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eIF4E: New Family Members, New Binding Partners, New Roles*

Eukaryotic initiation factor 4E (eIF4E) has long been known as the cap-binding protein that participates in recruitment of mRNA to the ribosome. A number of recent advances have not only increased our understanding of how eIF4E acts in translation but also uncovered non-translational roles. New structures have been determined for eIF4E in complex with various ligands and for other cap-binding proteins. We have also learned that most eukaryotic organisms express multiple eIF4E family members, some involved in general translation but others having specialized functions, including repression of translation. A number of new eIF4E-binding proteins have been reported, some of which tether it to specific mRNAs.

Eukaryotic initiation factor 4E (eIF4E) has long been known as the cap-binding protein that participates in recruitment of mRNA to the ribosome. A number of recent advances have not only increased our understanding of how eIF4E acts in translation but also uncovered non-translational roles. New structures have been determined for eIF4E in complex with various ligands and for other cap-binding proteins. We have also learned that most eukaryotic organisms express multiple eIF4E family members, some involved in general translation but others having specialized functions, including repression of translation. A number of new eIF4E-binding proteins have been reported, some of which tether it to specific mRNAs.
The 7-methylguanosine-containing "cap" plays an essential role at each stage of the mRNA "life cycle": transcription, splicing, nuclear export, translation, translational repression, and degradation. This is mediated by specific cap-binding proteins, of which at least 10 have been discovered so far (supplemental Table S1). The most widely studied and best understood of these cap-binding proteins is eIF4E. 2 eIF4E was discovered as a protein that promotes translation initiation, and most of the work on its structure, function, and regulation has been performed with this role in mind. As a canonical initiation factor involved in recruitment of mRNA to the ribosome, eIF4E has the potential to influence expression of virtually every protein in the cell. It is against this backdrop of eIF4E serving as a translational enhancer that discoveries over the past decade have been so surprising, that eIF4E can also function as a translational repressor. Another unexpected result of recent research is that nearly all eukaryotes express multiple eIF4E family members and that they can have different functions in the cell. A third research trend has been the discovery of new eIF4E-binding partners, some of which tether eIF4E to specific mRNAs. This minireview touches on all of these new directions in eIF4E research.

Role of eIF4E in Translation
Recruitment of mRNA to the 43 S initiation complex to form the 48 S initiation complex requires eIF3, the poly(A)-binding protein, and the eIF4 proteins ( Fig. 1B) (1). The eIF4 factors consist of eIF4A, a 46-kDa RNA helicase; eIF4B, a 70-kDa RNA-binding and RNA-annealing protein; eIF4E, a 25-kDa cap-binding protein; eIF4H, a 25-kDa protein that acts with eIF4B to stimulate eIF4A helicase activity; and eIF4G, a 185-kDa protein that co-localizes all of the other proteins involved in mRNA recruitment on the 40 S subunit. 3 At least four cis-acting elements affect the efficiency of mRNA translation: the cap, the poly(A) tail, sequence elements in the 5Ј-UTR, and sequence elements in the 3Ј-UTR. It is well established that eIF4E stimulates translation of capped mRNAs, but mRNAs differ in their dependence on eIF4E. For instance, mRNAs with extensive 5Ј-UTR secondary structure have a greater requirement for the eIF4A-based unwinding machinery recruited by eIF4E ( Fig. 1B) (2).
The tertiary structures of mouse, yeast, human, and wheat eIF4Es have been solved (3)(4)(5)(6). The specificity of eIF4E interaction with the cap results primarily from cationbond stacking between Trp-56 and Trp-102 and H-bonds between Glu-103 and the N-1 and N-2 protons of 7-methylguanine. 4 Following the initial structure determination of eIF4E, structures have emerged for a number of non-eIF4E cap-binding proteins (supplemental Table S1). Interestingly, the cationbond interaction between the m 7 G moiety and aromatic amino acid residues is a feature shared by most of these proteins. An exception is the decapping enzyme Dcp2, which, unlike eIF4E, binds the cap only when attached to the RNA body (7).

eIF4E Family Members
It is common to have protein families with multiple members, but eIF4E was initially assumed (albeit tacitly) to be a single protein, in part because only a single 25-kDa polypeptide was obtained from mammalian sources by affinity chromatography. When the Ravel laboratory made the unexpected discovery that wheat germ contained two versions of eIF4E (8), termed eIF4E and eIF(iso)4E, it seemed to be a peculiarity of this organism. More than 10 years later, it was reported almost simultaneously that Arabidopsis thaliana expresses not only eIF4E and eIF(iso)4E but also nCBP (9); Homo sapiens expresses a second family member, 4EHP (10); and Caenorhabditis elegans expresses three eIF4E family members, IFE-1, -2, and -3 (11). It is now recognized that the wheat germ "oddity" is the norm; virtually all eukaryotes express multiple eIF4E family members. Multiple family members have been found for other initiation factors as well, e.g. eIF4A and eIF4G.
Joshi et al. (12) combined published eIF4E sequences with sequences mined from GenBank TM to identify 411 eIF4E family members from 230 species. An unusual feature of the eIF4E sequence is the high content of Trp residues. Some of these are involved in the binding of the cap (supplemental Table S1) and eIF4G (3)(4)(5)13). These Trp residues allowed Joshi et al. (12) to discern a core region with consensus sequence H(X 5 )W(X 2 )W(X 8 -12 )W-(X 9 )F(X 5 )FW(X 20 )F(X 7 )W(X 10 )W(X 9 -12 )W(X 34 -35 )W(X 32-34 )H in seven well established eIF4Es. They used this to subdivide eIF4E family members into three classes according to the residues corresponding to Trp-43 and Trp-56 of H. sapiens eIF4E-1. Class I members contain Trp at both positions; Class II members, e.g. 4EHP, nCBP, IFE-4, and d4EHP, contain Tyr, Phe, or Leu at the first position and Tyr or Phe at the second position; and Class III members contain Trp at the first position and Cys or Tyr at the second position.

Binding Partners for eIF4E
eIF4E was initially discovered as a single polypeptide, but when it was subsequently found in complexes with eIF4G, it was redefined as a subunit of a new initiation factor in mammals termed eIF4F. However, the pioneering work of the Sonenberg and Lawrence laboratories showed that eIF4E can have other binding partners besides eIF4G, namely 4E-BP1, -2, and -3 (49,50). The number of known eIF4E-binding partners has grown substantially over the past decade ( Table 2). Many of the new roles that have been discovered for eIF4E are mediated by its interaction with these new binding partners. eIF4E participates in mRNA recruitment through specific and high affinity binding to eIF4G (Fig. 1B). X-ray crystallogra-  (17). A similar mechanism may occur with p20 and Gemin5 (Table  2). B, eIF4E in the translationally active 48 S initiation complex (1). The interactions among eIF1, eIF2, eIF3, eIF4A, eIF4E, eIF4G, eIF5, Mnk (mitogen-activated protein kinase-interacting kinase), poly(A)-binding protein (PABP), mRNA, and the 40 S ribosomal subunit are shown. C, eIF4E in a translationally inactive complex with Maskin, bound through CPEB to a CPE-containing mRNA (19). Similar mechanisms may occur with Cup, Bicoid, Brat, Neuroguidin, and CYFIP1 ( Table 2).  (14) Class III M. musculus eIF4E-3 Binds to eIF4G but not 4E-BPs (14) phy, sequence comparisons, and mutational studies have shown that the interaction between eIF4E and either eIF4G or the 4E-BPs involves the sequence motif YXXXXL⌽, where X is any amino acid and ⌽ is Leu, Met, or Phe (13). Most other eIF4E-binding partners appear to utilize similar motifs ( Table  2). Two conserved Trp residues in eIF4E are important for the interaction with eIF4G, Trp-43 and Trp-73, 4 the latter being found within a phylogenetically conserved sequence, (S/T)V(E/ D)(E/D)FW (12). eIF4G and 4E-BP1 bind the dorsal surface of eIF4E, opposite to the cap-binding pocket, and undergo a disordered-ordered transition upon binding to eIF4E (13). The solution structure of the complex between Saccharomyces cerevisiae eIF4E⅐m 7 GDP and eIF4G-(393-490) revealed that a coupled folding transition occurs for both proteins, with the eIF4G domain wrapping around the N terminus of eIF4E (18).
Binding of eIF4E to either the 4E-BPs or eIF4G is mutually exclusive (17). Sequestration of eIF4E from the 48 S initiation complex (Fig. 1A) inhibits translation of mRNAs that require the eIF4A-driven unwinding machinery, e.g. those mRNAs with extensive 5Ј-UTR secondary structure (2). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation in response to polypeptide hormones such as insulin that activate the mTOR pathway (17).
The next major development in our understanding of eIF4Ebinding partners came from the Richter laboratory, which showed that an eIF4E-binding protein, Maskin, can be tethered to a specific mRNA by virtue of a 3Ј-UTR sequence motif (Fig.  1C) (19). This provides a mechanism for mRNA-specific translational repression. Xenopus laevis oocytes arrested in meiotic progression contain silent maternal mRNAs with short poly(A) tails. Upon exposure to progesterone, the poly(A) is elongated and translation begins, which is a requirement for maturation. These "masked messengers" contain a CPE in their 3Ј-UTRs, which regulates poly(A) length by binding CPEB, the poly(A) polymerase Gld2, and the poly(A)-specific ribonuclease PARN. Translational repression is due to both the short poly(A) tail and sequestration of eIF4E by Maskin. Progesterone initiates a signaling cascade that results in phosphorylation of CPEB, leading to dissociation of PARN, polyadenylation by Gld2, displacement of Maskin from eIF4E, and initiation of translation. Piqué et al. (20) have recently found that the timing of polyadenylation is governed by a "combinatorial code" of CPEs and Pumilio-binding elements in the 3Ј-UTRs of these mRNAs.
A similar mechanism appears to operate during neurogenesis, in which Neuroguidin binds to eIF4E and represses translation of CPE-containing mRNAs (21). The protein is detected in axons and dendrites of the mammalian nervous system and contains three eIF4E-binding motifs ( Table 2). Neural tube closure and neural crest migration are arrested in X. laevis embryos if Neuroguidin is depleted, suggesting that Neuroguidin regulates the translation of CPE-containing mRNAs to direct neural development. Another eIF4E-binding protein that functions in neuronal dendrites is CYFIP1 (22). FMRP is an RNA-binding protein that is important for synaptic maturation. FMRP recognizes mRNAs through G quartets and U-rich sequences as well as through small noncoding RNAs. A binding partner of FMRP, CYFIP1, is also an eIF4E-binding protein ( Table 2). Stimulation of neurons by neurotrophic factors causes CYFIP1 to dissociate from eIF4E at synapses, thereby resulting in translation of mRNAs previously repressed by FMRP.
eIF4E-binding proteins are also involved in embryogenesis. In the early Drosophila melanogaster embryo, Nanos protein represses the translation of hunchback mRNA, permitting posterior development. The 3Ј-UTR of nanos mRNA contains stem/loop structures that repress the translation of unlocalized nanos mRNA. Two of these stem/loops bind Smaug, a sequence-specific RNA-binding protein that functions as a translational repressor. Nelson et al. (23) showed that Smaug interacts with Cup and that Cup is an eIF4E-binding protein ( Table 2). Nakamura et al. (24) also found Cup to be an eIF4Ebinding partner, but in this case mediating the repression of oskar mRNA translation during its localization to the posterior of the oocyte. Repression is mediated by Bruno, a protein that binds the 3Ј-UTR of oskar mRNA. Likewise, Zappavigna et al. (25), studying oogenesis in D. melanogaster, showed that Cup was an eIF4E-binding partner.
The VPg proteins of plant potyviruses and human caliciviruses are covalently linked to the 5Ј-end of the viral genome but also act as eIF4E-binding proteins (26,27). This apparently pro- Represses translation of mRNAs that bind FMRP LLLDKRKRSEC (22) a These were determined from sequence alignments. The residues in italics align with Tyr of H. sapiens eIF4G-1 shown in the first row. The residues shown underlined and in boldface are implicated in eIF4E binding by mutational studies. b aa, amino acid(s); TuMV, turnip mosaic virus; IRES, internal ribosome entry site.
vides the virus with a translational advantage over the host (Table 2). Naturally occurring potyviral resistance has been mapped to the genes for either eIF4E or eIF(iso)4E, depending on the species (28).

Physiological Roles for Specific eIF4E Family Members
Hernández and Vazquez-Pianzola (29) have suggested that there is one eIF4E family member in each organism that is ubiquitously and constitutively expressed to carry out general translation and that others are involved in specialized functions, both translational and non-translational. Examples of this are beginning to emerge (Table 1).
IFE-1 and Spermatogenesis-IFE-1, one of five eIF4E family members in C. elegans (11,16), is enriched in the germ line. It is the only one of the five to bind the P granule-resident protein PGL-1, which localizes IFE-1 to P granules (30). Depletion of IFE-1 shows that it is required for spermatogenesis, specifically progression through the meiotic divisions and production of functional sperm, in both hermaphrodites and males, whereas oogenesis is not affected. Sequestration of IFE-1 in P granules may regulate its level or function during spermatogenesis.

IFE-4 and Egg
Laying-IFE-4, a Class II eIF4E family member in C. elegans, is expressed in neurons, muscle, spermatheca, and vulva (31). Knock-out of ife-4 produces a pleiotropic phenotype that includes a defect in egg laying. A global analysis of the polysomal distribution of individual mRNAs revealed that translation of only a small subset of mRNAs is affected by ife-4 loss. The steady-state levels of proteins encoded by these mRNAs are correspondingly reduced, the functions of which can explain some of the phenotypic traits of ife-4 knock-out mutants.
IFE-2 and Aging-A third C. elegans family member, IFE-2, is expressed in somatic tissues. Knock-out of the ife-2 gene reduces global protein synthesis, protects from oxidative stress, and extends life span (32). The signaling through IFE-2 appears to be different from that of previously discovered longevity pathways. A model was proposed in which down-regulation of protein synthesis in the soma facilitates cellular maintenance and repair by diminishing the large energy requirement of translation, thus delaying the decline of somatic functions and senescence.
eIF4E-1B and Oogenesis-A study by Minshall et al. (33) combined two themes covered above: a specific eIF4E family member (Table 1) and an eIF4E-binding partner tethered to 3Ј-UTR sequence motifs (Table 2). These authors found that the oocyte-specific X. laevis eIF4E-1B is responsible for repressing translation of CPE-containing mRNAs early in oogenesis when Maskin is absent. They found that CPEB specifically interacts with an RNA helicase (Xp54), two RNA-binding proteins (Pat1 and RAP55), an eIF4E-binding partner (4E-T), and eIF4E-1B. Further evidence for eIF4E-1B-mediated translational repression comes from a very recent study by Evsikov and Márin de Evsikova (34), who depleted eIF4E-1B in fully grown stage VI oocytes of Xenopus tropicalis and observed acceleration of oocyte maturation after progesterone induction.
d4EHP and Embryonic Patterning-Another example of a specific eIF4E family member being tethered to a specific 3Ј-UTR sequence motif occurs with D. melanogaster 4EHP (Table 1). In the D. melanogaster embryo, Caudal protein is synthesized asymmetrically because translation of its mRNA is inhibited in the anterior region by Bicoid. Cho et al. (35) found that the Class II eIF4E family member d4EHP, which binds the cap but not eIF4G, specifically interacts with Bicoid to suppress caudal mRNA translation. The inhibition is dependent on the Bicoid-binding region present in the 3Ј-UTR of caudal mRNA. In another study, Cho et al. (36) found that translation of hunchback mRNA is regulated by the same eIF4E family member, d4EHP, but in this case, the eIF4E-binding partner is Brat ( Table 2). This system appears similar to the Maskin model (Fig. 1C).

Conclusions and Future Directions
This minireview has attempted to highlight several recent trends in research involving eIF4E: discovery of new eIF4E family members, discovery of new eIF4E-binding partners, and the involvement of both in developmental processes. One is struck, however, by the growing number of eIF4E family members (umbicc3-215.umbi.umd.edu/iisstart.asp) and the rapidly expanding number of eIF4E-binding partners compared with the paucity of well understood mechanisms for how they act, suggesting there is still much to learn. Furthermore, a new chapter in the regulation of translation is opening with the discovery of microRNAs that interfere with initiation of translation in an mRNA-specific manner (37). eIF4E family members and eIF4E-binding partners could well play a role in microRNA-mediated translational control. eIF4E should also prove to be central to our understanding of P-bodies and mRNA degradation because P-bodies lack ribosomes and all translation initiation factors except eIF4E (37). The involvement of eIF4E in malignant transformation and metastasis is the subject of intense research (38), including its utility as a marker for cancer stage and potential to serve as a target for novel anticancer therapies (39,40). Further knowledge of the factors that influence eIF4E function should aid in our understanding of malignant transformation and in the development of therapeutic strategies.