InteractiveFly: GeneBrief

eukaryotic translation initiation factor 4E1: Biological Overview | References

Gene name - eukaryotic translation initiation factor 4E1

Synonyms -

Cytological map position - 67B4-67B4

Function - translation initiation factor

Keywords - 5' cap-binding protein - regulation of translation initiation, splicing cofactor, regulation of cell growth, oncogene

Symbol - eIF4E1

FlyBase ID: FBgn0015218

Genetic map position - chr3L:9,392,718-9,395,237

Classification - Translation initiation factor 4E

Cellular location - cytoplasmic and nuclear

NCBI links: EntrezGene

eIF4E1 orthologs: Biolitmine

Recent literature
Peter, D., Igreja, C., Weber, R., Wohlbold, L., Weiler, C., Ebertsch, L., Weichenrieder, O. and Izaurralde, E. (2015). Molecular architecture of 4E-BP translational inhibitors bound to eIF4E. Mol Cell 57: 1074-1087. PubMed ID: 25702871
The eIF4E-binding proteins (4E-BPs) represent a diverse class of translation inhibitors that are often deregulated in cancer cells. 4E-BPs inhibit translation by competing with eIF4G for binding to eIF4E through an interface that consists of canonical and non-canonical eIF4E-binding motifs connected by a linker. The lack of high-resolution structures including the linkers, which contain phosphorylation sites, limits understanding of how phosphorylation inhibits complex formation. Furthermore, the binding mechanism of the non-canonical motifs is poorly understood. This study presents structures of human eIF4E bound to 4E-BP1 and fly eIF4E bound to Thor (4E-BP), eIF4E-Transporter (4E-T), and eIF4G. These structures reveal architectural elements that are unique to 4E-BPs and provide insight into the consequences of phosphorylation. Guided by these structures, a 4E-BP mimic was designed and crystallized that shows increased repressive activity. These studies pave the way for the rational design of 4E-BP mimics as therapeutic tools to decrease translation during oncogenic transformation.
Cha, I. J., Lee, D., Park, S. S., Chung, C. G., Kim, S. Y., Jo, M. G., Kim, S. Y., Lee, B. H., Lee, Y. S. and Lee, S. B. (2020). Ataxin-2 Dysregulation Triggers a Compensatory Fragile X Mental Retardation Protein Decrease in Drosophila C4da Neurons. Mol Cells 43(10): 870-879. PubMed ID: 33115979
Dendrites require precise and timely delivery of protein substrates to distal areas to ensure the correct morphology and function of neurons. Many of these protein substrates are supplied in the form of ribonucleoprotein (RNP) complex consisting of RNA-binding proteins (RBPs) and mRNAs, which are subsequently translated in distal dendritic areas.This study investigated how Drosophila sensory neurons respond to the dysregulation of a disease-associated RBP, Ataxin-2 (ATX2), which leads to dendritic defects. ATX2 plays a crucial role in spacing dendritic branches for the optimal dendritic receptive fields in Drosophila class IV dendritic arborization (C4da) neurons, where both expression level and subcellular location of ATX2 contribute significantly to this effect. Translational upregulation through the expression of eukaryotic translation initiation factor 4E (eIF4E) further enhanced the ATX2-induced dendritic phenotypes. Additionally, this study found that the expression level of another disease-associated RBP, fragile X mental retardation protein (FMRP), decreased in both cell bodies and dendrites when neurons were faced with aberrant upregulation of ATX2. Finally, this study revealed that the PAM2 motif of ATX2, which mediates its interaction with poly(A)-binding protein (PABP), is potentially necessary for the decrease of FMRP in certain neuronal stress conditions. Collectively, these data suggest that dysregulation of RBPs triggers a compensatory regulation of other functionally-overlapping RBPs to minimize RBP dysregulation-associated aberrations that hinder neuronal homeostasis in dendrites.
Santalla, M., García, A., Mattiazzi, A., Valverde, C. A., Schiemann, R., Paululat, A., Hernandez, G., Meyer, H. and Ferrero, P. (2022). Interplay between SERCA, 4E-BP, and eIF4E in the Drosophila heart. PLoS One 17(5): e0267156. PubMed ID: 35588119
Appropriate cardiac performance depends on a tightly controlled handling of Ca2+ in a broad range of species, from invertebrates to mammals. The role of the Ca2+ ATPase, SERCA, in Ca2+ handling is pivotal, and its activity is regulated, inter alia, by interacting with distinct proteins. This study gives evidence that 4E binding protein (4E-BP) is a novel regulator of SERCA activity in Drosophila melanogaster during cardiac function. Flies over-expressing 4E-BP showed improved cardiac performance in young individuals associated with incremented SERCA activity. Moreover, it was demonstrated that SERCA interacts with translation initiation factors eIF4E-1, eIF4E-2 and eIF4E-4 in a yeast two-hybrid assay. The specific identification of eIF4E-4 in cardiac tissue leads to a proposal that the interaction of elF4E-4 with SERCA may be the basis of the cardiac effects observed in 4E-BP over-expressing flies associated with incremented SERCA activity.
Layana, C., Vilardo, E. S., Corujo, G., Hernandez, G. and Rivera-Pomar, R. (2023). Drosophila Me31B is a Dual eIF4E-Interacting Protein. J Mol Biol 435(5): 167949. PubMed ID: 36638908
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). This study showed that Drosophila eIF4E-1 and eIF4E-3 occur in PBs along the DEAD-box RNA helicase Me31B. This study shows 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.


In female fruit flies, Sex-lethal (Sxl) turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2) mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors -- the U1/U2 snRNP protein Sans-fils (Snf), the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2)d--that have been directly implicated in Sxl splicing regulation (Graham, 2011).

Translation initiation is mediated by the binding of a pre-initiation complex to the 5' cap of the mRNA (reviewed in Merrick, 1996; Gingras, 1999) that in turn recruits the small subunit of the 40S ribosome to the mRNA. The pre-initiation complex consists of the cap binding protein, eIF4E, and a scaffolding protein, eIF4G, which mediates interactions with various components of the 40S initiation complex. In many organisms there is also a third protein in the complex, eIF4A, an ATP dependent RNA helicase. Modulating eIF4E activity appears to be a key control point for regulating translation. One of the most common mechanisms of regulation is by controlling the association eIF4E with eIF4G. Factors such as poly-A binding protein that promote the association between eIF4E and eIF4G activate translation initiation, while factors such as the 4E-binding proteins (4E-BPs; see Drosophila 4E-BP) that block their association, inhibit initiation (Graham, 2011 and references therein).

Although eIF4E's primary function in the cell is in regulating translation initiation, studies over the past decade have revealed unexpected activities for eIF4E at steps prior to translation. Among the more surprising findings is that there are substantial amounts of eIF4E in eukaryotic nuclei. One role for eIF4E in the nucleus is the transport of specific mRNAs, like cyclin D1, to the cytoplasm (Rousseau, 1996). This eIF4E activity is distinct from translation initiation since an eIF4E mutation that prevents it from forming an active translation complex still allows cyclin D1 mRNA transport. The transport function of eIF4E is modulated by at least two other proteins, PML and PRH (Topisirovic, 2002; Topisirovic, 2003). While PML seems to be ubiquitously expressed, PRH is found only in specific tissues. In addition, the intracellular distribution of eIF4E exhibits dynamic changes during Xenopus development (Strudwick, 2002). These observation raise the possibility that eIF4E might have additional functions in the nucleus during development. Consistent with this idea, this study shows that eIF4E plays a novel role in the process of sex determination in Drosophila (Graham, 2011).

Sex determination in the fly is controlled by the master regulatory switch gene Sex-lethal (Sxl). The activity state of the Sxl gene is selected early in development by an X chromosome counting system. The target for the X/A signaling system is the Sxl establishment promoter, Sxl-Pe. When there are two X chromosomes, Sxl-Pe is turned on, while it remains off when there is a single X chromosome. Sxl-Pe mRNAs encode RRM type RNA binding proteins which mediate the transition from the initiation to the maintenance mode of Sxl regulation by directing the female-specific splicing of the first pre-mRNAs produced from a second, upstream promoter, the maintenance promoter, Sxl-Pm. Sxl-Pm is turned on before the blastoderm cellularizes, just as Sxl-Pe is being shut off. In the presence of Sxl-Pe proteins, the first Sxl-Pm transcripts are spliced in the female-specific pattern in which exon 2 is joined to exon 4 (see Model of the alternatively spliced region of Sxl ). The resulting Sxl-Pm mRNAs encode Sxl proteins that direct the female specific splicing of new Sxl-Pm pre-mRNAs and this establishes a positive autoregulatory feedback loop that maintains the Sxl gene in the 'on' state for the remainder of development. In male embryos, which lack the Sxl-Pe proteins, the Sxl-Pm pre-mRNAs are spliced in the default pattern, incorporating the male specific exon 3. This exon has several in-frame stop codons that prematurely truncate the open reading frame so that male specific Sxl-Pm mRNAs produce only small non-functional polypeptides. As a consequence the Sxl gene remains off throughout development in males (Graham, 2011).

In females, Sxl orchestrates sexual development by regulating the alternative splicing of transformer (tra) pre-mRNAs. Like Sxl, functional Tra protein is only produced by female-specific tra mRNAs, while mRNAs spliced in the default, male pattern encode non-functional polypeptides. Sxl also negatively regulates the dosage compensation system, which is responsible for hyperactivating X-linked transcription in males, by repressing male-specific lethal-2 (msl-2). Sxl represses msl-2 by first blocking the splicing of an intron in the 5' UTR of the msl-2 pre-mRNA, and then by inhibiting the translation of the mature mRNA. In addition, there are two other known targets for Sxl translational repression. One is the Sxl mRNA itself. Sxl binds to target sequences in the Sxl 5' and 3' UTRs and downregulates translation. It is thought that this negative autoregulatory activity provides a critical homeostasis mechanism that prevents the accumulation of excess Sxl protein. This is important as too much Sxl can disrupt development and have female lethal effects. The other known target is the Notch (N) mRNA (Penn, 2007). Sxl-dependent repression of N mRNA translation is important for the elaboration of sexually dimorphic traits in females. Like msl-2 and Sxl, translational repression appears to be mediated by Sxl binding to sites in the N UTRs (Graham, 2011).

Translational repression of msl-2 mRNA by Sxl is thought to involve two separate mechanisms acting coordinately. Binding sites for Sxl in the unspliced intron in the 5' UTR and in the 3'UTR of msl-2 are required for complete repression. Sxl binding to the 5'UTR blocks recruitment of the 40S pre-initiation complex (. While factors that act with Sxl at the 5'UTR of msl-2 have yet to be identified, repression by the 3'UTR requires Sxl, PABP and a co-repressor UNR. Somewhat unexpectedly, this complex does not affect recruitment of eIF4E or eIF4G to the 5' end. Instead it prevents ribosomes that do manage to attach to the msl-2 mRNA from scanning (Graham, 2011).

Although eIF4E does not appear to be a key player in the translational repression of msl-2 mRNAs, this study reports that it has an important role in the process of sex determination in Drosophila. eIF4E activity is required in females to stably activate and maintain the Sxl positive autoregulatory feedback loop and to efficiently repress msl-2. Surprisingly, this requirement for eIF4E activity in fly sex determination is in promoting the female-specific splicing of the Sxl and msl-2 transcripts, not in translational regulation (Graham, 2011).

The RNA binding protein Sxl orchestrates sexual development by controlling gene expression post-transcriptionally at the level of splicing and translation. To exert its different regulatory functions Sxl must collaborate with sex-non-specific components of the general splicing and translational machinery. In this study evidence is presented that one of the splicing co-factors is the cap binding protein eIF4E. eif4e was initially identified in a screen for mutations that dominantly suppress the male lethal effects induced by ectopic expression of a mutant Sxl protein, Sx-N, which lacks part of the N-terminal domain. The Sx-N protein is substantially compromised in its splicing activity, but appears to have closer to wild type function in blocking the translation of the Sxl targets msl-2 and Sxl-Pm. As the male lethal effects of Sx-N (in an Sxl- background) are due to its inhibition of Msl-2 expression, it is anticipated that general translation factors needed to help Sxl repress msl-2 mRNA would be recovered as suppressors in the screen. Indeed, one of the suppressors identified was eif4e. However, consistent with in vitro experiments, which have shown that Sxl dependent repression of msl-2 mRNA translation is cap independent, this study found that eif4e does not function in Sxl mediated translational repression of at least one target mRNA in vivo. Instead, the results indicate that eif4e is needed for Sxl dependent alternative splicing, and it is argued that it is this splicing activity that accounts for the suppression of male lethality by eif4e mutations. In wild type females, Sxl protein blocks the splicing of a small intron in the 5' UTR of the msl-2 pre-mRNA. This is an important step in msl-2 regulation because the intron contains two Sxl binding sites that are needed by Sxl to efficiently repress translation of the processed msl-2 mRNA. When this intron is removed repression of msl-2 translation by Sxl is incomplete and this would enable eif4e/+ males to escape the lethal effects of the Sx-N transgene (Graham, 2011).

Several lines of evidence support the conclusion that eif4e is required for Sxl dependent alternative splicing. One comes from the analysis of the dominant maternal effect female lethal interactions between eif4e and Sxl. The initial activation of the Sxl positive autoregulatory feedback loop in early embryos can be compromised by a reduction in the activity of splicing factors like Snf, Fl(2)d, and U1-70K, and mutations in genes encoding these proteins often show dose sensitive maternal effect, female lethal interactions with Sxl. Like these splicing factors, maternal effect female lethal interactions with Sxl are observed for several eif4e alleles. Moreover, these female lethal interactions can be exacerbated when the mothers are trans-heterozygous for mutations in eif4e and the splicing factors snf or fl(2)d. Genetic and molecular experiments indicate that female lethality is due to a failure in the female specific splicing of Sxl-Pm mRNAs. First, female lethality can be rescued by gain-of-function Sxl mutations that are constitutively spliced in the female mode. Second, transcripts expressed from a Sxl-Pm splicing reporter in the female Sxl-/+ progeny of eif4e/+ mothers are inappropriately spliced in a male pattern at the time when the Sxl positive autoregulatory loop is being activated by the Sxl-Pe proteins. While splicing defects are evident in these embryos at the blastoderm/early gastrula stage, obvious abnormalities in expression of Sxl protein are not observed until several hours later in development (Graham, 2011).

Though this difference in timing would favor the idea that eif4e is required for splicing of Sxl-Pm transcripts rather than for the export or translation of the processed Sxl-Pm mRNAs, the possibility cannot be excluded that there are subtle defects in the expression of Sxl protein at the blastoderm/early gastrula stage that are sufficient to disrupt splicing regulation during the critical activation phase yet aren't detectable in the antibody staining experiments. However, evidence from two different experimental paradigms using adult females indicates that this is likely not the case. In the first, it was found that reducing eif4e activity in a sensitized snf1621 Sxlf1/++ background can compromise Sxl dependent alternative splicing even though there is no apparent reduction in Sxl protein accumulation. In this experiment advantage was taken of the fact that once the positive autoregulatory feedback loop is fully activated a homeostasis mechanism (in which Sxl negatively regulates the translation of Sxl-Pm mRNAs) ensures that Sxl protein is maintained at the same level even if there are fluctuations in the amount of female spliced mRNA. While only a small amount of male spliced Sxl-Pm mRNAs can be detected in snf1621 Sxlf1/++ females, the level increases substantially when eif4e activity is reduced. Since these synergistic effects occur even though Sxl levels in the triply heterozygous mutant females are the same as in the control snf1621 Sxlf1/++ females, it is concluded that the disruption in Sxl dependent alternative splicing of Sxl-Pm transcripts in this context (and presumably also in early embryos) can not be due to a requirement for eif4e in either the export of Sxl mRNAs or in their translation. Instead, eif4e activity must be needed specifically for Sxl dependent alternative splicing of Sxl-Pm pre-mRNAs. Consistent with a more general role in Sxl dependent alternative splicing, there is a substantial increase in msl-2 mRNAs lacking the first intron when eif4e activity is reduced in snf1621 Sxlf1/++ females. In the second experiment the splicing was examined of pre-mRNAs from the endogenous Sxl gene and from a Sxl splicing reporter in females heterozygous for two hypomorphic eif4e alleles. Male spliced mRNAs from the endogenous gene and from the splicing reporter are detected the eif4e/+ females, but not in wild type females. Moreover, the effects on sex-specific alternative splicing seem to be specific for transcripts regulated by Sxl as no male spliced dsx mRNAs were seen in eif4e/+ females (Graham, 2011).

Two models could potentially explain why eif4e is needed for Sxl dependent alternative splicing. In the first, eif4e would be required for the translation of some critical and limiting splicing co-factor. When eif4e activity is reduced, insufficient quantities of this splicing factor would be produced and this, in turn, would compromise the fidelity of Sxl dependent alternative splicing. In the second, the critical splicing co-factor would be eif4e itself. It is not possible to conclusively test whether there is a dose sensitive requirement for eif4e in the synthesis of a limiting splicing co-factor. Besides the fact that the reduction in the level of this co-factor in flies heterozygous for hypomorphic eif4e alleles is likely to be rather small, only a subset of the Sxl co-factors have as yet been identified. For these reasons, the first model must remain a viable, but unlikely possibility. As for the second model, the involvement of a translation factor like eif4e in alternative splicing is unexpected if not unprecedented. For this to be a viable model, a direct role for eif4e must be consistent with what is known about the dynamics of Sxl pre-mRNA splicing and the functioning of the Sxl protein. The evidence that the second model is plausible is detailed below (Graham, 2011).

Critical to the second model is both the nuclear localization of eIF4E and an association with incompletely spliced Sxl pre-mRNAs. Nuclear eIF4E has been observed in other systems, and this was confirmed for Drosophila embryos. It was also found that eIF4E is bound to Sxl transcripts in which the regulated exon2-exon3-exon4 cassette has not yet been spliced. In contrast, it is not associated with incompletely processed transcripts from the tango gene that are constitutively spliced. With the caveat that only one negative control is available, it is not surprising that Sxl transcripts might be unusual in this respect. There is growing body of evidence that splicing of constitutively spliced introns is co-transcriptional. However, recent in vivo imaging experiments have shown that the splicing of the regulated Sxl exon2-exon3-exon4 cassette is delayed until after the Sxl transcript is released from the gene locus in female, but not in male cells. These in vivo imaging studies also show that, like bulk pre-mRNAs, the 1st Sxl intron is spliced co-transcriptionally in both sexes. Consistent with a delay in the splicing of the regulated cassette, it has been previously reported that polyadenylated Sxl RNAs containing introns 2 and 3 can be readily detected by RNase protection, whereas other Sxl intron sequences are not observed. The delay in the splicing of the regulated Sxl cassette until after transcription is complete and the RNA polyadenylated could provide a window for exchanging eIF4E for the nuclear cap binding protein (Graham, 2011).

To function as an Sxl co-factor, eIF4E would have to be associated with the pre-mRNA-spliceosomal complex before or at the time of the Sxl dependent regulatory step. There is still a controversy as to exactly which step in the splicing pathway Sxl exerts its regulatory effects on Sxl-Pm pre-mRNAs and two very different scenarios have been suggested. The first is based on an in vitro analysis of Sxl-Pm splicing using a small hybrid substrate consisting of an Adenovirus 5' exon-intron fused to a short Sxl-Pm sequence spanning the male exon 3' splice site. These in vitro studies suggest that Sxl acts very late in the splicing pathway after the 1st catalytic step, which is the formation of the lariat intermediate in the intron between exon 2 and the male exon. According to these experiments Sxl blocks the 2nd catalytic step, the joining of the free exon 2 5' splice site (or Adeno 5' splice site) to the male exon 3' splice site. It is postulated that this forces the splicing machinery to skip the male exon altogether and instead join the free 5' splice site of exon 2 to the downstream 3' splice site of exon 4. Since this study has shown that eIF4E binds to Sxl-Pm pre-mRNAs that have not yet undergone the 1st catalytic step, it would be in place to influence the splicing reaction if this scenario were correct (Graham, 2011).

The second scenario is more demanding in that it proposes that Sxl acts during the initial assembly of the spliceosome. Evidence for Sxl regulation early in the pathway comes from the finding that Sxl and the Sxl co-factor Fl(2)d show physical and genetic interactions with spliceosomal proteins like U1-70K, Snf, U2AF38 and U2AF50 that are present in the early E and A complexes and are important for selecting the 5' and 3' splice sites. In addition to these proteins, Sxl can also be specifically cross-linked in nuclear extracts to the U1 and U2 snRNAs. Formation of the E complex depends upon interactions of the U1 snRNP with the 5' splice site, and this is thought to be one of the first steps in splicing. The other end of the intron is recognized by U2AF, which recruits the U2 snRNP to the 3' splice site. After the base pairing of the U2 snRNP with the branch-point to generate the A complex the next step is the addition of the U4/U5/U6 snRNPs to form the B complex. However, Sxl and Fl(2)d are not found associated with components of the splicing apparatus like U5-40K, U5-116K or SKIP that are specific for complexes B and B*, or the catalytic C complex. Nor can Sxl be cross-linked to the U4, U5 or U6 snRNAs. If Sxl and Fl(2)d dissociated from the spliceosome before U4/U5/U6 are incorporated into the B complex, then they must influence splice site selection during the formation/functioning of the E and/or A complex. (Since the transition from the E to the A complex has been shown to coincide with an irreversible commitment to a specific 5'—3' splice site pairing, Sxl would likely exerts its effects in the E complex when splice site pairing interactions are known to still be dynamic. If this is scenario is correct, eIF4E would have to be associated with factors present in the earlier complexes in order to be able to promote Sxl regulation. This is the case. Thus, eIF4E is found in complexes containing the U1 snRNP protein U1-70K, the U1/U2 snRNP protein Snf, and the two U2AF proteins, U2AF38 and U2AF50. With the exception of the Snf protein bound to the U2 snRNP, all of these eIF4 associated factors are present in the early E or A complexes, but are displaced from the spliceosome together with the U1 and U4 snRNPs when the B complex is rearranged to form the activated B* complex. This would imply that eIF4E is already in place either before or at the time of B complex assembly. Arguing that eIF4E associates with these E/A components prior to the assembly of the B complex is the finding that eIF4E is also in complexes with both Sxl and Fl(2)d. Thus, even in this more demanding scenario for Sxl dependent splicing, eIF4E would be present at a time when it could directly impact the regulatory activities of Sxl and its co-factor Fl(2)d (Graham, 2011).

Taken together these observations would be consistent with a Sxl co-factor model. While further studies will be required to explain how eIF4E helps promote female specific processing, an intriguing possibility is suggested by the fact that hastening the nuclear export of msl-2 in females would favor the female splice (which is no splicing at all). Hence, one idea is that eIF4E binding to the pre-mRNA provides a mechanism for preventing the Sxl regulated splice sites from re-entering the splicing pathway, perhaps by constituting a 'signal' that blocks the assembly of new E/A complexes. A similar post-transcriptional mechanism could apply to female-specific splicing of the regulated Sxl exon2-exon3-exon4 cassette. The binding of eIF4E (and PABP) to incompletely processed Sxl transcripts after transcription has terminated in females would prevent the re-assembly of E/A complexes on the two male exon splice sites, and thus promote the formation of an A complex linking splicing factors assembled on the 5' splice sites of exons 2 and on the 3' splice site of exon 4 (Graham, 2011).

Patterning of the Drosophila oocyte by a sequential translation repression program involving the d4EHP and Belle translational repressors

During Drosophila development, translational control plays a crucial role in regulating gene expression, and is particularly important during pre-patterning of the maturing oocyte. A critical step in translation initiation is the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the mRNA cap structure, which ultimately leads to recruitment of the ribosome. d4EHP is a translational repressor that prevents translation initiation by out-competing eIF4E on the cap structure for a subset of mRNAs. However, only two examples of mRNAs subject to d4EHP translation repression in Drosophila are known. This study shows that the belle (bel) mRNA is translationally repressed by the d4EHP protein in the Drosophila ovary. Consistent with this regulation, d4EHP overexpression in the ovary phenocopies the bel mutant. Evidence is provided that the Bel protein binds to eIF4E and may itself function as a translation repressor protein, with bruno as a potential target for Bel repression in the oocyte. Bruno is known to repress the mRNA of the key oocyte axis determinant oskar (osk) during oogenesis, and this study found that an increase in the level of Bruno protein in bel mutant ovaries is associated with a reduction in Osk protein. Overall, these data suggest that a translational regulatory network exists in which consecutive translational repression events act to correctly pattern the Drosophila oocyte (Yarunin, 2011).

Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila

Animals use the insulin/TOR signaling pathway to mediate their response to fluctuations in nutrient availability. Energy and amino acids are monitored at the single-cell level via the TOR branch of the pathway and systemically via insulin signaling to regulate cellular growth and metabolism. Using a combination of genetics, expression profiling, and chromatin immunoprecipitation, this study examined nutritional control of gene expression and identified the transcription factor Myc as an important mediator of TOR-dependent regulation of ribosome biogenesis. myc was also identified as a direct target of FOXO, and genetic evidence is provided that Myc has a key role in mediating the effects of TOR and FOXO on growth and metabolism. FOXO and TOR also converge to regulate protein synthesis, acting via 4E-BP and Lk6, regulators of the translation factor eIF4E. This study uncovers a network of convergent regulation of protein biosynthesis by the FOXO and TOR branches of the nutrient-sensing pathway (Teleman, 2008).

The global transcriptional analysis reported in this study has revealed a surprising degree of interconnectedness between the two branches of the nutrient-sensing pathway. Insulin, acting through PI3K and Akt, feeds into the FOXO and TORC1 branches of the pathway, whereas energy levels (AMP/ATP) and amino acids act directly on the TORC1 branch. How are these inputs integrated to maintain energy balance? It was previously known that 4E-BP is transcriptionally regulated by FOXO and posttranslationally regulated by TOR. This study has identified the protein kinase Lk6 as a second direct FOXO target. Thus, there appear to be two parallel, independent mechanisms by which the TOR and FOXO branches of the insulin signaling pathway converge to regulate eIF4E activity and hence cellular protein translation. This 'belt and suspenders' approach to translational control might be important to make the system robust (Teleman, 2008).

A key finding of this study is the identification of Myc as a point of convergent regulation by the FOXO and TOR branches of the pathway. myc mRNA levels are controlled by FOXO in a tissue-specific manner. In addition, Myc protein levels are dependent on TORC1. Why use two independent means to control Myc levels? Transcription alone would limit the speed with which the system can respond to changing nutritional conditions. This might be detrimental, particularly as conditions worsen. Regulation of Myc activity by TORC1 permits a rapid response to changes in energy levels or amino acid availability and could serve to fine tune the nutritional response in the cell by controlling translational outputs. This parallels the situation with 4E-BP, albeit with a slightly different logic. Reduced insulin signaling allows FOXO to enter the nucleus and increase 4E-BP expression and at the same time alleviates TORC1-mediated inhibition of the existing pool of 4E-BP. A subsequent increase in energy or amino acid levels would permit rapid reinhibition of 4E-BP and thus allow a flexible response during the time needed for the pool of protein elevated in response to reduced insulin levels to decay (Teleman, 2008).

In yeast, TORC1 is known to regulate ribosome biogenesis through different nuclear RNA polymerases. It has been shown that yeast TORC1 can bind DNA directly at the 35S rDNA promoter and activate Pol I-mediated transcription in a rapamycin-sensitive manner. Moreover, yeast TORC1 is known regulate Pol II-dependent RP gene expression by controlling the nuclear localization of the transcription factor SFP1 and CRF1, a corepressor of the forkhead transcription factor FHL1. In Drosophila, TORC1 has recently been reported to regulate a set of protein-coding genes involved in ribosome assembly. This study has identified Myc as the missing link mediating TORC1-dependent regulation of this set of genes. Indeed, the fact that more than 90% of TORC1-activated genes contain E boxes suggests that Myc might be the main mediator of this transcriptional program. This connection suggests that expression of Myc targets as a whole should be responsive to nutrient conditions. Indeed, this study found that 33% of direct Myc targets -- defined as genes reported to be bound by Myc when assayed by DNA adenine methyltransferase ID (DamID) in Kc cells and to be regulated by myc overexpression in larvae -- are downregulated upon nutrient deprivation. This is a significant enrichment of 4-fold relative to all genes in the genome, despite the comparison being based on correlating data from different tissue types (Teleman, 2008).

It seems reasonable that cellular translation rates need to be dampened if the TOR branch of the pathway senses low amino acid levels. As ribosome biogenesis is energetically expensive, it may be advantageous to link ribosome biogenesis and translational control via TORC1. This dual regulation is well reflected in tissue growth, since this study observed that Myc, the regulator of ribosome biogenesis, is essential for tissue growth driven by the TOR pathway but not sufficient to drive growth in the absence of TOR activity. The FOXO branch of the pathway senses reduced insulin or mitogen levels. FOXO is also highly responsive to oxidative and other stresses and would integrate this information into the cellular control of translation. The data support the notion of a network in which TOR and FOXO regulate protein biosynthesis by converging on Myc to regulate ribosome biogenesis and on eIF4E activity via 4E-BP and Lk6 to regulate translation initiation (Teleman, 2008).

The work presented in this study complements a previous study in which larvae were either starved completely or starved for amino acids only, while having a supply of energy in the form of sugar. A significant and positive correlation (~0.4) indicates general agreement between the two data sets, but they differ in two ways. The current goal was to explore the regulatory network by which insulin controls cellular transcription. Individual tissues were isolated rather than assaying the whole animal. Genes found to be regulated in a previous but not in the current assays may be regulated in tissues other than muscle or adipose tissue. Conversely, genes identified only by the current study might be regulated oppositely in different tissues or might only be regulated in a subset of tissues and so be missed in a whole-animal analysis.

Is Myc also involved in nutritional signaling networks in mammals? No similar rapid downregulation of c-myc was seen in response to rapamycin in human cell lines, suggesting that the mechanism by which TOR signaling controls gene expression may differ between phyla. This is further supported by the fact that the sets of genes reported to be rapamycin regulated also appear to be largely distinct in Drosophila and mammalian cells, with the caveat that different cell types were used in the two analyses. Although the mechanism does not appear to be identical in mammals, there are several suggestions in the literature of a connection between c-Myc and nutritional signaling. For example, dMyc and c-Myc share the ability to regulate ribosome biogenesis, although the specific target genes through which they do so are different. There is also evidence that mammalian c-myc expression in liver is regulated by nutrition and that transgenic expression of c-myc in liver affects metabolism, i.e., glucose uptake and gluconeogenesis. Furthermore, it has been reported that FOXO3 represses Myc activity in colon cancer cells by inducing members of the Mad/Mxi family, which are known to antagonize Myc. The current data suggest that Max and Mnt are not transcriptionally regulated by insulin or FOXO in Drosophila, whereas myc is. This is similar to what has been reported in murine lymphoid cells, in which c-myc expression is regulated by the FOXO homolog FKHRL1. These parallels between the fly and mammalian systems suggest a broader connection between insulin signaling and activity of the Myc/Mnt/Max network. Although some features may be different in the two systems, the similarities merit further investigation (Teleman, 2008).

Finally, this work has revealed a surprising amount of tissue specificity in the transcriptional response to insulin signaling. Roughly half of the genes regulated by insulin in adipose tissue or in muscle were not significantly regulated in the other tissue. Furthermore, 155 genes were differentially regulated in the two tissues (i.e., upregulated in one tissue and downregulated in the other). This likely reflects the roles of the different tissues in the organism's response to nutrient deprivation. Further work will elucidate the underlying molecular mechanisms (Teleman, 2008).

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). This study examined the role of key functional amino acid residues of eIF4E in the recruitment of this protein to cytoplasmic foci. Tryptophan residues required for mRNA cap recognition are not required for the recruitment of eIF4E to SG or PB. A tryptophan residue required for protein-protein interactions is essential for the accumulation of eIF4E in granules. Moreover, by the analysis of two Drosophila eIF4E isoforms, it was shown 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. It is concluded that protein-protein interactions rather than interactions with the mRNA are essential for the recruitment of eIF4E and for a putative nucleation function (Ferrero, 2012).

An important consequence of these results is a re-evaluation of the role of eIF4E. Cap binding is absolutely required to initiate translation but, more importantly, the tethering of eIF4E to the mRNA is required to bring other translations factors to the 5' end of the mRNA. The recruitment of other translation factors is mediated by a domain that, modified in 4E-HP, is not required for the localization in cytoplasmic foci. These data support the notion of a dual role for eIF4E, namely a function on translation that requires the cap-binding activity and the recruitment to PB or SG that is independent of the cap binding activity. It is proposed that the removal of the translation machinery and the assembly of PB-specific factors such as Rck/p54, eIF4E-T, and others would take over the translation-related eIF4E interactors. The notion is supported that eIF4E participates in the exchange of factors via protein-protein interactions that require W117. This agrees with the observation that human eIF4E simultaneously interacts with the translation repressor eIF4E-T and the helicase rck/p54 in PB of HeLa cells. This does not rule out a role for eIF4E in preventing decapping as it has been postulated. Previous work suggested that there is a hierarchy of factors required for sequential assembly of PB formation in which the lack of eIF4E, Rck/p54 or eIF4E-T prevented the recruitment of Dcp-1, Xrn-1, Ccr-4 and other processing factors. Recent evidence showed that Hsp90 also plays a role in PB formation and that is required for the presence of eIF4E and eIF4E-T. Taken together, the current evidence supports the notion that during mRNP remodeling, eIF4E is maintained in the mRNP complex independently of the binding to the cap, and that protein–protein interactions agglutinate the components that forms the cytoplasmic granules (Ferrero, 2012).

GW182 proteins cause PABP dissociation from silenced miRNA targets in the absence of deadenylation

GW182 family proteins interact with Argonaute proteins and are required for the translational repression, deadenylation and decay of miRNA targets. To elicit these effects, GW182 proteins interact with poly(A)-binding protein (PABP) and the CCR4-NOT deadenylase complex. Although the mechanism of miRNA target deadenylation is relatively well understood, how GW182 proteins repress translation is not known. This study demonstrates that GW182 proteins decrease the association of eIF4E, eIF4G and PABP with miRNA targets. eIF4E association is restored in cells in which miRNA targets are deadenylated, but decapping is inhibited. In these cells, eIF4G binding is not restored, indicating that eIF4G dissociates as a consequence of deadenylation. In contrast, PABP dissociates from silenced targets in the absence of deadenylation. PABP dissociation requires the interaction of GW182 proteins with the CCR4-NOT complex. Accordingly, NOT1 and POP2 cause dissociation of PABP from bound mRNAs in the absence of deadenylation. These findings indicate that the recruitment of the CCR4-NOT complex by GW182 proteins releases PABP from the mRNA poly(A) tail, thereby disrupting mRNA circularization and facilitating translational repression and deadenylation (Zekri, 2013).

Regulation of cell growth by Notch signaling and its differential requirement in normal vs. tumor-forming stem cells in Drosophila

Cancer stem cells (CSCs) are postulated to be a small subset of tumor cells with tumor-initiating ability that shares features with normal tissue-specific stem cells. The origin of CSCs and the mechanisms underlying their genesis are poorly understood, and it is uncertain whether it is possible to obliterate CSCs without inadvertently damaging normal stem cells. This study shows that a functional reduction of eukaryotic translation initiation factor 4E (eIF4E) in Drosophila specifically eliminates CSC-like cells in the brain and ovary without having discernable effects on normal stem cells. Brain CSC-like cells can arise from dedifferentiation of transit-amplifying progenitors upon Notch hyperactivation. eIF4E is up-regulated in these dedifferentiating progenitors, where it forms a feedback regulatory loop with the growth regulator dMyc to promote cell growth, particularly nucleolar growth, and subsequent ectopic neural stem cell (NSC) formation. Cell growth regulation is also a critical component of the mechanism by which Notch signaling regulates the self-renewal of normal NSCs. These findings highlight the importance of Notch-regulated cell growth in stem cell maintenance and reveal a stronger dependence on eIF4E function and cell growth by CSCs, which might be exploited therapeutically (Song, 2011).

The differential cell growth rates observed between ectopic NBs and normal or primary NBs and the correlation between cell growth defects and NB fate loss prompted a test of whether slowing down cell growth might selectively affect the formation of ectopic NBs. Attenuation of TOR signaling, a primary mechanism of cell growth regulation, through NB-specific overexpression of TSC1/2, a strong allele of eIF4E antagonist 4EBP [4EBP(LL)s], or a dominant-negative form of TOR (TOR.TED) all partially suppressed ectopic NB formation in α-adaptin (ada) mutants without affecting normal or primary NBs. Interestingly, RNAi-mediated knockdown of eIF4E, a stimulator of oncogenic transformation (Lazaris-Karatzas, 1990) and a downstream effector of TOR signaling (Mamane, 2004), showed a better suppression than manipulating other TOR pathway components, suggesting that eIF4E might play a more important role in ectopic NB formation. Strikingly, the brain tumor phenotypes caused by overactivation of N signaling - as in lethal giant larvae (lgl) mutant, aPKCCAAX overexpression, or N overexpression conditions - were also fully suppressed by eIF4E knockdown. Furthermore, the brain tumor phenotypes of brat mutants were also completely rescued by eIF4E RNAi (Song, 2011).

In contrast, normal NB formation or maintenance was not affected by eIF4E knockdown. NBs with eIF4E knockdown remained highly proliferative, as evidenced by the mitotic figures, and displayed relatively normal apical basal cell polarity. There are several other eIF4E-like genes in the fly genome (Hernandez, 2005), which may play partially redundant roles in normal NB maintenance. eIF4E knockdown appeared to specifically block ectopic NB formation caused by the dedifferentiation of IPs in type II NB lineages, since it did not affect ectopic type I NB formation in cnn or polo mutants that are presumably caused by symmetric divisions of type I NBs. In addition, cell fate transformation induced by N overactivation in the SOP lineage was not affected by eIF4E RNAi, supporting the idea that eIF4E is particularly required for type II NB homeostasis. Supporting the specificity of the observed eIF4E RNAi effect, another eIF4E RNAi transgene (eIF4E-RNAi-s) also prevented ectopic NB formation. Moreover, a strong loss-of-function mutation of eIF4E also selectively eliminated ectopic NBs induced by N overactivation without affecting normal NBs, reinforcing the hypothesis that ectopic NBs exhibit higher dependence on eIF4E (Song, 2011).

To further support the notion that the ectopic NBs are particularly vulnerable to eIF4E depletion, a conditional expression experiment was carried out in which eIF4E-RNAi-s was turned on in brat mutants using the 1407ts system, after ectopic NBs had been generated. Whereas the brain tumor phenotype exacerbated over time in the brat mutants, 1407-GAL4-driven eIF4E-RNAi-s expression in brat mutants effectively eliminated ectopic NBs, leaving normal NBs largely unaffected (Song, 2011).

In normal type II NB lineage, eIF4E protein was enriched in the NBs. Ectopic NBs induced by N overactivation in ada mutants also expressed eIF4E at high levels, whereas spdo mutant NBs exhibited reduced eIF4E expression. Thus, eIF4E up-regulation correlates with N-induced ectopic NB formation in a dedifferentiation process that likely involves elevated cell growth (Song, 2011).

Given the coincidence of nucleolar size change with ectopic NB formation, the involvement of the growth regulator dMyc was tested. dMyc protein levels were up-regulated in normal or N overactivation-induced ectopic NBs, but were down-regulated in spdo mutant NBs. Furthermore, dMyc transcription, as detected with a dMyc-lacZ transcriptional fusion reporter, was also up-regulated in both normal and ectopic NBs in ada mutants. A previous study in Drosophila S2 cells identified dMyc as a putative N target. In vivo chromatin immunoprecipitation (ChIP) experiments were carried out to assess whether dmyc transcription is directly regulated by N signaling in NBs. Using chromatin isolated from wild-type larval brains and a ChIP-quality antibody against the N coactivator Suppressor of Hairless [Su(H)], specific binding was demonstrated of Su(H) to its putative binding sites within the second intron of dmyc (dmyc-A). No binding to an internal negative control region proximal to the first exon of dmyc (dmyc-B) or to the promoter region of the rp49 gene was detected. N signaling thus directly activates dMyc transcription in the NBs. Similar to eIF4E RNAi, knockdown of dMyc strongly suppressed ectopic NB formation induced by Brat or Ada inactivation or N overactivation. Intriguingly, the strong tumor suppression effect of eIF4E knockdown was partially abolished by dMyc overexpression. Furthermore, dMyc function, as reflected by its promotion of nucleolar growth in IPs, was attenuated by eIF4E RNAi, although eIF4E RNAi alone had no obvious effect. Different from the reported eIF4E regulation of Myc expression in mammalian cells (Lin, 2008), dMyc promoter activity or protein levels remained unaltered under eIF4E RNAi conditions, suggesting that eIF4E may modulate dMyc activity without altering its expression. One possibility is that eIF4E may enter the nucleus to interact with Myc and promote its transcriptional activity. To test this hypothesis, HEK293T cells were transfected with Flag-tagged human eIF4E alone or in combination with HA-tagged dMyc. Indeed, both Drosophila dMyc and endogenous human c-Myc specifically coimmunoprecipitated with human eIF4E from nuclear extracts, indicating a conserved interaction between eIF4E and Myc within the nuclei of proliferating cells. Consistent with these biochemical data, dMyc transcriptional activity within NBs, which could be monitored with an eIF4E-lacZ reporter, was drastically reduced upon eIF4E knockdown (Song, 2011).

In contrast, eIF4E transcription, as detected with an eIF4E-lacZ transcriptional fusion reporter, as well as eIF4E protein levels detected by immunostaining were up-regulated upon dMyc overexpression and down-regulated by dMyc RNAi. It is unlikely that the changes in eIF4E-lacZ activity were due to global increases or decreases in β-galactosidase (β-gal) translation caused by altered dMyc levels, since lacZ expression from a dMyc-lacZ reporter was unaffected under similar conditions. Furthermore, like dMyc protein, eIF4E-lacZ reporter expression was up-regulated in normal NBs or ectopic NBs in ada mutants, further supporting the notion that dMyc may up-regulate eIF4E transcription. Moreover, ChIP experiments using chromatins isolated from wild-type larval brains and a ChIP-quality antibody against dMyc demonstrated specific binding of dMyc to an eIF4E promoter region harboring a cluster of adjacent noncanonical E boxes, supporting a direct regulation of eIF4E transcription by dMyc. dMyc and eIF4E thus appeared to form a regulatory feedback loop that promoted NB growth and renewal. Consistent with this model, while knocking down either dMyc or eIF4E had no noticeable effect on type II NB maintenance and only a mild effect on NB nucleolar size in the case of dMyc RNAi, their simultaneous knockdown led to a significant reduction in nucleolar size, premature neuronal differentiation, and loss of NBs (Song, 2011).

If the dMyc-eIF4E axis of cell growth control is a crucial downstream effector of N signaling in regulating NB maintenance, its up-regulation might be able to rescue the type II NB depletion phenotype resulting from reduced N signaling. Indeed, the loss of NBs associated with reduced Notch signaling was preventable when cell growth was boosted by dMyc overexpression. Thus, while N-IR directed by 1407-GAL4 led to complete elimination of type II NBs, the coexpression of dMyc, but not CD8-GFP or Rheb, an upstream component of the TOR pathway, resulted in the preservation of approximately half of type II NBs with apparently normal cell sizes, cell fate marker expression, and lineage composition. A similar effect was observed when dMyc was coexpressed with N-IR using the conditional 1407ts system, with transgene expression induced at the larval stage. While both dMyc and Rheb promote cell growth, they do so through distinct mechanisms, with the former increasing nucleolar size and the latter expanding cytoplasmic volume. These results thus provide compelling evidence that control of cell growth, particularly nucleolar growth, is a critical component in the maintenance of NB identity by N signaling (Song, 2011).

The differential responses of normal and tumor-initiating stem cells to functional reduction of eIF4E prompted a test of whether chemicals that specifically inhibit eIF4E function might have therapeutic potential in preventing CSC-induced tumorigenesis. Indeed, the brain tumor phenotypes induced by N overactivation or ada loss of function were effectively suppressed by feeding animals with fly food containing Ribavirin, an eIF4E inhibitor that interferes with eIF4E binding to mRNA 5' caps and promotes the relocalization of eIF4E from the nucleus to the cytoplasm (Kentsis, 2004; Assouline, 2009) (Song, 2011).

The CSC hypothesis was initially developed based on studies in mammalian systems. Various studies have supported the notion that CSCs share many functional features with normal stem cells, such as signaling molecules, pathways, and mechanisms governing their self-renewal versus differentiation choice. However, the cellular origin of CSCs and the molecular and cellular mechanisms underlying their development or genesis remain poorly understood. It has been proposed that CSCs could arise from (1) an expansion of normal stem cell niches, (2) normal stem cells adapting to different niches, (3) normal stem cells becoming niche-independent, or (4) differentiated progenitor cells gaining stem cell properties. This study has showen that in the Drosophila larval brain, CSCs can arise from the dedifferentiation of transit-amplifying progenitor cells back to a stem cell-like state. Importantly, eIF4E was identified as a critical factor involved in this dedifferentiation process. More significantly, it was shown that reduction of eIF4E function can effectively prevent the formation of CSCs without affecting the development or maintenance of normal stem cells. This particular dependence on eIF4E function by CSCs appears to be a general theme, as reduction of eIF4E function also effectively prevented the formation of CSCs, but not normal GSCs, in the fly ovary. These findings may have important implications for stem cell biology and cancer biology, in terms of both mechanistic understanding and therapeutic intervention (Song, 2011).

This study also offers mechanistic insights into the cellular processes leading to the dedifferentiation of progenitors back to stem cells. In Drosophila type II NB clones with overactivated N signaling, ribosome biogenesis within ectopic NBs appears to be faster than in normal NBs, as shown by the fact that the ratio of nucleolar to cellular volume of the ectopic NBs is approximately fivefold higher than that of normal NBs. The faster growth rate is accompanied by the up-regulation of dMyc and eIF4E and appears to be essential for transit-amplifying progenitors to undergo complete dedifferentiation back to a stem cell-like state. When the function of cell growth-promoting factors such as eIF4E is attenuated, the faster cell growth of ectopic NBs can no longer be sustained and the dedifferentiation process stalls. As a result, brain tumor formation caused by uncontrolled production of ectopic NBs is suppressed. In contrast, normal NBs, which presumably have relatively lower requirements for cell growth and hence eIF4E function, maintain their stem cell fate and development under similar conditions. Therefore, a potential key to a successful elimination of CSC-induced tumors would be to find the right level of functional reduction in eIF4E, which causes minimal effects on normal stem cells but effectively obliterates CSCs. An ongoing clinical trial with Ribavirin in treating acute myeloid leukemia (AML) (Assouline, 2009), a well-characterized CSC-based cancer, demonstrated exciting proof of principle that such a strategy is feasible. The current version of Ribavirin, however, has certain limitations, such as its poor specificity and the high dosage (micromolar range) required for effective treatment. Thus, more specific and effective eIF4E inhibitors are urgently needed. The drug treatment experiments with Ribavirin validated Drosophila NBs as an excellent CSC model for searching further improved drugs. More importantly, the nuclear interaction between eIF4E and Myc unraveled by this biochemical analysis not only provides a new mechanistic explanation for the synergistic effects of eIF4E and Myc in tumorigenesis (Ruggero, 2004; Wendel, 2007), but also sheds new light on how to rationally optimize drug design and therapy for treating CSC-based cancer (Song, 2011).

The results offer new information on how N signaling helps specify and maintain NSC fate. N signaling regulates stem cell behavior in various tissues of diverse species. However, it remains unclear how differential N signaling determines distinct cell fate within the stem cell hierarchy. This study demonstrates that N signaling maintains Drosophila NSC fate at least in part through promoting cell growth. The following evidence supports that cell growth, but not cell fate, change is the early and primary effect of N signaling inhibition in type II NBs: (1) Pros expression is not immediately turned on in spdo mutant NBs with reduced cell sizes. Instead, it gradually increases during the course of spdo mutant NB divisions. (2) Up-regulation of Pros is not the cause of stem cell fate loss in spdo mutant NBs, as shown by spdo pros double-mutant analysis. (3) Cell growth defects precede the up-regulation of Ase expression in aph-1 mutant NBs. (4) Promotion of cell growth, and particularly nucleolar growth, by dMyc is sufficient to prevent NB loss caused by N inhibition. At the molecular level, N signaling appears to regulate the transcription of dMyc, which in turn up-regulates the transcription of eIF4E. Such a transcriptional cascade and feedback regulation of dMyc activity by eIF4E may help to sustain and amplify the activity of the Notch-dMyc-eIF4E molecular circuitry. Hence, differential N signaling within the lineage can lead to different cell growth rates, which partially determine differential cell fates. Consistent with this notion, knockdown of both eIF4E and dMyc results in defects of NB cell growth and loss of stem cell fate (Song, 2011).

While many signaling pathways and molecules have been implicated in the maintenance of stem cell identity, the question of how a stem cell loses its 'stemness' at the cellular level remains poorly understood. A stem cell may lose its stem cell fate by undergoing a symmetric division to yield two daughter cells that are both committed to differentiation or through cell death. Earlier studies provided intriguing hints that cell growth and translational regulation could influence stem cell maintenance in the Drosophila ovary. This study usded detailed clonal analyses of NSCs over multiple time points to provide direct evidence that a NSC with impaired N signaling will gradually lose its identity due to a gradual slowing down of cell growth and loss of cell mass. Remarkably, such loss of stem cell fate can be prevented when cell growth is restored by dMyc, but not Rheb, overexpression, demonstrating the functional significance of regulated cell growth, particularly nucleolar growth, in stem cell maintenance. More importantly, this information offers clues on how to specifically eliminate tumor-initiating stem cells. These studies suggest that a stem cell, normal or malignant, has to reach a certain growth rate in order to acquire and maintain its stemness, presumably because when the stem cell grows below such a threshold, its proliferative capacity becomes too low, whereas the concentration of differentiation-promoting factors becomes too high to be compatible with the maintenance of stem cell fate. Consistent with this notion are the strong correlation between the expression of ribosomal proteins and cellular proliferation (van Riggelen, 2010) as well as the correlation between the reduction of NB sizes and the up-regulation of differentiation-promoting factor Pros or Ase in different developmental contexts (Song, 2011).

Crystal structure of a minimal eIF4E-Cup complex reveals a general mechanism of eIF4E regulation in translational repression

Cup is an eIF4E-binding protein (4E-BP) that plays a central role in translational regulation of localized mRNAs during early Drosophila development. In particular, Cup is required for repressing translation of the maternally contributed oskar, nanos, and gurken mRNAs, all of which are essential for embryonic body axis determination. This study presents a 2.8 Å resolution crystal structure of a minimal eIF4E-Cup assembly, consisting of the interacting regions of the two proteins. In the structure, two separate segments of Cup contact two orthogonal faces of eIF4E. The eIF4E-binding consensus motif of Cup (YXXXXLΦ) binds the convex side of eIF4E similarly to the consensus of other eIF4E-binding proteins, such as 4E-BPs and eIF4G. The second, noncanonical, eIF4E-binding site of Cup binds laterally and perpendicularly to the eIF4E β-sheet. Mutations of Cup at this binding site were shown to reduce binding to eIF4E and to promote the destabilization of the associated mRNA. Comparison with the binding mode of eIF4G to eIF4E suggests that Cup and eIF4G binding would be mutually exclusive at both binding sites. This shows how a common molecular surface of eIF4E might recognize different proteins acting at different times in the same pathway. The structure provides insight into the mechanism by which Cup disrupts eIF4E-eIF4G interaction and has broader implications for understanding the role of 4E-BPs in translational regulation (Kinkelin, 2012).

The results also provide new insights into how the evolutionarily conserved tripartite motif and Ncl-1, HT2A, and Lin-41 (TRIM-NHL) domain proteins regulate stem cell homeostasis. The TRIM-NHL protein family, to which Brat and Mei-P26 belong, include evolutionarily conserved stem cell regulators that prevent ectopic stem cell self-renewal by inhibiting Myc. However, the downstream effectors of the TRIM-NHL proteins remain largely unknown. This study identified eIF4E as such a factor. NB-specific knockdown of eIF4E completely suppresses the drastic brain tumor phenotype caused by loss of Brat. Interestingly, eIF4E knockdown is even more effective than dMyc knockdown in this regard. N signaling and Brat have been proposed to act in parallel in regulating Drosophila type II NB homeostasis. However, at the molecular level, how deregulation of these two rather distinct pathways causes similar brain tumor phenotypes remain largely unknown. The current results suggest that these two pathways eventually converge on the dMyc-eIF4E regulatory loop to promote cell growth and stem cell fate. N overactivation and loss of Brat both result in up-regulation of eIF4E and dMyc in transit-amplifying progenitors, accelerating their growth rates and helping them acquire stem cell fate. Consistent with a general role of eIF4E and dMyc in stem cell regulation, it was shown that partial reduction of eIF4E or dMyc function in the Drosophila ovary effectively rescues the ovarian tumor phenotype due to the loss of Mei-P26. The vertebrate member of the TRIM-NHL family, TRIM32, is shown to suppress the stem cell fate of mouse neural progenitor cells, partially through degrading Myc (Schwamborn, 2009). Whether eIF4E acts as a downstream effector of TRIM32 in balancing stem cell self-renewal versus differentiation in mammalian tissues awaits future investigation (Song, 2011).

MicroRNAs block assembly of eIF4F translation initiation complex in Drosophila

miRNAs silence their complementary target mRNAs by translational repression as well as by poly(A) shortening and mRNA decay. In Drosophila, miRNAs are typically incorporated into Argonaute1 (Ago1) to form the effector complex called RNA-induced silencing complex (RISC). Ago1-RISC associates with a scaffold protein GW182, which recruits additional silencing factors. Previously studies have shown that miRNAs repress translation initiation by blocking formation of the 48S and 80S ribosomal complexes. However, it remains unclear how ribosome recruitment is impeded. This study examined the assembly of translation initiation factors on the target mRNA under repression. Ago1-RISC was shown to induce dissociation of eIF4A, a DEAD-box RNA helicase, from the target mRNA without affecting 5' cap recognition by eIF4E in a manner independent of GW182. In contrast, direct tethering of GW182 promotes dissociation of both eIF4E and eIF4A. It is proposed that miRNAs act to block the assembly of the eIF4F complex during translation initiation (Fukaya, 2014).

MicroRNAs (miRNAs) silence their complementary target mRNAs via formation of the effector ribonucleoprotein complex called RNA-induced silencing complex (RISC). The core component of RISC is a member of the Argonaute (Ago) proteins. In Drosophila, miRNAs are sorted into two functionally distinct Ago proteins, Ago1 and Ago2, according to their structural features and the identity of the 5' end nucleotides. Compared to fly Ago2, fly Ago1 shares more common features with mammalian Ago1-4, making it a suitable model for investigating miRNA-mediated gene silencing in animals. Ago1-RISC mediates translational repression as well as shortening of the poly(A) tail followed by mRNA decay (Behm-Ansmant, 2006). While deadenylation per se disrupts the closed-loop configuration of mRNA and leads to inhibition of translation initiation, Ago1-RISC can repress translation independently of deadenylation (Fukaya and Tomari, 2011). Such a deadenylation-independent 'pure' translational repression mechanism seems to be widely conserved among species (Bazzini, 2012, Bethune, 2012, Mishima, 2012 and Iwakawa and Tomari, 2013; Fukaya, 2014 and references therein).

Ago is not the only protein involved in the miRNA-mediated gene silencing pathway. In flies, a P-body protein GW182 specifically interacts with Ago1, but not with Ago2, through the N-terminal glycine/tryptophan (GW) repeats and provides a binding platform for PAN2-PAN3 and CCR4-NOT deadenylase complexes (Braun, 2011; Chekulaeva, 2011). This protein interaction network is conserved in animals including zebrafish, nematodes, and humans (Fabian, 2011; Kuzuoglu-Ozturk, 2012; Mishima, 2012). Accordingly, GW182 is essential for shortening of the poly(A) tail by miRNAs. In contrast, recent studies revealed that miRNA-mediated translational repression occurs in both GW182-dependent and -independent manners (Fukaya, 2012; Wu, 2013). Previous sedimentation analysis on sucrose density gradient suggested that both of the two translational repression mechanisms block recruitment of the ribosomal 43S preinitiation complex to the target mRNA independently of deadenylation (Fukaya, 2012; Fukaya, 2014 and references therein).

In eukaryotes, recruitment of the 43S preinitiation complex is initiated by the formation of eukaryotic translation initiation factor 4F (eIF4F). eIF4F is a multiprotein complex composed of the cap-binding protein eIF4E, which recognizes the 7-methyl guanosine (m7G) structure of the capped mRNA; the scaffold protein eIF4G, which interacts with 40S ribosome-associated eIF3 and bridges the mRNA and the 43S preinitiation complex; and the DEAD-box RNA helicase eIF4A, which plays a pivotal role in translation initiation supposedly through unwinding the secondary structure of the 5' UTR for landing of the 43S complex. In addition, the poly(A)-binding protein PABP stimulates translation initiation through its direct interaction with eIF4G. miRNAs likely block one (or more) of these steps to repress translation initiation. It was recently proposed that, in mammals, preferential recruitment of eIF4AII (one of the two eIF4A paralogs) is required for miRNA-mediated translational repression (Meijer, 2013). This model postulates that eIF4AII acts to inhibit rather than activate translation, unlike its major counterpart eIF4AI. However, the role of eIF4AII in translation remains largely unexplored, as opposed to eIF4AI's well-established function to promote translation. Moreover, invertebrates have only one eIF4A, making this model incompatible in flies. Thus, it still remains unclear how miRNAs repress translation initiation. This is largely due to technical limitations in directly monitoring the assembly of the translation initiation complex specifically on the mRNA targeted by miRNAs (Fukaya, 2014).

Using site-specific UV crosslinking this study examined the association of translation initiation factors on the target RNA under repression. Fly Ago1-RISC specifically induces dissociation of eIF4A from the target mRNA without affecting the 5' cap recognition by eIF4E in a manner independent of GW182 or PABP. On the other hand, direct tethering of GW182 to the target mRNA promotes dissociation of both eIF4E and eIF4A. It is proposed that miRNAs act to block assembly of the eIF4F complex during translation initiation, in addition to their established role in deadenylation and decay of their target mRNAs (Fukaya, 2014).

Although eIF4G could not be detected via any of the crosslinking positions spanning from 2 nt to 13 nt downstream of the cap, previous studies have shown that noncanonical translation driven by direct tethering of eIF4G to the 5' UTR was fully susceptible to translational repression by Ago1-RISC (Fukaya, 2012). Therefore, it was reasoned that Ago1-RISC directly targets eIF4A rather than eIF4E or eIF4G. In the accompanying paper, Fukao (2014) revealed that human Ago2-RISC specifically induces dissociation of eIF4A-both eIF4AI and eIF4AII-without affecting eIF4E or eIF4G in a cell-free system deriving from HEK293F cells (Fukao, 2014). Thus, eIF4A is likely a target of miRNA action conserved among species. In agreement with this model, miRNA-mediated gene silencing is cancelled by the eIF4A inhibitors silvestrol (Fukao, 2014), hippuristanol, or pateamine A (Leung, 2011; Meijer, 2013) in human cells (Fukaya, 2014).

GW182 is a well-known interactor of miRNA-associated Ago proteins and is a prerequisite for miRNA-mediated deadenylation/decay of target mRNAs (Behm-Ansmant, 2006). GW182 directly binds to both NOT1 and CAF40/CNOT9, thereby recruiting the CCR4-NOT deadenylase complex to the target mRNA. It has been suggested that the CCR4-NOT complex not only shortens the poly(A) tail but also plays a role in miRNA-mediated translational repression, because direct tethering of the CCR4-NOT complex was capable of inducing translational repression independently of deadenylation. It was originally proposed that, in humans, the CCR4-NOT complex specifically binds to eIF4AII (but not to eIF4AI) to repress translation. However, this model was challenged by recent studies showing that, although the MIFG4 domain of human CNOT1 structurally resembles the middle domain of eIF4G, it does not bind eIF4AI or II but instead partners with the DEAD-box RNA helicase DDX6, which has been implicated in repression of translation initiation and/or translation elongation as well as activation of decapping. Given that miRNAs mediate gene silencing via multiple different pathways, recruitment of DDX6 by GW182 via the CCR4-NOT complex may well play a role in inhibiting protein synthesis from miRNA targets. Indeed, this study observed strong dissociation of both eIF4E and eIF4A by direct tethering of GW182. However, at the physiological stoichiometry between Ago1 and GW182 in S2 cell lysate, eIF4A was specifically dissociated without apparent effect on eIF4E by canonical miRNA targeting, which is in agreement with the result of the reporter assay in S2 cells depleted of each eIF4F component. It is envisioned that, although GW182 is clearly essential for miRNA-mediated deadenylation, the degree of contribution of GW182 to translational repression can vary in different cell types and conditions, depending on the concentrations of GW182 and Ago proteins, as well as their protein interaction networks that are subject to regulation by extracellular signaling. In this regard, direct tethering of GW182 may potentially overestimate its role in miRNA-mediated translational repression (Fukaya, 2014).

How could Ago1-RISC specifically dissociate eIF4A from the initiation complex? Previous work has shown that none of GW182, the CCR4-NOT complex, or PABP is required for translational repression by Ago1-RISC (Fukaya, 2012). The current data extend these findings to reveal that Ago1-RISC can induce dissociation of eIF4A independently of GW182 or PABP. It is tempting to speculate that an as-yet-unidentified factor associated with Ago1-RISC, or perhaps Ago1-RISC itself, blocks the interaction between eIF4G and eIF4A (e.g., similarly to Programmed Cell Death 4 [PDCD4] whose tandem MA-3 domains compete with the MA-3 domain of eIF4G to bind the N-terminal domain of eIF4A, thereby displacing eIF4A from the eIF4F initiation complex). Alternatively, Ago1-RISC might directly or indirectly inhibit the ATP-dependent RNA-binding activity of eIF4A, which is tightly regulated by its accessory proteins eIF4B and eIF4H (Abramson, 1988; Richter, 1999). Future studies are warranted to determine how miRNAs block the assembly of the eIF4F translation initiation complex (Fukaya, 2014).

4E-BPs require non-canonical 4E-binding motifs and a lateral surface of eIF4E to repress translation

eIF4E-binding proteins (4E-BPs; see Drosophila Thor) are a widespread class of translational regulators that share a canonical (C) eIF4E-binding motif (4E-BM) with eIF4G. Consequently, 4E-BPs compete with eIF4G for binding to the dorsal surface on eIF4E to inhibit translation initiation. Some 4E-BPs contain non-canonical 4E-BMs (NC 4E-BMs), but the contribution of these motifs to the repressive mechanism-and whether these motifs are present in all 4E-BPs-remains unknown. This study shows that the three annotated Drosophila melanogaster 4E-BPs contain NC 4E-BMs. These motifs bind to a lateral surface on eIF4E that is not used by eIF4G. This distinct molecular recognition mode is exploited by 4E-BPs to dock onto eIF4E-eIF4G complexes and effectively displace eIF4G from the dorsal surface of eIF4E. These data reveal a hitherto unrecognized role for the NC4E-BMs and the lateral surface of eIF4E in 4E-BP-mediated translational repression, and suggest that bipartite 4E-BP mimics might represent efficient therapeutic tools to dampen translation during oncogenic transformation (Igreja, 2014).

Drosophila argonaute1 and argonaute2 employ distinct mechanisms for translational repression

microRNAs induce translational repression by binding to partially complementary sites on their target mRNAs. An in vitro system was established that recapitulates translational repression mediated by the two Drosophila Argonaute (Ago) subfamily proteins, Ago1 and Ago2. Ago1-RISC (RNA-induced silencing complex) was shown to represses translation primarily by ATP-dependent shortening of the poly(A) tail of its mRNA targets. Ago1-RISC can also secondarily block a step after cap recognition. In contrast, Ago2-RISC competitively blocks the interaction of eIF4E with eIF4G and inhibits the cap function. The finding that the two Ago proteins in flies regulate translation by different mechanisms may reconcile previous, contradictory explanations for how miRNAs repress protein synthesis (Iwasaki, 2009).

This study shows that in flies both Ago1 and Ago2 can induce translational repression. These findings contradict a previous proposal that only Ago1 can repress translation (Förstemann, 2007). This notion was based on the observation that in S2 cells a central-bulged reporter for endogenous miR-277, which partitions into both Ago1 and Ago2, was derepressed when Ago1, but not Ago2, was absent. However, in flies Ago1- and Ago2-RISC-loading pathways compete in vitro and in vivo; when one Ago is absent, another Ago can function more strongly with more small RNAs loaded. Because the translational repression by Ago1 is stronger, up to ∼8-fold repression for Ago1 and up to ∼2.5-fold repression for Ago2 in an in vitro system) and presumably more irreversible than that of Ago2, it is possible that, in the case of miR-277, the Ago1-mediated repression was enhanced to compensate for the loss of Ago2. In contrast, this study programmed Ago1-RISC or Ago2-RISC in an essentially exclusive manner, which allowed discrimination of their functions (Iwasaki, 2009).

Drosophila Ago1 shortens the poly(A) tail of the target mRNA, and miRNA-directed deadenylation has been reported in other organisms. Because miRNAs can shorten the poly(A) tail even when translation is blocked, deadenylation is not a consequence of translational repression, but a process independent of it. This study found that deadenylation by Ago1 requires ATP, even though ATP is dispensable for the cleavage activity of Ago1-RISC and for the association of Ago1 with GW182, Pop2/Caf1, and Ccr4. Deadenylation catalyzed by the Ccr4-Not complex is per se ATP independent. Thus, the ATP-dependent step after target recognition remains to be identified for Ago1-directed mRNA deadenylation. Such ATP-dependent deadenylation is reminiscent of regulation of the Drosophila mRNA nanos (Iwasaki, 2009).

Kinetic modeling analyses have shown that differences in the rate-limiting steps of translation can have large effects on the outcome of repression. The current data show that poly(A) mRNAs are less efficiently repressed by Ago1 compared with poly(A)+ mRNAs, agreeing with previous reports on miRNA-mediated translational repression in mammals. This can be simply explained by the fact that poly(A)+ mRNAs are better translated than poly(A) mRNAs. At the same time, this is also consistent with the idea that poly(A) and poly(A)-binding protein (PABP) influence the rate-limiting steps in translation, which can affect the ability of repressors to limit translation. Indeed, PABP is involved in multiple key steps of translation initiation, including cap recognition by eIF4E, 48S, and 80S formation and ribosome recycling. Some literature concludes that poly(A) tail is hardly required for miRNA-mediated repression, but the reason for the apparent discrepancy on the poly(A) dependence of repression is obscure. Perhaps repression at a step after cap recognition is significantly stronger and/or translation itself is less dependent on the poly(A) length under these assay protocols compared with other protocols. Such variations can be caused by a number of factors, including time scale of analysis and stoichiometry of RISC, target mRNA, and translational machinery (Iwasaki, 2009).

Most importantly, the current data show that, even when the same protocols and substrates were used (i.e., under conditions with identical rate-limiting steps in translation), there are striking differences between Ago1- and Ago2-mediated translational repression. (1) Repression by Ago1 is accompanied by deadenylation of the target mRNA, whereas repression by Ago2 has no such effect on the target mRNA. (2) Ago1 requires GW182 for translational repression and ATP-dependent deadenylation, whereas Ago2 represses translation independent of GW182. (3) Ago1-RISC blocks a step after cap recognition, whereas Ago2-RISC binds to eIF4E and specifically blocks eIF4E-eIF4G interaction. Of note is that the affinity of Ago2 to eIF4E is dramatically enhanced when Ago2-RISC binds to a target mRNA, which should, in theory, ensure that the Ago2-mediated repression is limited to translation of the cognate target. Because Ago2 exerts no influence on the target mRNA quantitatively and qualitatively, it is tempting to speculate that Ago2 provides rapid, short-lived repression that preserves the target mRNA, allowing its expression to be reactivated later, whereas repression mediated by Ago1 is more irreversible in cells where deadenylation triggers mRNA decay. Although it is not known whether mammalian Ago proteins similarly act by distinct mechanisms, these findings may reconcile previous, contradictory explanations for how miRNAs repress protein synthesis (Iwasaki, 2009).

The extracellular-regulated kinase effector Lk6 is required for Glutamate receptor localization at the Drosophila neuromuscular junction

The proper localization and synthesis of postsynaptic glutamate receptors are essential for synaptic plasticity. Synaptic translation initiation is thought to occur via the target of rapamycin (TOR) and mitogen-activated protein kinase signal-integrating kinase (Mnk) signaling pathways, which is downstream of extracellular-regulated kinase (ERK). This study used the model glutamatergic synapse, the Drosophila neuromuscular junction, to better understand the roles of the Mnk and TOR signaling pathways in synapse development. These synapses contain non-NMDA receptors that are most similar to AMPA receptors. The data show that Lk6, the Drosophila homolog of Mnk1 and Mnk2, is required in either presynaptic neurons or postsynaptic muscle for the proper localization of the GluRIIA glutamate receptor subunit. Lk6 may signal through eukaryotic initiation factor (eIF) 4E to regulate the synaptic levels of GluRIIA as either interfering with eIF4E binding to eIF4G or expression of a nonphosphorylatable isoform of eIF4E resulted in a significant reduction in GluRIIA at the synapse. It was also found that Lk6 and TOR may independently regulate synaptic levels of GluRIIA. (Hussein, 2016).

This study is the first to provide information on the properties and regulation of the Drosophila protein kinase LK6. Its catalytic domain is strikingly similar to those of mammalian Mnks; similar to them, in mammalian cells LK6 can bind to ERK, can be activated by ERK signalling and can phosphorylate eIF4E. This occurs at the physiological site, Ser209. The MAPK-binding motif of LK6 is of the type previously shown to bind ERK but not p38 MAPK. Consistent with this, when expressed in mammalian cells, LK6 is not activated by stimuli that turn on p38 MAPK (Hussein, 2016).

It is more challenging to perform similar experiments in Drosophila cells owing to the difficulty in transfecting, e.g. S2 cells with high efficiency. However, importantly, this study shows that LK6 also interacts with the ERK homologue Rolled, but not with the Drosophila p38 homologue. The results, furthermore, show that LK6 is activated by Phorbol myristate acetate (PMA), but not by arsenite, which activates p38 MAPK. The regulatory properties of LK6 thus appear to be similar in mammalian and Drosophila cells, indicating that the specificity of the MAPK-interaction motifs is probably similar in both mammals and Diptera. Similar to Mnk1 and Mnk2a, LK6 is primarily, if not exclusively, cytoplasmic. It does contain a basic region of the type that, in Mnk1 and Mnk2, can bind to the nuclear shuttling protein importin-α. It therefore seems probable that either (1) it contains an NES, which ensures its efficient re-export from the nucleus, or (2) the basic region is not accessible to importin-α. The lack of effect of LMB on the localization of LK6 rules out the operation of a CRM1-type NES of the kind found in Mnk1, although the very long C-terminal extension of LK6 might contain an LMB-insensitive NES (Hussein, 2016).

By analogy with the Mnks, it is probable that the N-terminal polybasic region of LK6 mediates its binding to eIF4G and could also interact with importin-α. Given that full-length LK6 shows less efficient binding to eIF4G when compared with Mnk1, it also seems possible that it binds importin-α less efficiently, which may contribute to the finding that LK6 is cytoplasmic. It has been shown previously that even the much shorter C-terminus of Mnk2a impedes access to the N-terminal basic region in that protein, so it is entirely possible that the much larger C-terminal part of LK6 has a similar effect. This could explain why the fragment of LK6 that lacks the C-terminus bound better to eIF4G than did the full-length protein. It may also be that the low degree of binding reflects the fact that the association of LK6 with the heterologous human protein was being studied, rather than with Drosophila eIF4G. Repeated attempts have been made to use the available antisera to examine the association of LK6 with eIF4G in S2 cells, but without success. Comparison of the polybasic region of LK6 with those of Mnk1 and Mnk2a (which do bind eIF4G and importin-α), and recent results for mutants with alterations in these features, do not reveal any difference that might obviously explain the decreased ability of LK6 to bind mammalian eIF4G. As argued above, the C-terminus of LK6 may also impair its activation by ERK, based on the observation that the catalytic domain is more effectively activated than a mutant of the full-length protein that also lacks the ERK-binding motif (Hussein, 2016).

The results support the idea that LK6 is a Drosophila eIF4E kinase. LK6 can phosphorylate eIF4E in vitro and its overexpression in cells leads to increased phosphorylation of endogenous eIF4E. Furthermore, the activation of LK6 by ERK signalling but not by p38 MAPK signalling correlates well with the observed behaviour of the phosphorylation of eIF4E in PMA- or arsenite-treated Drosophila cells, and the fact that LK6 is activated by stimuli that stimulate ERK but is not activated by stimuli that activate p38 MAPK, in HEK-293 cells. The ability of LK6 to bind eIF4G also supports the contention that it can act as an eIF4E kinase in vivo (Hussein, 2016).

The observation that phosphorylation of the endogenous eIF4E in S2 cells is increased by PMA but not by arsenite is consistent with the regulatory properties of LK6 and with the notion that LK6 may phosphorylate eIF4E in these cells. The fact that it is the only close homologue of the Mnks in the fruitfly genome is also consistent with this notion. Phosphorylation of eIF4E has previously been shown to play an important role in growth in this organism and in its normal development. The current data show that LK6 can phosphorylate Drosophila eIF4E in vitro, consistent with the idea that LK6 acts as an eIF4E kinase in this organism. The dsRNAi data that was obtained, which show that two different interfering dsRNAs directed against LK6 each markedly decrease eIF4E phosphorylation in S2 cells, offer strong support to the conclusion that LK6 acts as an eIF4E kinase in Drosophila. Unfortunately, the poor quality of the available anti-LK6 antisera prevented assessing whether the incomplete nature of the loss of phosphorylation of eIF4E reflects incomplete elimination of LK6 expression (Hussein, 2016).

Previous genetic studies have linked LK6 to Ras signalling in Drosophila. This agrees very well with the finding that LK6 is activated by ERK signalling, since ERK lies downstream of Ras. LK6 was first identified as interacting with microtubules and centrosomes. Overexpression of LK6 led to defects in microtubule organization, indicative of their increased stability. The connections between the phosphorylations of eIF4E and microtubules are not immediately obvious. However, it is entirely possible that LK6 has additional substrates that interact with microtubules or are components of centrosomes and their phosphorylation may be important in the regulation of, for example, mitosis. Numerous microtubule-associated proteins are indeed phosphorylated. Microtubules undergo massive reorganization during mitosis and this involves an array of phosphorylation events and protein kinases. It may therefore be relevant that LK6 is activated by mitogenic signalling (i.e. through ERK and thus Ras) (Hussein, 2016).

Functional analysis of seven genes encoding eight translation initiation factor 4E (eIF4E) isoforms in Drosophila

The Drosophila genome-sequencing project has revealed a total of seven genes encoding eight eukaryotic initiation factor 4E (eIF4E) isoforms. Four of them (eIF4E-1,2, eIF4E-3, eIF4E-4 and eIF4E-5) share exon/intron structure in their carboxy-terminal part and form a cluster in the genome. All eIF4E isoforms bind to the cap (m7GpppN) structure. All of them, except eIF4E-6 and eIF4E-8 are able to interact with Drosophila eIF4G or eIF4E-binding protein (4E-BP). eIF4E-1, eIF4E-2, eIF4E-3, eIF4E-4 and eIF4E-7 rescue a yeast eIF4E-deficient mutant in vivo. Only eIF4E-1 mRNAs and, at a significantly lower level, eIF4E3 and eIF4E-8 are expressed in embryos and throughout the life cycle of the fly. The transcripts of the remaining isoforms are detected from the third instar larvae onwards. This indicates the cap-binding activity relies mostly on eIF4E-1 during embryogenesis. This agrees with the proteomic analysis of the eIF4F complex purified from embryos and with the rescue of l(3)67Af, an embryonic lethal mutant for the eIF4E-1,2 gene, by transgenic expression of eIF4E-1. Overexpression of eIF4E-1 in wild-type embryos and eye imaginal discs results in phenotypic defects in a dose-dependent manner (Hernandez, 2005; full text of article).

The ability of the eight Drosophila eIF4E isoforms to bind to cap, to eIF4G and to 4E-BP, and to support cell growth in a yeast mutant deficient for eIF4E, demonstrate that eIF4E-1, eIF4E-2, eIF4E-3, eIF4E-4 and eIF4E-7 support translation initiation and suggest that they may be functional equivalents in vivo. On contrary, eIF4E-6 and eIF4E-8 do recognize cap with lower efficiency than other eIF4Es, and did not interact with eIF4G nor were able to support cell growth in the yeast mutant. They are the unique isoforms carrying nonconservative amino acid substitutions (two in the case of eIF4E-6 and six in the case of eIF4E-8) at important residues for the interaction of eIF4E with eIF4G. In conclusion, eIF4E-6 and eIF4E-8 may be either negative regulators of translation or simply non-functional proteins. In contrast, serine 251 of eIF4E-1 is critical for the function of eIF4E-1 in Drosophila (Lachance, 2002), but eIF4E-3 that lacks this serine is able to support cell growth in the yeast eIF4E-mutant. Since yeast eIF4E also lacks this serine it is supposed that there is no requirement for it in the function of eIF4E from this organism (Hernandez, 2005).

In spite of the existence of eight isoforms for eIF4E in Drosophila, this study has shown that in Drosophila embryos the cap-dependent translation relies mainly on eIF4E-1 and that the activity of this factor is essential throughout embryogenesis. This has important implications for development. During oogenesis, the repression of oskar mRNA in the posterior pole of the oocyte is essential for germ line formation and patterning. During early embryogenesis, repression of caudal mRNA expression in the anterior part of the embryo is required for appropriate assembly of the head segments. Both maternal mRNAs are regulated by the interaction of their repressors Cup and Bicoid with eIF4E-1 (Nakamura, 2004; Niessing, 1999, 2002; Wilhelm, 2003). This study shows that the likely isoform to be involved in translation of early mRNAs is eIF4E-1. The analysis of the Me31B complex from ovary extracts presented by Nakamura (2004) showing that only eIF4E-1 is enriched in the oskar mRNA repression complex also supports the idea of a key role for eIF4E-1 in early embryogenesis (Hernandez, 2005).

In spite of the evolutionary conservation of the translational machinery across the eukaryotic phyla, only some eIF4E isoforms can complement for the lack of the yeast eIF4E. They include human eIF4E-1, A. thaliana eIF4E, zebrafish eIF4E-1A, and five Drosophila eIF4E isoforms. In contrast, other initiation factors involved in mRNA recruitment and scanning do not allow for cross-complementation: neither mouse eIF4A nor Drosophila factors eIF4A, eIF4B or eIF4G substitute for their yeast counterparts. This suggests that the pathways regulating eIF4E activity may be universally more conserved than those for other factors. It will be worthwhile to test whether other initiation factors (e.g. eIF3, eIF2, etc.) are able to substitute for their homologs, and whether eIF4E is interchangeable between phyla other than yeast (e.g., between plants and mammals) (Hernandez, 2005).

Increased eIF4F formation is closely related to enhanced protein synthesis and thus to cell growth. eIF4E is referred to as the limiting factor in the formation of eIF4F complex (Sonenberg, 1998) because it is less abundant in some mammalian cells than eIF4G. However, in most cases, overexpression of eIF4E in mammal cells leads to malignant transformation and non-controlled cell proliferation only when overexpressed in cell cycle-sensitized cells or when co-expressed together with other proto-oncogenes. In S. cerevisiae, even 100 fold overexpression of eIF4E had only a minor effect on growth rates. In Xenopus and S. pombe, overexpression of either of the two eIF4Es modestly increases translation in oocytes or had not affected cell growth, respectively. This study observed that the overexpression of eIF4E-1 transgene produces phenotypic defects in early embryos (a time when endogenous eIF4E-1 is expressed most strongly) or when it was expressed in more than one copy in the developing eye. Since it has recently been demonstrated for Drosophila eIF4B (Hernandez, 2004), this study provides in vivo evidence for phenotypic defects produced by changes in eIF4E levels in a non-oncogenic or cell cycle-sensitized genetic background, both in embryo and in the developing eye. These defects are produced in a dose-dependent manner (Hernandez, 2005).

Altogether, these data suggest that in a wild type genetic background only a very high level of overexpression of eIF4E-1 might result in phenotypic effects. This would explain why no phenotypic defects were obtained by overexpression of Drosophila eIF4E-1 in previous studies (Lachance, 2002; Zhang, 2000). It seems likely that those cells having an excess of eIF4E over eIF4G, like in rabbit reticulocyte lysates and Drosophila embryos, are less sensitive to a further increase in the amount of free eIF4E. This implies that eIF4G, not eIF4E, is the limiting factor in the formation of eIF4F during the initiation of translation (Hernandez, 2005 and references therein).

The Bin3 RNA methyltransferase is required for repression of caudal translation in the Drosophila embryo

Bin3 was first identified as a Bicoid-interacting protein in a yeast two-hybrid screen. In human cells, a Bin3 ortholog (BCDIN3) methylates the 5' end of 7SK RNA, but its role in vivo is unknown. This study shows that in Drosophila, Bin3 is important for dorso-ventral patterning in oogenesis and for anterior-posterior pattern formation during embryogenesis. Embryos that lack Bin3 fail to repress the translation of caudal mRNA and exhibit head involution defects. bin3 mutants also show (1) a severe reduction in the level of 7SK RNA, (2) reduced binding of Bicoid to the caudal 3' UTR, and (3) genetic interactions with bicoid, and with genes encoding eIF4E, Larp1, polyA binding protein (PABP), and Ago2. 7SK RNA coimmunoprecipitated with Bin3 and is present in Bicoid complexes. These data suggest a model in which Bicoid recruits Bin3 to the caudal 3' UTR. Bin3's role is to bind and stabilize 7SK RNA, thereby promoting formation of a repressive RNA-protein complex that includes the RNA-binding proteins Larp1, PABP, and Ago2. This complex would prevent translation by blocking eIF4E interactions required for initiation. These results, together with prior network analysis in human cells, suggest that Bin3 interacts with multiple partner proteins, methylates small non-coding RNAs, and plays diverse roles in development (Singh, 2011).

The human homolog of Bin3, also called BCDIN3 or methylphosphate capping enzyme (MePCE), was shown to methylate the 5' γ-phosphate on 7SK RNA and to stabilize 7SK RNA in cell culture. This study found that Bin3 associates with and stabilizes 7SK RNA in ovaries and embryos. And, as in human cells, Bin3 activity was specific for 7SK RNA and did not affect U3 RNA or another RNA pol III product, U6 RNA, both of which are methylated by distinct mechanisms. It seem likely, therefore, that Drosophila Bin3 has a similar biochemical activity to its human counterpart despite differing in size and sequence outside the AdoMet binding domain and the highly conserved Bin3-homology domain. Prior attempts to demonstrate protein-arginine methyltransferase activity of Bin3 were negative, consistent with Bin3 methylating RNA rather than protein. In Drosophila, there are two other Bin3-like genes, CG11342 and CG1239, but each is more divergent from the human BCDIN3 within the conserved motif architecture (24% and 39% identity, respectively) than Bin3. It is possible that CG1239, which is expressed in early embryos, could have partially overlapping functions with Bin3 that might contribute to the incomplete penetrance of the bin3 mutations (Singh, 2011).

Putative Bin3 orthologs containing the two conserved motifs are found in at least 70 eukaryotic organisms ranging from the yeast, Schizosaccharomyces pombe to humans, and including Caenorhabditis elegans, Arabidopsis thaliana, Xenopus laevis, and Danio rerio. It is not known what any of these genes do, with the possible exception of the zebrafish bin3 gene which was shown by morpholino knockdown to be important for anterior development and to display RNA splicing defects. Similar defects were sought in splicing of bicoid, caudal, eIF4E, d4EHP, and a control gene, taf1, known to show alternative splicing. No splicing defects were found using a sensitive qRT-PCR approach. It is possible that the splicing defects in zebrafish result from aberrant 5' capping of non-coding RNAs important for splicing (Singh, 2011).

Mammalian 7SK RNA has been studied extensively, but Drosophila 7SK RNA has only been annotated, and prior to this study has not been characterized. This study shows that 7SK RNA is highly expressed in ovaries and embryos and is regulated by Bin3 in a manner similar to that in humans (by BCDIN3). 7SK RNA can be coimmunoprecipitated with Bin3 and Bicoid and may work as a scaffold in translation repression. This is the first indication that 7SK RNA has a function apart from its role in the regulation of the pTEFb transcription elongation factor. While this study focused on Bicoid-dependent regulation, it is likely that 7SK RNA also functions in transcription elongation in other stages of development. Indeed, it was found that Drosophila 7SK RNA mutants showed larval lethality at later stages of development (Singh, 2011).

Bin3 seems to play no role in Bicoid's gene activation function, but instead is crucial for Bicoid-dependent repression of caudal mRNA. Bin3 seems to stabilize Bicoid at the caudal BRE via a mechanism that involves 7SK RNA. As suggested by genetic interaction data, the Bicoid/Bin3/7SK RNA complex may include Larp1, PABP, and Ago2, and target the eIF4E initiation factor (Singh, 2011).

La-related proteins are not restricted to control of transcription elongation. In C. elegans, a Larp1 homolog was shown to be important for downregulation of translation of mRNAs in the Ras-MAPK pathway and to localize to P-bodies, known sites of mRNA degradation, while in mammalian cells, LARP4B plays a stimulatory role in translation initiation. In Drosophila, it has been shown that Larp1 associates directly with PABP independent of RNA and double mutants show enhanced lethality, suggesting that Larp1 facilitates mRNA translation. It is not surprising, therefore, that genetic interactions were observed between bin3 and larp1, as well as with pAbp in the context of caudal translation regulation. Note that it is though that PABP (and Larp1) plays a negative role in translation initiation, as does PABP in the repression of msl-2 mRNA by Sex-lethal (Singh, 2011).

In human cells, BCDIN3 and LARP7 interact cooperatively with 7SK RNA forming a stable core complex that associates transiently with HEXIMS, hnRNPs and the P-TEFb elongation complex (see Drosophila Hexim). An emerging theme is that 7SK RNA serves as a scaffold for stable association of protein partners. In fact, there is evidence that 5' γ-methylation of 7SK RNA by BCDIN3 may occur co-transcriptionally, but that the modified RNA remains associated with both BCDIN3 and LARP7, which bind 7SK RNA cooperatively. It is proposed, therefore, that Bin3 and Larp1 are associated with 7SK RNA at the caudal BRE, but that 5'-methylation does not necessarily occur there. Consistent with the idea of cooperative binding to 7SK RNA, it was found that larp1 mutation enhanced the bin3 mutant phenotype (Singh, 2011).

Some of the phenotypes observed for bin3 mutants were also observed in mutants of the microRNA miR-184, including oogenesis defects and a cellularization defect. This was the rationale behind including ago2 in the genetic analysis. However, no effect was found of bin3 mutation on levels of several miRNAs, including miR-184, it was surprising to observe a genetic enhancement (albeit mild) of the bin3 phenotype when combined with an ago2 mutation. Ago2 has been shown to bind eIF4E and interfere with mRNA circularization mediated by PABP. However, this occurs in the context of the miRNA/RISC complex, so whether and how Ago2 participates in Bicoid-Bin3 repression is not clear, but it could potentially involve the 7SK RNA component (Singh, 2011).

Finally, no interaction was detected between bin3 and D4EHP, which encodes a previously identified partner of Bicoid important for repressing caudal translation. D4EHP interacts with Bicoid and is thought to directly bind the m7G cap of caudal mRNA, thereby displacing eIF4E and blocking all subsequent steps of initiation. Perhaps the Bin3 mechanism works redundantly with the D4EHP mechanism or perhaps Bin3 helps recruit D4EHP, and so that mutation of bin3 would preclude binding of D4EHP to the initiation complex. Thus, bin3 mutation would be epistatic to the D4EHPCP53 mutation. Further investigation will be needed to determine relationship between these two pathways (Singh, 2011).

Bin3 is unlikely to be a dedicated Bicoid interactor and probably has roles as an RNA methyltranferase in many distinct pathways throughout development. In adults, quantitative trait transcript analysis linked bin3 with sleep-wake cycling. While studying Bin3's role in embryonic patterning, strong oogenesis defects were observed, particularly in bin3 null mothers, although other allelic combinations also revealed similar defects, especially at 29°C. Specifically, bin3 loss-of-function mutants showed dorsalized egg shell phenotypes. Conversely, bin3 overepressing lines showed strong ventralized egg shell patterns that appear to result from a failure of the dorsal appendage primordium to resolve into two domains along dorsal midline. These defects are similar to those of early D-V patterning mutations in the grk pathway, and probably do not result from defects that occur in later during morphogenesis step (Singh, 2011).

bin3 loss-of-function mutants resembled mutations in capicua, squid, cup and fs(K10), among others, while bin3 overexpressing lines resembled grk and pAbp mutations. Interestingly, mechanisms for translation repression of unlocalized grk mRNA feature prominently in the D-V patterning pathway, with squid and cup playing a critical role in repression via interaction with eIF4E, and PABP55 being important for release of that repression. Staining of bin3 mutant ovaries revealed a delocalized signal for Gurken protein but not for grk mRNA. Given the role of Bin3 in translation regulation, and the egg shell phenotypes of bin3 mutations, it seems plausible that Bin3 plays a role in negative regulation of grk translation (Singh, 2011).

Results presented in this study show that Bin3 plays a critical role during both oogenesis and embryonic development. In embryos, Bin3 is required for Bicoid to establish the Caudal protein gradient. Bin3 binds 7SK RNA and likely works by methylating 7SK RNA and stabilizing a repressive complex that assembles on the Bicoid-response element in the 3' UTR of caudal mRNA. Bin3's role during oogenesis is less clear, but based on the observed eggshell phenotypes in bin3 mutants, and gurken expression, Bin3 could play a similar role to help ensure that grk mRNA is translated only in the anterior-dorsal region of the oocyte (Singh, 2011).

Drosophila Lk6 kinase controls phosphorylation of eukaryotic translation initiation factor 4E and promotes normal growth and development

Eukaryotic initiation factor 4E (eIF4E) controls a crucial step of translation initiation and is critical for cell growth. Biochemical studies have shown that it undergoes a regulated phosphorylation by the MAP-kinase signal-integrating kinases Mnk1 and Mnk2. Although the role of eIF4E phosphorylation in mammalian cells has remained elusive, recent work in Drosophila has established that it is required for growth and development (Lachance, 2002). This study demonstrates that a previously identified Drosophila kinase called Lk6 is the functional homolog of mammalian Mnk kinases. lk6 loss-of-function alleles were generated and it was found that eIF4E phosphorylation is dramatically reduced in lk6 mutants. Importantly, lk6 mutants exhibit reduced viability, slower development, and reduced adult size, demonstrating that Lk6 function is required for organismal growth. Moreover, it is shown that uniform lk6 expression rescues the lethality of eIF4E hypomorphic mutants in an eIF4E phosphorylation site-dependent manner and that the two proteins participate in a common complex in Drosophila S2 cells, confirming the functional link between Lk6 and eIF4E. This work demonstrates that Lk6 exerts a tight control on eIF4E phosphorylation and is necessary for normal growth and development (Arquier, 2005).

To investigate the regulation of eIF4E phosphorylation in the physiological context of a developing organism, the Drosophila genome was scanned for kinases related to the vertebrate Mnk proteins. The best match corresponded to the sequence of a predicted kinase encoded by the lk6 gene. The Lk6 kinase was previously identified as a putative centrosomal, microtubules-associated protein, although a clear centrosomal function for Lk6 has not been established (Kidd, 1997). Alignment of the Lk6 and human Mnk1/2 protein sequences shows a high level of similarity in the kinase domain (65%-76%), as well as the conservation of specific features of the Mnk kinases, such as the presence of three regulatory threonines, an N-terminal stretch of basic amino acids involved in eIF4G binding in vertebrate Mnks, and a conserved MAPK binding sequence. It is therefore proposed that Lk6 is the Drosophila Mnk ortholog (Arquier, 2005).

To investigate the function of lk6, the P element EP3333, inserted in the first intron of the gene, was mobilized. Two imprecise excisions were obtained that delete part of the lk6 locus and thus may constitute lk6 loss-of-function alleles. lk61 contains a 3 kb deletion, which removes an alternative 5′ exon but leaves intact the rest of the coding sequence, allowing the production of lk6-A but not lk6-B transcripts. Homozygous lk61 mutant flies show reduced viability (20% lethality) and are fertile. Adult lk61 flies emerge with a 1-2 day developmental delay and a reduction of mass of 13% in males and 20% in females compared to the wild-type. lk62 deletes all lk6 exons downstream of the EP3333 insertion site, as well as sequences 5′ of the neighboring gene CG6923. Homozygous lk62 animals die as young second-instar larvae with dramatic growth defects. To exclude the possibility that some of these defects may result from the deletion of functional sequences in CG6923, the lk61/lk62 heteroallelic combination, which shows a more severe phenotype than lk61 homozygotes, was examined. lk61/lk62 flies have reduced viability (45% lethality) and a 20% reduction in mass in adult males and 25% in females as compared to heterozygous controls. lk61/lk62 mutant larvae present growth defects, as manifested in endoreplicating tissues such as the fat body and the salivary glands (30% size reduction for fat body cells). When measured in the adult wing, growth reduction (12%) appears primarily caused by a defect in cell number with barely any effect on cell size, indicative of a balanced reduction of cell growth and cell proliferation. The stronger phenotype of lk61/lk62 compared with lk61 homozygotes confirms that lk61 is a partial loss-of-function allele. Ubiquitous da-GAL4-driven expression of an lk6wt construct rescued the lethality, the delayed development, and most of the size reduction associated with lk61/lk62 combination, confirming that mutant animals are indeed deficient for lk6 function (Arquier, 2005).

lk61/lk62 mutants present a mass reduction, development delay, and lethality comparable to eIF4E mutants expressing a nonphosphorylatable form of eIF4E (eIF4ESer251Ala) from a minigene (Lachance, 2002). This and the similarity between Lk6 and mammalian Mnks suggested that lk6 mutants might be deficient in eIF4E phosphorylation. To test this possibility, the level of eIF4E phosphorylation was compared in wild-type and lk61/lk62 mutant ovaries after metabolic labeling with 32P-orthophosphate of eIF4E. eIF4E immunoprecipitated from mutant ovaries incorporates only 10% of the [32P]-orthophosphate found in the control, revealing a 90% reduction of eIF4E kinase activity in the mutant. Thus, Lk6 kinase is required for physiological levels of eIF4E phosphorylation in flies, suggesting that Lk6 acts as an eIF4E kinase in vivo (Arquier, 2005).

To further confirm the link between eIF4E and Lk6, possible genetic interplay between the two genes was examined during development. eIF4E mutants arrest larval growth at various stages depending on allele strength and never reach late larval stages or pupariate. eIF4E67Af mutants exhibit development arrest at the first larval instar, whereas homozygous eIF4EScim-a animals survive to the third instar (Lachance, 2002). Although uniform lk6 expression causes no growth phenotype on its own, it suppressed the larval lethality of an eIF4EScim-a/eIF4E67Af hypomorphic combination. Interestingly, expression of a modified Lk6 protein (Lk6T424D), mimicking a constitutively active version of mammalian Mnk1 (T332D) (Waskiewicz, 1999), better rescued growth in eIF4E mutant larvae, allowing development to small pharate adults. Remarkably, expression of lk6T424D in the weaker eIF4EScim-a/eIF4EScim-a homozygous combination led to the emergence of viable, albeit small, adults. These results indicate that the major growth and developmental defects resulting from reduced eIF4E function are efficiently compensated by increased Lk6 kinase activity (Arquier, 2005).

If lk6T424D expression rescues eIF4E mutants through the ability of the kinase to interact with phosphorylatable eIF4E, it should not further rescue an eIF4E mutant expressing a nonphosphorylatable form of eIF4E (eIF4ESer251Ala). This hypothesis was tested by expressing a UAS-eIF4ESer251Ala construct under the control of the ubiquitous daughterless-Gal4 driver in an eIF4EScim-a/eIF4E67Af background. In these conditions, eIF4ESer251Ala expression only weakly rescues the larval lethality of the mutant, whereas eIF4Ewt expression provides a complete rescue. Coexpression of lk6T424D with eIF4ESer251Ala did not provide a better rescue, indicating that Lk6 cannot act through nonphosphorylatable eIF4ESer251Ala. Overall, these data demonstrate that Lk6 positively controls eIF4E function through its phosphorylation and is required for growth and development (Arquier, 2005).

In support of this conclusion, it was found that, once expressed in Drosophila S2 cells, tagged Lk6 coimmunoprecipitated endogenous eIF4E. Reciprocally, tagged eIF4E also coimmunoprecipitated endogenous Lk6. The presence of excess eIF4E correlated with accumulation of endogenous Lk6, suggesting that Lk6 could be stabilized upon association with eIF4E-containing complexes. These results demonstrate that Lk6 and eIF4E are partners in a common protein complex in insect cells. In this respect, it is worth noting that Lk6 has a conserved eIF4G binding motif analogous to mammalian Mnks, possibly enabling it to bind to the initiation complex (Arquier, 2005).

This work establishes that loss of lk6 function compromises eIF4E phosphorylation and normal growth and development in flies. lk6 expression efficiently compensates for a reduction in eIF4E function, and the two proteins are part of a common biochemical complex in vivo. Overall, this supports the notion that Lk6 controls organismal growth through eIF4E phosphorylation. lk6 mutant flies have reduced wing size caused by reduced cell number. This phenotype is slightly different from what is observed in eIF4ESer251Ala-rescued eIF4E mutants, in which growth reduction in the adult eye results mostly from reduced ommatidial size and only slightly from a reduction in their number (Lachance, 2002). This difference could be explained if Lk6 acts on other targets in addition to eIF4E (Arquier, 2005).

In mammalian cells, Mnks are phosphorylated by Erk and p38 kinases on two conserved Threonine residues in their catalytic domain (Fukunaga, 1997; Waskiewicz, 1997). Whereas Mnk2a exhibits a nonregulated high basal activity, phosphorylation of Mnk1 by MAP kinases greatly enhances its eIF4E kinase activity (for review see Scheper, 2002b). Interestingly, the Erk/p38-targeted residues, as well as the MAPK binding domain present in Mnks, are conserved in Lk6. This, as well as the fact that Lk6 was found in an overexpression screen for modifiers of the ras-signaling pathway in the Drosophila eye (Huang, 2000), suggests that, as in the case of Mnks, Lk6 activity is linked to the ras/MAP kinase cascade in vivo (Arquier, 2005).

Although phosphorylation of eIF4E has been consistently associated with activation of protein synthesis, studies in mammalian tissue culture cells have yielded contradictory results concerning the functional significance of eIF4E phosphorylation during translation initiation. In particular, recent biophysical data established that phosphorylation of eIF4E reduces its affinity for capped mRNA (Scheper, 2002a), suggesting a model in which eIF4E phosphorylation by Mnk kinases triggers its release from the cap structure, therefore allowing recycling and further recruitment of a new initiation complex on the cap. Facilitation of translation initiation through phosphorylation of eIF4E is in agreement with the demonstration of a positive role for Lk6 activity in controlling cell and tissue growth. According to this model, forced eIF4E phosphorylation might cause constitutive destabilization of cap complex and thus be detrimental to translation initiation, as already suggested in mammalian cells (Knauf, 2001). Indeed, in the course of the current experiments, it was observed that strong Lk6 overexpression in the eye disc leads to subtle growth impairments. This suggests that a precise control of eIF4E phosphorylation is necessary for optimal translation and growth machinery function in vivo (Arquier, 2005).

Recent genetic analysis of double Mnk1/Mnk2 knockout mice has revealed that both kinases are dispensable for growth and development (Ueda, 2004). This difference with results in the fly could be because of redundancies or compensatory mechanisms taking place in the regulatory circuitry of mammals. The Drosophila system might thus provide a simpler version of an integrated developing model, allowing important cell regulatory mechanisms to be uncovered (Arquier, 2005).

Diet-dependent effects of the Drosophila Mnk1/Mnk2 homolog Lk6 on growth via eIF4E

The control of cellular growth is tightly linked to the regulation of protein synthesis. A key function in translation initiation is fulfilled by the 5' cap binding eukaryotic initiation factor 4E (eIF4E), and dysregulation of eIF4E is associated with malignant transformation and tumorigenesis. In mammals, the activity of eIF4E is modulated by phosphorylation at Ser209 by mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2), which themselves are activated by ERK and p38 MAPK in response to mitogens, cytokines or cellular stress. Whether phosphorylation of eIF4E at Ser209 exerts a positive or inhibitory effect on translation efficiency has remained controversial. This study provides a genetic characterization of the Drosophila homolog of Mnk1/2, Lk6. Lk6 function is dispensable under a high protein diet, consistent with the recent finding that mice lacking both Mnk1 and Mnk2 are not growth-impaired (Ueda, 2004). Interestingly, loss of Lk6 function causes a significant growth reduction when the amino acid content in the diet is reduced. Lk6 expression has also shown to be upregulated upon starvation during larval development (Zinke, 2002). Overexpression of Lk6 also results in growth inhibition in an eIF4E-dependent manner. A model of eIF4E regulation is provided that may reconcile the contradictory findings with regard to the role of phosphorylation by Mnk1/2 (Reiling, 2005).

Evidence is provided that Lk6 exerts its function via phosphorylation of eIF4E because the effects of Lk6 overexpression are strictly dependent on the presence of Ser251 in eIF4E. This conclusion is strongly supported by the finding that eIF4E phosphorylation is diminished in ovaries of Lk6 mutant flies. Therefore, flies lacking Lk6 function can be expected to display the same phenotype as eIF4E mutant flies rescued by a P{eIF4ESer251Ala transgene. However, the rescued eIF4E mutants grow to a smaller size even under standard culture conditions. Although they contain significantly fewer cells, the size reduction is predominantly caused by smaller cells. In contrast, the loss of Lk6 function primarily affects cell number. Whether these discrepancies reflect a qualitative difference between eIF4E mutant flies rescued by a P{eIF4ESer251Ala} transgene and Lk6 mutants is currently unknown (Reiling, 2005).

It is speculated that the net result of Lk6/Mnk activity (i.e., whether translation is inhibited or promoted) is not determined by the absolute levels of Lk6/Mnk, but rather by the ratio of activated Lk6/Mnk and free eIF4E (i.e., not bound by 4E-BPs), the limiting factor for translation initiation. Under standard culture conditions (high protein), a larger fraction of eIF4E assembles into functional eIF4F complexes because of high TOR activity, thereby promoting translation. Under reduced conditions (e.g., 30% yeast), TOR pathway activity is lowered, and, thus, more 4E-BP binds and inhibits eIF4E, which dampens the rate of translation. It is likely that the predominant mechanism of eIF4E regulation is achieved by TOR/4E-BP activity, and that the phosphorylation of eIF4E by Lk6/Mnk imposes a translational fine-tuning that becomes rate limiting only under adverse food conditions. Lacking Lk6 function in addition to diminished eIF4E availability impinges on translation efficiency, which results in the observed body size reduction (Reiling, 2005).

Alternatively, high TOR activity caused by a diet rich in amino acids could enable the activation of another (unidentified) eIF4E kinase that acts redundantly to Lk6/Mnk. However, this is rather unlikely because mice lacking Mnk1 and Mnk2 do not show any residual eIF4E-Ser209 phosphorylation, strongly arguing against an uncharacterized eIF4E kinase (Reiling, 2005).

Overexpression of Lk6 under standard food conditions consistently resulted in a suppression of growth. Furthermore, another EP insertion in the Lk6 locus (EP3344), which promotes lower expression levels as compared to EPLk6, yielded qualitatively similar but milder phenotypes, suggesting that the dosage of Lk6 expression is important for its ability to regulate growth. Concentration-dependent effects of Mnk1 have also been described by Knauf (2001), who suggested a negative role of Mnk1/2 for cap-dependent translation. It is conceivable that overexpressed Lk6 exerts a dominant-negative effect on translation efficiency by reducing the affinity of phosphorylated eIF4E for capped mRNA, leading to a precocious disassembly of the eIF4F complex (Reiling, 2005).

Reducing the amino acid supply abolished the negative effects of Lk6 overexpression on growth, suggesting that the activity of Lk6 is also regulated in response to nutrients. The mechanism for this additional layer of regulation is unknown but is likely to involve phosphorylation by the upstream kinases ERK and/or p38. Consistently, the p38 homolog in fission yeast, Sty1/Spc1, is regulated in response to nutrient limitation and osmotic stress (Reiling, 2005).

The effects of Lk6 activity are therefore context dependent: They lead either to growth stimulation or growth inhibition. It is proposed that (1) the timing, (2) the amount of eIF4E phosphorylation during 48S complex assembly, and (3) nutrient (amino acid) availability are critical parameters for the modulation of growth by Lk6 (Reiling, 2005).

The results underscore the importance of the diet composition for growth studies. In fact, under the standard food conditions used, the Lk6 mutant growth phenotype would have escaped detection, demonstrating that different food compositions are likely to result in qualitatively different outcomes of otherwise identical experiments. Researchers in the growth field, ourselves included, have not paid sufficient attention to what their flies actually eat. Therefore, it is proposed that Drosophila geneticists should always describe the composition of the fly food when reporting on growth-related experiments. Ideally, a global standard fly medium should be defined (Reiling, 2005).

The relationship between nutrition and growth has also been appreciated by others. It has been estimated that 14%-20% of all cancer deaths in the US are attributable to overweight and obesity. Therefore, understanding the role of diet in cancer development will represent a crucial task in the future (Reiling, 2005).

LK6 is regulated by ERK and phosphorylates the eukaryotic initiation factor eIF4E in vivo

In Drosophila cells, phosphorylation of eIF4E (eukaryotic initiation factor 4E) is required for growth and development. In Drosophila, LK6 is the closest homologue of mammalian Mnk1 and Mnk2 [MAPK (mitogen-activated protein kinase) signal-integrating kinases 1 and 2 respectively] that phosphorylate mammalian eIF4E. Mnk1 is activated by both mitogen- and stress-activated signalling pathways [ERK (extracellular-signal-regulated kinase) and p38 MAPK], whereas Mnk2 contains a MAPK-binding motif that is selective for ERKs. LK6 possesses a binding motif similar to that in Mnk2. The present study shows that LK6 can phosphorylate eIF4E at the physiological site. LK6 activity is increased by the ERK signalling pathway and not by the stress-activated p38 MAPK signalling pathway. Consistent with this, LK6 binds ERK in mammalian cells, and this requires an intact binding motif. LK6 can bind to eIF4G in mammalian cells, and expression of LK6 increases the phosphorylation of the endogenous eIF4E. In Drosophila S2 Schneider cells, LK6 binds the ERK homologue Rolled, but not the p38 MAPK homologue. LK6 phosphorylates Drosophila eIF4E in vitro. The phosphorylation of endogenous eIF4E in Drosophila cells is increased by activation of the ERK pathway but not by arsenite, an activator of p38 MAPK. RNA interference directed against LK6 significantly decreases eIF4E phosphorylation in Drosophila cells. These results show that LK6 binds to ERK and is activated by ERK signalling and it is responsible for phosphorylating eIF4E in Drosophila (Parra-Palau, 2005; full text of article).

Cup is an eIF4E binding protein that associates with Bruno and recruits Barentsz in the regulation of oskar mRNA translation in oogenesis

Cup has been identified as a component of an eight-protein complex that contains oskar mRNA (Wilhelm, 2000). cup is also required for oskar mRNA localization and is necessary to recruit the plus end-directed microtubule transport factor Barentsz to the complex. eIF4E is localized within the oocyte in a cup-dependent manner and binds directly to Cup in vitro. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. It is proposed that Cup coordinates localization with translation (Wilhelm, 2003).

Because btz mutants display a late stage oskar mRNA localization defect similar to that of cup mutants (van Eeden, 2001), the effect of cup mutants on the distribution of Btz was examined. Normally, Btz protein is present on the nuclear envelope in nurse cells and colocalizes with oskar mRNA in the oocyte. However, in cup1/cup4506 egg chambers, the accumulation of Btz protein within in the oocyte is greatly reduced from stage 1 onward, whereas the Btz present on the nuclear envelope in the nurse cells is unaffected. The failure in the transport of Btz to the oocyte is not due to a general defect in assembly of the oskar RNP since cup1/cup4506 egg chambers localize Yps and oskar mRNA normally during early oogenesis. Thus, Cup is specifically required to localize Btz to the oocyte. This result, together with the findings that Cup and Btz colocalize as well as share similar oskar mRNA localization defects, argues that cup mutants fail to localize oskar mRNA because Cup is required to recruit Btz to the complex (Wilhelm, 2003).

Since all mutations isolated to date that disrupt oskar mRNA localization also block oskar translation, the role of cup in oskar translation was examined. Surprisingly, Oskar protein accumulated prematurely in the oocyte during stages 6 and 7 in cup1/cup4506 egg chambers, indicating that cup is required to translationally repress oskar mRNA during these stages. It is also worth noting that in cup mutants accumulation of Oskar protein was observed at only those sites where oskar mRNA is most enriched. This may be due to the fact that the cup alleles used in this study are hypomorphic alleles. The effects of cup are specific for oskar mRNA since the localized translation of gurken mRNA at the dorsal anterior region of the oocyte during stage 9 is unaffected in a cup1/cup4506 mutant background. Thus, cup is not a general translational regulator of localized messages (Wilhelm, 2003).

To better understand the role of Cup in maintaining the translational repression of oskar mRNA, attempts were made identify components of the translation machinery that were present in the complex by testing likely candidates. Immunoprecipitation of GFP-Exu and Yps show that eIF4E, the 5' cap binding component of the translation initiation complex, is specifically associated with these components of the oskar RNP complex. eIF4E and other components of the translation initiation machinery are generally thought of as being homogenously distributed due to their critical role in translation throughout the cell. Surprisingly, eIF4E is localized in a dynamic pattern within the oocyte. eIF4E is localized to the posterior of the oocyte early in oogenesis during stages 1-6. At stages 7 and 8, eIF4E redistributed to the anterior of the oocyte, and during stages 9 and 10, eIF4E accumulated at the posterior of the oocyte. This pattern of localization was also observed with a GFP-eIF4E protein trap line. Thus, eIF4E localizes in a temporal-spatial pattern identical to that of Cup, suggesting that it is a component of the complex in vivo (Wilhelm, 2003).

Since Cup is required for the correct localization of Btz to the oocyte, whether Cup is required for eIF4E localization was investigated. Immunostaining of cup1/cup4506 mutant egg chambers reveals that Cup is required for localization of eIF4E to the posterior of the oocyte from stage 1 onward. Disruption of cup function does not significantly affect the level of unlocalized eIF4E, indicating that the defect is primarily in the recruitment of eIF4E to the complex (Wilhelm, 2003).

Because Cup shares limited homology with 4E-T, a known eIF4E binding protein and a translational repressor in mammals, whether Cup binds to eIF4E was tested using a two-hybrid interaction assay. This assay showed a direct interaction between Cup and eIF4E. Cup interacted equally with both isoforms of eIF4E. Deletion analysis of Cup using the two-hybrid assay identified an eIF4E interaction domain that contains a canonical eIF4E binding motif. This motif is found in eIF4G as well as translational repressors (e.g., 4E-T) that block translation by preventing the eIF4E-eIF4G interaction. Thus, Cup is an eIF4E binding protein that acts directly to repress oskar translation (Wilhelm, 2003).

Thus, the assignment of Cup as a novel component of the oskar RNP complex is based on a number of findings: (1) Cup copurifies with both Exu and Yps, which have both been shown to be in a biochemical complex with oskar mRNA; (2) Cup protein exhibits the same dynamic localization pattern as that seen for oskar mRNA as well as other components of the complex; (3) Cup colocalizes with Yps and Btz particles, indicating that this these proteins form a complex in vivo; (4) the relevance of the biochemical association is supported by genetic studies of cup function, demonstrating a role for cup in translational repression of oskar mRNA as well as recruitment of Btz and eIF4E to the RNP complex (Wilhelm, 2003).

Because Cup is a translational repressor that is also required to assemble the oskar mRNA localization machinery, it is proposed that the coupling between localization and translation occurs by regulating these two functions of Cup. In this model, Cup is required early in the assembly of the transport complex in order to recruit components, such as Btz, that will later be used to dock to kinesin. This is consistent with the results that cup is required to localize Btz to the posterior pole and that cup mutants exhibit oskar mRNA localization defects comparable to those observed in btz mutants. The fact that mammalian Btz and 4E-T are nucleocytoplasmic shuttling proteins suggests that the defect in particle assembly in cup mutants may occur in the nucleus rather than in the cytoplasm. However, further studies will be necessary to determine the site of assembly (Wilhelm, 2003).

Because Btz is normally part of the transport complex throughout oogenesis even though it is only required for the kinesin-mediated transport step during stages 9 and 10, it is further proposed that the complex undergoes rearrangement in order to activate Btz and switch from minus end-directed transport to kinesin-mediated transport. Since the direct binding of Cup to Btz or Btz to kinesin has not yet been established it is unclear how many components of the complex may be involved in this reorganization (Wilhelm, 2003).

Once the complex reaches the posterior pole, it is argued that the localization machinery is disassembled and the interaction between Cup and eIF4E is broken to allow translational activation. Because Cup is stably maintained at the posterior pole after stage 9, whereas Btz is not, it is proposed that the trigger that disrupts the binding of Cup to eIF4E also leads to partial disassembly of the localization machinery via Cup. The molecular trigger for such rearrangements is unknown, however, the ability of 4E-T to bind eIF4E is regulated by phosophorylation (Pyronnet, 2001). Studies directed at identifying regulators of the Cup-eIF4E interaction might lead to greater mechanistic insights into the coupling mechanism (Wilhelm, 2003).

One of the attractive features of this model is that it suggests how coupling might be accomplished in other systems. Recent work in neurons on the translational regulator CPEB suggests that it can promote the transport of mRNA into dendrites. Since CPEB represses translation by recruiting the eIF4E binding protein, maskin, to transcripts, it is possible that the observed transport effect is due to a requirement for maskin to assemble the localization machinery. Thus, Cup may be representative of a general class of eIF4E binding proteins whose role is to couple mRNA localization to translational activation (Wilhelm, 2003).

Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis

Translational control is a critical process in the spatio-temporal restriction of protein production. In Drosophila oogenesis, translational repression of oskar1 (osk) RNA during its localization to the posterior pole of the oocyte is essential for embryonic patterning and germ cell formation. This repression is mediated by the osk 3' UTR binding protein Bruno (Bru), but the underlying mechanism has remained elusive. An ovarian protein, Cup, is required to repress precocious osk translation. Cup binds the 5'-cap binding translation initiation factor eIF4E through a sequence conserved among eIF4E binding proteins. A mutant Cup protein lacking this sequence fails to repress osk translation in vivo. Furthermore, Cup interacts with Bru in a yeast two-hybrid assay, and the Cup-eIF4E complex associates with Bru in an RNA-independent manner. These results suggest that translational repression of osk RNA is achieved through a 5'/3' interaction mediated by an eIF4E-Cup-Bru complex (Nakamura, 2004).

In a search for new components of the oskar RNP complex, this study identified the 147-kD protein of this complex as the product of the female sterile gene cup. Surprisingly, cup is required both for translational repression and localization of oskar mRNA. Cup was found to bind to eukaryotic initiation factor 4E (eIF4E) and is necessary to recruit the localization factor Barentsz to the complex. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. Because of its interactions with both the localization and translational control complexes, it is proposed that Cup is a likely regulatory target for the coupling machinery (Nakamura, 2004).

During localization, osk RNA forms cytoplasmic granules in both nurse cells and the oocyte. The granules contain several proteins, including the DEAD-box protein Maternal expression at 31B (Me31B), the Y-box protein Ypsilon schachtel (Yps), and Exuperantia (Exu). Genetic evidence has shown that Exu is involved in the proper localization of bcd and osk RNAs in oogenesis, although the molecular function of Exu remains unknown. Both Yps and Me31B are involved, directly or indirectly, in the translational silencing of osk RNA in oogenesis. Yps antagonizes Orb, a positive regulator of osk RNA localization and translation. In egg chambers lacking me31B, osk RNA is prematurely translated in early oogenesis (Nakamura, 2001). These data indicate that the granules are maternal ribonucleoprotein (RNP) complexes containing proteins required for both RNA localization and translational control. The complex is highly enriched in eIF4E and a germline protein, Cup. Cup is required to repress osk translation. Evidence is provided that Cup-mediated translational repression is achieved by preventing the assembly of the eIF4F complex at the 5' end of osk RNA, and that Cup acts together with Bru to repress osk translation (Nakamura, 2004).

To identify new proteins in the Me31B complex, ovarian extracts from wild-type females were immunoprecipitated on a preparative scale using an affinity-purified anti-Me31B antibody (α-Me31B). α-Me31B specifically coprecipitatesmany proteins from the extracts. Mass spectrometric analyses of these proteins revealed that both Exu and Yps, the known components in the Me31B complex (Nakamura, 2001), are present in the immunoprecipitates. The analyses also revealed that the 35 kDa protein was eIF4E and the 150 kDa protein is Cup, a germline-specific protein required for oogenesis. Cup is expressed from early oogenesis and present until the blastoderm stage of embryogenesis. Numerous cup alleles have been isolated as female sterile mutants, which show a wide range of phenotypes. However, the biochemical function of Cup has remained elusive (Nakamura, 2004).

To examine the association among Me31B, eIF4E and Cup in vivo, ovaries expressing a GFP-Me31B fusion protein were stained for eIF4E and Cup. The GFP-Me31B form cytoplasmic particles in the germline, and the distribution patterns of the fusion protein are indistinguishable from those of endogenous Me31B (Nakamura, 2001). α-eIF4E stains cytoplasmic particles that are positive for GFP-Me31B. This colocalization is observed throughout oogenesis. Cup colocalized with GFP-Me31B is also found throughout oogenesis. Thus, eIF4E, Cup, and Me31B all form a complex during oogenesis (Nakamura, 2004).

To better understand the interactions between Me31B, eIF4E, and Cup, ovarian extracts were immunoprecipitated by α-Me31B and α-eIF4E, and the precipitates were analyzed by Western blotting. α-Me31B coprecipitates eIF4E and Cup, and α-eIF4E coprecipitates Me31B and Cup, indicating that they all form a complex. However, in the presence of RNase during immunoprecipitation, α-Me31B fails to coprecipitate eIF4E or Cup. Thus, the Me31B-eIF4E and the Me31B-Cup interactions are indirect and probably mediated through RNA in the complex. In contrast, α-eIF4E coprecipitates Cup even in the presence of RNases, suggesting a direct interaction between eIF4E and Cup in vivo (Nakamura, 2004).

The interaction of Cup and eIF4E in vitro was studied using a GST pull-down assay. GST-eIF4E pulls down Cup synthesized in vitro. The association is unaffected by RNase. These results indicate that Cup associates with eIF4E in vitro and that the interaction is RNA independent (Nakamura, 2004).

The results show that Cup is an eIF4E binding protein that is involved in translational repression of osk RNA during oogenesis. The conserved YxxxxLφ motif in Cup is important for eIF4E binding and Cup and eIF4G are likely to bind the same surface of eIF4E. These results suggest that Cup competes with eIF4G for eIF4E binding, and hence inhibits translation initiation. CupΔ212 protein, which lacks the conserved eIF4E binding sequence, is unable to bind eIF4E in vivo, and fails to repress osk translation. These results strongly suggest that the Cup-eIF4E interaction is essential for the Cup-mediated repression of osk translation, although it is possible that other of Cup's functions are also affected in the cupΔ212 mutant. Furthermore, Cup was found to interact with Bru in a yeast two-hybrid assay and that the Cup-eIF4E complex associates with Bru in an RNA-independent manner. Based on these results, it is speculated that the Bru-mediated repression of osk translation is operated, at least in part, through the interaction with Cup, which binds eIF4E and prevents the eIF4E-eIF4G interaction at the 5' end of osk RNA (Nakamura, 2004).

Cup is an eIF4E-binding protein that functions in Smaug-mediated translational repression

Translational regulation plays an essential role in development and often involves factors that interact with sequences in the 3' untranslated region (UTR) of specific mRNAs. For example, Nanos protein at the posterior of the Drosophila embryo directs posterior development, and this localization requires selective translation of posteriorly localized nanos mRNA. Spatial regulation of nanos translation requires Smaug protein bound to the nanos 3' UTR; binding represses the translation of unlocalized nanos transcripts. While the function of 3' UTR-bound translational regulators is, in general, poorly understood, they presumably interact with the basic translation machinery. Smaug is shown to interact with the Cup protein and Cup is an eIF4E-binding protein that blocks the binding of eIF4G to eIF4E. Cup mediates an indirect interaction between Smaug and eIF4E, and Smaug function in vivo requires Cup. Thus, Smaug represses translation via a Cup-dependent block in eIF4G recruitment (Nelson, 2004),

To understand the mechanisms that underlie Smg's ability to repress translation, attempts were made to identify Smg-binding proteins. Initial work focused on proteins that would interact with amino acids 583-763. This region contains the Smg SAM domain, which is the protein's RNA-binding domain. An affinity resin carrying covalently coupled GST-Smg583-763 was mixed with early embryo extracts. After extensive washing, bound proteins were eluted and detected via silver staining following SDS-PAGE. Several proteins were eluted from both the GST-Smg583-763 resin and a resin carrying covalently coupled GST-Smg179-307. However, an `80 kDa protein and an `140 kDa protein were specifically eluted from the Smg583-763 resin. Both proteins were subjected to MALDI-TOF mass spectrometry, and while the smaller protein was not identified the larger was identified as Cup, which plays an essential but ill-defined role during oogenesis and early embryogenesis. To confirm that Cup interacts with Smg583-763, Cup was generated via in vitro translation in rabbit reticulocyte lysate. This protein interacted with GST-Smg583-763, as assayed by capture of Cup on glutathione agarose in the presence of GST-Smg583-763 (Nelson, 2004),

Biochemical and genetic evidence is presented that is consistent with Cup functioning as an eIF4E-binding protein that mediates an interaction between Smg and eIF4E. Cup blocks the eIF4E/eIF4G interaction, suggesting that Smg-dependent translational repression of SRE-containing mRNAs results from a Cup-mediated block in the recruitment of eIF4G. Cup's role in Smg function is therefore similar to that played by Maskin in translational repression mediated by CPEB. Given that Maskin and Cup are not homologous, this suggests that other undiscovered adaptor eIF4E-binding protein/3' UTR-binding protein pairs will employ this mechanism to regulate translation (Nelson, 2004),

Cup interacts with eIF4E using both an eIF4E-binding motif and a second site that interacts with eIF4E through a distinct mechanism. Despite this difference, the second site is still able to inhibit the eIF4E/eIF4G interaction in vitro. Further work will be required to assess the significance of this site to Cup function in vivo (Nelson, 2004),

This model for Cup suggests that Smg represses translation at the level of initiation. However, the association of repressed nos mRNA with polysomes indicates that translational repression is achieved at a step after initiation. This apparent contradiction may reflect the fact that repression of nos translation is mediated by at least two trans-acting factors: Smg and a yet to be identified factor that functions through sequences in the nos 3' UTR that are distinct from the SREs. Thus, while Smg regulates translation at the level of initiation, additional factors may function at other levels. Similarly, Smg itself may utilize multiple mechanisms to repress nos expression, only one of which is Cup dependent (Nelson, 2004),

Regulation of translation during development often involves both translational repression and translational activation. The combination of these controls can spatially or temporally restrict the expression of an mRNA, thereby directing the proper development of a cell type or tissue. For example, nos translation is spatially regulated allowing for the proper development of the posterior of the Drosophila embryo. Smg plays an essential role in this process by repressing the translation of unlocalized nos mRNA, while nos mRNA localized to the posterior escapes this repression allowing for the accumulation of Nos protein specifically at the posterior. Given that Smg protein is distributed throughout the embryo, this suggests that Smg function must be over-ridden at the posterior. Cup is also distributed throughout the embryo, suggesting that spatial regulation of nos translation may involve disrupting Cup and/or Smg function specifically at the posterior. Osk protein, which is localized to the posterior, is required for nos translation and Osk interacts with Smg. Thus translational activation could involve Osk binding to Smg thereby blocking Smg function. Interestingly, Cup and Osk interact with the same region of the Smg protein. This might imply that Osk's interaction with Smg could disrupt the Cup/Smg complex and in so doing play a role in activating nos translation at the posterior (Nelson, 2004),

In Xenopus, temporal regulation of translation involves Maskin-mediated repression of target mRNAs in immature oocytes. Upon oocyte maturation, this repression is disrupted resulting in the activation of translation. This activation of translation involves a CPEB-mediated increase in the length of the transcript's poly(A) tail and subsequent recruitment of poly(A)-binding protein (PABP) to the message. PABP brings eIF4G to the mRNA, which in turn disrupts the Maskin/eIF4E complex resulting in translational activation. Measurement of the length of the nos poly(A) tail suggests that regulation of nos translation does not involve changes in poly(A) tail length. Thus, activation of nos translation does not likely involve disruption of the Cup/eIF4E complex through poly(A)-dependent eIF4G recruitment. Taken together, these results also suggest that the use of adaptor proteins such as Cup in translational regulation mediated by sequence-specific RNA-binding proteins is not restricted to mRNAs whose translation is regulated through their poly(A) tail (Nelson, 2004),

The data demonstrate that the same region of Smg that has previously been shown to function in sequence-specific RNA binding also interacts with Cup. The model therefore suggests that this region of the protein would be sufficient to repress translation. However, a transgene that expresses the Smg RNA-binding domain plus a short carboxy-terminal extension fails to rescue the smg mutant phenotype. These results would suggest that Smg has other essential functions in the early embryo in addition to Cup-dependent translational repression. Smg has been suggested to induce the degradation of target mRNAs in a process that may be distinct from its ability to repress translation. Perhaps this ability to induce mRNA degradation is essential and requires regions of Smg outside of amino acids 583-763 (Nelson, 2004),

Phenotypic analysis of several cup mutant alleles highlights Cup's involvement in a number of different biological processes during oogenesis and early embryogenesis, including oocyte growth, maintenance of chromosome morphology, and the establishment of egg chamber polarity. However, the molecular mechanisms that underlie Cup function have not been characterized. The demonstration that Cup is an eIF4E-binding protein suggests that at least some of the defects associated with mutations in the cup gene result from misregulation of translation. Consistent with this possibility is the fact that Cup has been previously shown to interact with Nos protein, which is itself a translational repressor. Genetic experiments suggest that Cup negatively regulates Nos activity during oogenesis, but the molecular mechanisms are not understood. This contrasts with Cup's positive effect on Smg-mediated translational repression. Thus Cup might utilize different molecular mechanisms to influence different translational repressors. The pleiotropic nature of the cup mutant phenotype suggests that Cup may serve as an adaptor protein that is utilized by multiple translational repressors to interact with eIF4E (Nelson, 2004),

Cup is homologous to 4E-T, a human nucleocytoplasmic shuttling protein that employs an eIF4E-binding motif to transport eIF4E into the nucleus (Dostie, 2000). The similarity between these proteins may suggest that Cup also functions to transport eIF4E into the nucleus. Thus some of the phenotypes associated with cup mutants may be related to a defect in eIF4E shuttling during oogenesis. The similarity between Cup and 4E-T also suggests that 4E-T might function in translational repression as an adaptor protein that mediates interactions between eIF4E- and 3' UTR-binding proteins. Specifically, 4E-T could function in translational repression mediated by the human Smg homolog. Similarly, additional RNA-binding proteins that interact with other eIF4E-binding proteins could function to regulate translation spatially or temporally. These protein pairs could control the translation of different mRNAs in various cell types throughout development (Nelson, 2004),

Postsynaptic translation affects the efficacy and morphology of neuromuscular junctions

Long-term synaptic plasticity may be associated with structural rearrangements within the neuronal circuitry. Although the molecular mechanisms governing such activity-controlled morphological alterations are mostly elusive, polysomal accumulations at the base of developing dendritic spines and the activity-induced synthesis of synaptic components suggest that localized translation is involved during synaptic plasticity. This study shows that large aggregates of translational components as well as messenger RNA of the postsynaptic glutamate receptor subunit DGluR-IIA are localized within subsynaptic compartments of larval neuromuscular junctions of Drosophila. Genetic models of junctional plasticity and genetic manipulations using the translation initiation factors eIF4E and poly(A)-binding protein showed an increased occurrence of subsynaptic translation aggregates. This was associated with a significant increase in the postsynaptic DGluR-IIA protein levels and a reduction in the junctional expression of the cell-adhesion molecule Fasciclin II. In addition, the efficacy of junctional neurotransmission and the size of larval neuromuscular junctions were significantly increased. These results therefore provide evidence for a postsynaptic translational control of long-term junctional plasticity (Sigrist, 2000).

Translational control is primarily exerted by regulation of the initiation step of translation, which appears to be controlled by the rate-limiting initiation factor eIF4E. In addition, the interaction of the 5' cap bound eIF4E with the 3' end of mRNAs through a complex of other initiation factors and the poly(A)-binding protein (PABP) has been shown to synergistically facilitate translation initiation. To assess the potential role of regulated translation during the development of the larval neuromuscular junctions (NMJs) in Drosophila, the subcellular expression pattern of eIF4E and PABP were analyzed in filet preparations of third instar larvae. Both antigens showed a weak and ubiquitous expression in the cytoplasm of all larval cells, and they colocalized in strongly immunopositive aggregates up to 2microm in length close to NMJs. The specific localization of eIF4E/PABP aggregates close to and partially overlapping with junctional profiles revealed that eIF4E/PABP aggregates are positioned subsynaptically within or adjacent to the subsynaptic reticulum (SSR). No evidence was found for presynaptic or axonal localization of such aggregates. Therefore, the almost exclusive subsynaptic distribution of the eIF4E/PABP aggregates within larval muscles indicates that there may be a functional relationship between NMJs and the appearance of nearby eIF4E/PABP aggregates (Sigrist, 2000).

Ultrastructural examinations of larval NMJs revealed polysomal accumulations within and close to the SSR. According to their variable size, subsynaptic location and frequency of detection, the larger of these polysomal clusters are likely to represent the eIF4E/PABP aggregates detected by light microscopy. In addition, smaller polysomal aggregates were widely distributed in discrete membranous compartments throughout the SSR, whereas presynaptic and axonal profiles were free of polysomes. It is therefore concluded that mRNAs are translated within subsynaptic compartments of larval NMJs and that local centres of concentrated, subsynaptic translation are identified by large junctional eIF4E/PABP aggregates (Sigrist, 2000).

To assess whether junctional translation is subject to regulation, the number was quantified of synaptic specializations (boutons) per NMJ that were labelled by one or more translation aggregates. Animals that overexpressed PABP in larval muscles and larvae that were mutant in pabp showed a significantly increased occurrence of subsynaptic eIF4E/PABP aggregates and an unaltered level of muscular PABP staining. In addition, the total PABP levels in crude larval protein extracts were unaltered in all analysed genotypes, even when PABP mRNA levels were significantly increased or reduced under genetic gain-of-function or loss-of-function conditions, respectively. Such a homeostasis of total PABP levels is a well described phenomenon for PABP, and in crude protein extracts it might have masked the significant local increase in the number of PABP aggregates observable within subsynaptic compartments of NMJs. Although the exact reason for this increase in the occurrence of eIF4E/PABP aggregates is unknown, a local perturbation of PABP levels owing to a previously described overshooting compensation of the PABP-homeostasis mechanism might facilitate formation of subsynaptic translation aggregates (Sigrist, 2000).

A similar increase in the frequency of postsynaptic translation aggregates was also observed in two mutants representing well established genetic models of long-term synaptic plasticity in Drosophila, the hyperactive K+-channel mutant eag, Sh and the cAMP-phosphodiesterase mutant dunce. Thus, increased neuronal activity levels (in eag, Sh) as well as elevated cellular cAMP levels (in dunce) are capable of inducing subsynaptic translation aggregate formation. These findings are consistent with the hypothesis that synaptic activity can control synaptic translation (Sigrist, 2000).

To identify potential substrates and targets of subsynaptic translation at larval NMJs, quantitative immunostainings were performed of several junctionally expressed proteins, including the synaptic vesicle protein synaptotagmin, the junctional anti-horseradish peroxidase (HRP) epitope, the cell-adhesion molecule Fasciclin II (FasII), the postsynaptic glutamate receptor subunit DGluR-IIA and the conventional myosin as a nonsynaptic protein. No obvious differences were detected in the expression levels of myosin, synaptotagmin and the junctional anti-HRP immunoreactivity in all analysed genotypes; however, animals that showed elevated numbers of subsynaptic translation aggregates consistently displayed increased junctional levels of DGluR-IIA and an altered junctional distribution of FasII, which was associated with a significant reduction of synaptic FasII levels as compared with control animals. A similar FasII phenotype has been described in the plasticity models eag, Sh and dunce, and it has been shown that presynaptic FasII downregulation is essential for increased junctional outgrowth. Intriguingly, in Aplysia the FasII homologue apCAM is also presynaptically downregulated after treatments that increase synaptic efficacy and growth of new synaptic connections. This synaptic apCAM regulation is thought to be achieved by a protein-synthesis-dependent activation of an endocytic apCAM internalization. Given that FasII has been detected in membranes of a subset of presynaptic vesicles, it seems possible that subsynaptic protein synthesis affects junctional FasII levels through similar mechanisms to those in Aplysia (Sigrist, 2000).

The postsynaptic DGluR-IIA immunoreactivities were significantly stronger in translationally sensitized animals. This strong increase of synaptic DGluR-IIA expression was not due to transcriptional upregulation of dglur-IIA; the total amounts of DGluR-IIA mRNAs were unaltered or even reduced in the analysed genotypes as compared with controls. In situ hybridization experiments revealed that DGluR-IIA mRNA surrounds individual type-I boutons, with prominent staining of terminal and branch-point boutons and weak or absent staining within the SSR of interbouton connectives. Thus, the subsynaptically localized DGluR-IIA mRNA represents a direct substrate for the junctional translation machinery. These results can not exclude an extrajunctional contribution to the observed synaptic DGluR-IIA increase, but they suggest that this phenotype is due to an increased subsynaptic synthesis of DGluR-IIA in genotypes with a higher occurrence of junctional eIF4E/PABP aggregates (Sigrist, 2000).

To analyse the functional consequences of genetically modified subsynaptic translation, the strength of neurotransmission at NMJs was assessed on muscle 6 of third instar larvae. The average amplitudes of miniature excitatory junctional currents (mEJCs) and thus the quantal sizes were indistinguishable in all analysed genotypes. This finding indicates either that the additional receptor subunits that are synaptically localized may be functionally silent (for example, through physiological silencing or intracellular localization or that the amount of glutamate released from an individual quantum is not sufficient to saturate the postsynaptic receptors. In contrast, postsynaptic responses evoked by stimulation of motor nerve axons were substantially larger in all mutants exhibiting increased levels of subsynaptic translation. Thus, the derived quantal content was significantly increased above control values, suggesting that the observed larger amplitudes of evoked junctional responses arise from an increased number of released presynaptic vesicles per action potential (Sigrist, 2000).

To investigate whether the increase in junctional efficacy was due to a change in the number of synaptic specializations, the number of junctional boutons per NMJ was quantified. Genotypes that displayed an increased occurrence of subsynaptic translation aggregates had significantly larger NMJs and reduced junctional FasII levels. In addition, the junctional sizes of the analysed animals correlated in a highly significant manner with their estimated quantal contents, suggesting that junctional efficacy and the morphological elaboration of NMJs are tightly coupled. On the basis of light microscopic examinations of DGluR-IIA labelled NMJs, the density of synapses within NMJs of all mutant animals appeared similar to that of controls or even higher, indicating that the total number of synapses increased proportionally with the junctional size. This finding indicates that the increased quantal content in animals with facilitated subsynaptic translation may be because of an increase in the number of vesicle release sites per given stimulus (Sigrist, 2000).

In summary, this study has shown that translational machinery and mRNAs are associated with the subsynaptic reticulum of NMJs and that genetic manipulations that affect the occurrence of subsynaptic translation aggregates are accompanied by changes in the levels of synaptic proteins, such as DGluR-IIA and FasII. These same manipulations also affected the function and morphology of NMJs, suggesting that subsynaptic translation can instruct junctional growth and synaptic reorganization and thereby long-term functional changes. These results further suggest that subsynaptic translation can be regulated by altered levels of neuronal activity, indicating that the regulation of postsynaptic translation participates in activity-dependent junctional plasticity. Thus, the inducible recruitment of postsynaptic protein synthesis appears to render individual synapses competent to instruct long-term changes in their functions and morphological organization. Given that localized protein synthesis has been shown to act in a synapse specific stabilization of long-term facilitation in central neurons of Aplysia, it emerges that synaptic translation might represent a common principle of long-term alterations of neuronal function and connectivity (Sigrist, 2000).

The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila

The initiation factor 4E for eukaryotic translation (eIF4E) binds the messenger RNA 5'-cap structure and is important in the regulation of protein synthesis. Mammalian eIF4E activity is inhibited when the initiation factor binds to the translational repressors, the 4E-binding proteins (4E-BPS). The Drosophila 4E-BP (d4E-BP) is a downstream target of the phosphatidylinositol-3-OH kinase [PI(3)K] signal-transduction cascade, which affects the interaction of d4E-BP with eIF4E. Ectopic expression of a highly active d4E-BP mutant in wing-imaginal discs causes a reduction of wing size, brought about by a decrease in cell size and number. A marked reduction in cell size is also observed in post-mitotic cells. Expression of d4E-BP in the eye and wing together with PI(3)K or dAkt1, the serine/threonine kinase downstream of PI(3)K, results in suppression of the growth phenotype elicited by these kinases. These results support a role for d4E-BP as an effector of cell growth (Miron, 2001).

Drosophila 4E-BP (d4E-BP) was isolated by interaction cloning from a complementary DNA expression library using 32P-labelled deIF4EI. d4E-BP is identical to Drosophila Thor (Bernal, 2000) and homologous to 4E-BPs from other species. Phosphorylation sites in mammalian 4E-BP1 are conserved in d4E-BP, but the predicted eIF4E-binding motif in d4E-BP (YERAFMK) diverges from the canonical consensus sequence (Miron, 2001).

To examine the binding of d4E-BP to deIF4E, residues within the consensus eIF4E-binding site were mutated. Recombinant proteins were expressed in Escherichia coli, and far Western blotting was performed using 32P-labelled deIF4EI. Mutation of Tyr 54 to Ala (Y54A) or Phe (Y54F), and Met 59 to Ala (M59A) abrogates the interaction of d4E-BP with deIF4E. Mutation of Lys 60 to Ala (K60A) decreases deIF4E binding by 87%, indicating that Lys 60 contributes to deIF4E binding. However, when either Met 59 or Lys 60 are mutated to the consensus Leu, the interaction of d4E-BP with deIF4EI is 2.5-fold higher than with the wild type, and when both Met 59 and Lys 60 are so changed, deIF4E binding increases by 3.4-fold. These results indicate that d4E-BP interacts with deIF4E, albeit more weakly than previously characterized 4E-BPs, owing to its divergent eIF4E-binding motif (Miron, 2001).

4E-BP1 is hyperphosphorylated in response to insulin in many cell types. To test whether this response operates in Drosophila, Schneider-2 (S2) cells were deprived of serum and treated with insulin. Increasing levels of a slower migrating form of d4E-BP (d4E-BP) were observed consequent to insulin treatment. To determine whether the ß-form corresponds to phosphorylated d4E-BP, extracts from insulin-stimulated S2 cells were treated with either calf intestine alkaline phosphatase (CIP) or protein phosphatase 2A (PP2A). Untreated extracts (or extracts kept on ice) contain both the faster migrating alpha- and the slower migrating ß-forms. In contrast, phosphatase-treated extracts contained only the alpha-form (Miron, 2001).

LY294002 and rapamycin inhibit PI(3)K and target of rapamycin (TOR) activity, respectively, and block the insulin-induced hyperphosphorylation of 4E-BP1. Similarly, exposure of serum-deprived S2 cells to either drug before treatment with insulin, results in a dose-dependent decrease in d4E-BP phosphorylation. To determine whether phosphorylation of d4E-BP prevents its binding to deIF4E, m7GDP-agarose precipitation was performed. The alpha form is present primarily in the bound fraction, whereas the ß-form is found exclusively in the unbound fraction. These results show that d4E-BP is a downstream target of the PI(3)K pathway, and that the binding of d4E-BP to deIF4E is modulated by its phosphorylation state (Miron, 2001).

Assembly of eIF4F is essential for translational control, and overexpression of eIF4E in mammalian cells results in malignant transformation. To investigate whether eIF4F is also linked to growth control, eIF4F assembly was perturbed in Drosophila. UAS transgenic fly lines were generated that express wild-type d4E-BP or the mutant d4E-BP that binds deIF4E most strongly, d4E-BP(LL). Expression of d4E-BP was targeted to the wing-imaginal disc using MS1096-GAL4. The size and cell number of wings from males were measured. Expression of wild-type d4E-BP has no effect on wing size or pattern. However, expression of d4E-BP(LL) from one line [d4E-BP(LL)w] causes a marked reduction of wing size without affecting cell number. Another line, [d4E-BP(LL)s], which expresses d4E-BP(LL) more strongly, causes a larger reduction, which is partly due to a decrease in cell number. Since direct inhibition of cellular proliferation increases, rather than decreases, cell size, it is possible that d4E-BP(LL) also affects cell size directly, and cell proliferation as a consequence. This is supported by analysis of the effects of d4E-BP(LL) expression in larval-wing discs. Although discs from the d4E-BP(wt) and d4E-BP(LL)w lines are indistinguishable from control discs, d4E-BP(LL)s discs are 52% smaller. d4E-BP(LL)s males also required 1-2 days longer to eclose, which would account for the smaller decrease in adult wings (Miron, 2001).

Acridine-orange staining shows that d4E-BP(LL)s discs contain many apoptotic cells. Co-expression of p35, the baculovirus inhibitor of apoptosis, with d4E-BP(LL)s partially rescues the size of adult wings. To distinguish between apoptosis and decreased proliferation, cell clones expressing d4E-BP(LL), with or without p35, and co-expressing green fluorescent protein (GFP), were induced 72 h after egg deposition in developing wing discs using the flip-out technique. Clones expressing d4E-BP(LL)w contain fewer cells than wild-type clones, but co-expression of p35 with d4E-BP(LL)w does not affect the number of cells per clone, indicating that decreased proliferation, but not increased apoptosis, is the cause of reduction. Few clones expressing d4E-BP(LL)s are recovered, and they usually contain 1-2 cells. Co-expression of p35 greatly increases the number of clones recovered, but only marginally increases the number of cells per clone (1-4 cells) (Miron, 2001).

Direct interference with cell proliferation using string mutants results in increased cell size. To help distinguish effects on size from effects on proliferation, cell size was evaluated by flow cytometry (FACS). Mean forward-light scatter values for GFP-positive cells that expressed d4E-BP(LL) were reduced by 6%-8%. Because cells that expressed d4E-BP(LL) are smaller and proliferate more slowly than their wild-type counterparts, it is conceivable that d4E-BP(LL) directly affects cell growth and consequently retards proliferation, which would lead to reduced viability and ultimately apoptosis. Similar results were observed in dTOR mutants, and interpreted as a primary defect in cellular growth coupled with a consequent decrease in cell proliferation. The possibility that growth and proliferation are affected independently by d4E-BP(LL) expression cannot be excluded (Miron, 2001).

To exclude proliferation effects, the growth and viability of d4E-BP(LL) cells were examined in a post-mitotic tissue. Polyploid fat-body cells undergo successive rounds of DNA synthesis without mitoses. Cells that express d4E-BP(LL)s, induced 48 h after egg deposition in the fat body, are 45%-70% smaller than neighboring wild-type cells, but their frequency is much higher than in mitotically active tissues, such as the wing-imaginal disc. Thus, viability of cells that express d4E-BP(LL) is maintained in the absence of mitogenic signals, indicating that proliferation of wild-type neighboring cells is necessary to cause the disappearance of cells expressing d4E-BP(LL). In support of this notion is the finding that when d4E-BP(LL)s clones are induced during development of eye-imaginal discs, only the clones that are generated posterior to the morphogenetic furrow survive; the clones generated anterior to the furrow (that is, in mitotically active cells), are eliminated (Miron, 2001).

To study the possible role of d4E-BP as an effector of cell growth through the PI(3)K signaling pathway, potential interactions between d4E-BP and relevant signaling genes of this pathway were examined. Expression of wild-type d4E-BP in eye-imaginal discs, using GMR-GAL4, does not engender any discernible phenotype, whereas expression of dAkt1 results in an enlarged eye. However, co-expression of wild-type d4E-BP and dAkt1 partially suppresses the enlarged-eye phenotype, and fully suppresses the roughness induced by expression of dAkt1. Since d4E-BP by itself has no effect on eye size but is able to suppress the dAkt1 phenotype, there is a genuine epistatic relationship between d4E-BP and dAkt1 (Miron, 2001).

Other components of the PI(3)K pathway were also examined for potential epistatic interactions with d4E-BP in the wing, using dpp-GAL4 and 4E-BP(LL)s. Ectopic expression of Dp110 and dAkt1 causes an enlargement of the region encompassed by the third and fourth longitudinal veins, the anterior crossvein and wing margin. In contrast, expression of a dominant-negative mutant form of PI(3)K (Dp110D954A) or d4E-BP(LL)s results in reduction of the size of this region. Co-expression of d4E-BP(LL)s with Dp110 or dAkt1 suppresses the growth enhancement engendered by expression of these kinases, whereas co-expression of d4E-BP(LL)s with Dp110D954A results in further size reduction. Flies that lacked a copy of the gene encoding the adaptor protein p60 [the Drosophila homolog of mammalian PI(3)K subunit p85] are also reduced in size when d4E-BP(LL)s is co-expressed. These results provide genetic evidence that d4E-BP is a downstream effector of the PI(3)K pathway (Miron, 2001).

Null mutants of d4E-BP are viable and although their immune response is compromised (Bernal, 2000), they do not exhibit increased growth. Furthermore, ectopic expression of Drosophila eIF4E in a wild-type or d4E-BP null background fails to produce a growth-related phenotype. Therefore, an increase in eIF4E activity alone is not sufficient to promote cell growth in Drosophila imaginal discs. This is consistent with data in primary mouse-embryo fibroblasts, in which eIF4E overexpression leads only to oncogenic transformation when co-expressed with c-myc or E1A. Attempts were made to rescue the d4E-BP(LL)-induced growth defects in imaginal discs by co-expressing deIF4E. Unexpectedly, growth is further reduced. Thus, endogenous eIF4E expression levels are optimal for cell growth and proliferation, and in the absence of activation of the PI(3)K pathway, a further increase in eIF4E expression is either without effect or deleterious (Miron, 2001).

Many studies have shown that PI(3)K and TOR-mediated signaling is important for normal cell growth and proliferation. However, one downstream target of this pathway, S6K, regulates cell size but not proliferation. Constitutive expression of dS6K in dTOR mutants only partially suppresses the dTOR phenotype, indicating that S6K-independent targets operate downstream of dTOR. Regulation of eIF4E activity, independent of S6K, contributes to the control of cell size. In Drosophila, the activity of eIF4E is modulated through 4E-BP. Phosphorylation of eIF4E is correlated with increased translation rates. Mutation of the phosphorylation site in Drosophila eIF4E causes a cell size reduction. In summary, the results presented here show that d4E-BP acts as an important downstream effector of PI(3)K in the regulation of cell proliferation and growth, independent of S6K, and further underline the importance of translation initiation in the latter process (Miron, 2001).


Search PubMed for articles about Drosophila Eif4E

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