can bind capped mRNA and promote translation in vitro, it has reduced ability to bind an eIF4G-derived peptide.Vertebrates encode a novel Class I isoform called EIF4E1B with oocyte-specific expression and functions (Evsikov and Marín de Evsikova, 2009). Zebrafish (Danio rerio) EIF4E1B, with expression limited to muscle and reproductive tissue, has conserved residues identified as necessary for binding cap analog and eIF4G, yet fails to bind either and cannot functionally complement deletion of yeast (Saccharomyces cerevisiae) eIF4E (Robalino et al., 2004). In Xenopus spp. oocytes, the eIF4E1b protein was found to bind eIF4E transporter and cytoplasmic polyadenylation element binding protein to form a translation-repressing complex (Minshall et al., 2007). Drosophila species have undergone extensive expansion of EIF4E-encoding loci to as many as seven different Class I eIF4E isoforms (Tettweiler et al., 2012). The seven EIF4E isoforms of Drosophila melanogaster are differentially expressed, with only five able to bind to eIF4G and complement deletion of yeast eIF4E (Hernández et al., 2005). The eIF4E-3 isoform of D. melanogaster was recently described as having a specific role in spermatogenesis (Hernández et al., 2012).Upon completion of sequencing of the Arabidopsis (Arabidopsis thaliana) genome (Rhee et al., 2003), it was discovered that in addition to the conserved plant EIF4E (At4g18040) and EIFISO4E (At5g35620), there existed a tandem pair of genes of high sequence similarity on chromosome 1 that also encoded Class I eIF4E family proteins, EIF4E1B (At1g29550, also known as EIF4E3) and EIF4E1C (At1g29590, also known as EIF4E2). Published microarray and RNA Sequencing (RNA-Seq) data indicate little to no EIF4E1C gene expression; however, the EIF4E1B gene appears to be expressed at low levels in most tissues and enriched in tissues involved in reproduction. The protein sequences contain the residues predicted to be involved in regular eIF4E function but also showed some divergence at highly conserved residues of the canonical plant eIF4E. Genome sequencing data indicate that these genes are part of a divergent eIF4E clade specific to Brassicaceae.The biochemical properties of the eIF4E1b and eIF4E1c proteins were investigated in this work, and it was found that while they can bind mRNA cap analog and eIF4G and support translation in yeast lacking eIF4E, their eIF4G-binding and translation initiation enhancing capabilities in vitro were less robust when compared with the conserved Arabidopsis eIF4E. In addition, it appears that these EIF4E1B-type genes cannot substitute for EIF4E or EIFISO4E in planta because deletion of both of these genes appears to be lethal. Taken together, these findings indicate the EIF4E1B-type genes represent a divergent eIF4E whose roles should be considered separately from the canonical eIF4E in plant translation initiation.