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).

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).

In many circumstances, translation initiation is the rate-limiting step, and it is often the target of regulation. Translation initiation begins with the binding of the 43S preinitiation complex to the mRNA. In the cap-dependent mode of translation initiation, the m7GpppN (N, any nucleotide) cap structure (see mRNA capping at Genome Knowledge Base) at the 5' end of the mRNA attracts the eukaryotic initiation factor (eIF)4F complex to the mRNA (see Cap-dependent Translation Initiation at Genome Knowledge Base). This complex contains the cap binding protein eIF4E and the scaffold-like protein eIF4G. The cap-bound eIF4F directs the 43S complex to the 5' end of the mRNA through the interaction of eIF4G with eIF3, a multisubunit component of the 43S complex. Thus, the eIF4E-eIF4G interaction is crucial for cap-dependent translation initiation. The eIF4E binding proteins (4E-BPs) are well-characterized proteins that regulate translation initiation by blocking the eIF4E-eIF4G interaction in a reversible, growth signal-dependent manner (Gingras, 1999). 4E-BPs contain the conserved eIF4E binding sequence defined by YxxxxLphi (where x is any residue and phi is a hydrophobic residue), and compete with eIF4G to bind to the same region of eIF4E (Gingras, 1999; Marcotrigiano, 1999). 4E-BPs, however, do not discriminate among mRNAs but regulate global translation efficiency in response to external signals (Nakamura, 2004 and references therein).

Transcript-specific translational repression usually requires cis-acting sequences within the RNA, and one or more translational repressors that bind the sequences. These sequences are often found in the untranslated regions (UTRs) of the RNA. For instance, binding of the iron response element (IRE) binding protein to the IRE in the 5' UTR of ferritin mRNA prevents the interaction of the 43S complex with eIF4F by a steric hindrance mechanism. More commonly, translational repressors bind to the 3' UTR and somehow repress translation. While some repressors shorten the poly(A) tail, thereby lowering the efficiency of translational initiation, many 3' UTR binding repressors can act in a poly(A)-independent fashion. For poly(A)-independent repression, 3' UTR binding proteins must ultimately exert their function on the translation apparatus. However, only a few examples of this have been verified experimentally. Translational repression of caudal (cad) RNA in the Drosophila embryo is mediated by Bicoid (Bcd), which binds a specific sequence in the 3' UTR of cad RNA. Bcd also binds eIF4E, thus preventing the eIF4E-eIF4G interaction through a 4E-BP-like mechanism (Niessing, 2002). Similarly, in the Xenopus oocyte, Maskin represses cyclin-B1 translation by sequestering eIF4E. However, Maskin does not bind cyclin-B1 RNA directly. Instead, the target specificity of repression is provided by the interaction of Maskin with the cytoplasmic polyadenylation element (CPE) binding protein, CPEB, a factor that binds CPE in the 3' UTR of cyclin-B1 RNA (Nakamura, 2004 and references therein).

The translational control of localized RNAs is also striking in the oogenesis and embryogenesis of diverse organisms. In Drosophila, the localization of oskar (osk) RNA at the posterior pole of the oocyte leads to the assembly of a specialized cytoplasm, the pole plasm. Pole plasm contains factors required for embryonic posterior patterning and germ cell formation. The Drosophila oocyte develops in an egg chamber consisting of 15 nurse cells and an oocyte; all are interconnected through cytoplasmic bridges. From a very early stage of oogenesis, osk RNA is transcribed in the nurse cells and transported to the oocyte, where it accumulates. From late stage 8, osk RNA becomes localized to the posterior pole of the oocyte. Translation of osk RNA, however, is tightly repressed during translocation, and only when osk RNA is posteriorly localized is it translationally derepressed. Premature or ectopic translation of osk RNA causes severe developmental defects, indicating the essential role of the translational repression of osk RNA during its localization (Nakamura, 2004 and references therein).

The translational repression of osk RNA during its localization is mediated in part by the RNA binding protein Bruno (Bru), which binds specific repeated sequences in the 3' UTR of osk RNA, called the Bruno response elements (BREs). When BRE-mutated osk RNA is expressed during oogenesis, Osk protein is produced prematurely in the oocyte. The premature expression of Osk leads to a maternal-effect embryonic patterning defect. Thus, BRE-dependent translational repression of osk RNA prior to its posterior localization is essential for proper development. Direct evidence for a role of Bru in the repression of osk translation has come from the results of an in vitro translation system using Drosophila embryonic extract. Although embryonic extract lacking Bru efficiently translates BRE-containing RNA, the addition of recombinant Bru to the extract recapitulates BRE-mediated translational repression. However, the precise mechanism of the Bru-mediated translational repression remains elusive (Nakamura, 2004 and references therein).

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 (Keyes, 1997; Verrotti, 2000). Numerous cup alleles have been isolated as female sterile mutants, which show a wide range of phenotypes (Schüpbach, 1991; Keyes, 1997). 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 (Marcotrigiano, 1999). These results suggest that Cup competes with eIF4G for eIF4E binding, and hence inhibits translation initiation. Cup^Δ212 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 cup^Δ212 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).

The model of osk translational repression by the eIF4E-Cup-Bru interactions is similar to that of the Maskin-mediated repression of cyclin-B1 translation in the Xenopus oocyte (Stebbins-Boaz, 1999; Cao, 2002). In both models, Cup and Maskin play two crucial roles for achieving translational repression of a specific RNA. First, they themselves repress translation by preventing the eIF4F assembly at the 5' end of the RNA, and second, they act as adaptors to ensure target specificity by associating with specific 3' UTR binding proteins (Nakamura, 2004).

It is expected that translational corepressors that have the Cup/Maskin-like function are present in somatic cells as well, because 3' UTR-mediated translational control is a common strategy in a wide range of cell types. 3' UTR-mediated translational repression is essential for the asymmetric cell division of neuronal cells and probably for activity-regulated protein synthesis in dendrites governing synaptic plasticity. Importantly, the primary sequences of Cup and Maskin are not conserved, except for the eIF4E binding motif. Thus, such corepressors are likely to have the eIF4E binding motif but may be otherwise unrelated to Cup and Maskin (Nakamura, 2004 and references therein).

It has been reported that Bru-mediated translational repression might be independent of cap recognition, because in a cell-free translation system, Bru-mediated translational repression is effective, even in the presence of excess free cap analog. The current results strongly suggest that one major mechanism of Bru-mediated repression of osk translation is to inhibit the cap-dependent process. Bru may repress the translation of BRE-containing RNA by two discrete mechanisms: one directly interfering with the cap-dependent process and the other directing a cap-independent process. A similar situation has been described for Nanos/Pumilio-mediated repression of hunchback (hb). In this case, Nanos/Pumilio appears to repress hb translation by two mechanisms: underlying poly(A) tail removal and by a second poly(A)-independent mechanism. It seems likely that many translational repressors repress translation at multiple steps in order to exert their function properly. Supporting this idea, osk RNA has multiple BREs in its 3' UTR (Nakamura, 2004).

Several lines of evidence suggest that Bru also regulates grk translation in oogenesis. However, no defects were observed in grk RNA localization or Grk accumulation at the anterior-dorsal corner of the cup^Δ212 oocyte. Thus, the Bru-Cup-eIF4E interactions are dispensable for the regulation of grk translation, which may be controlled by redundant mechanisms. (Nakamura, 2004).

Although the eIF4E-Cup-Bru interactions shed light on the mechanism of the translational repression of osk RNA, it remains unanswered how osk translation is derepressed at the posterior pole of the oocyte. Nevertheless, the current findings suggest several potential targets for translational derepression. Studies of 4E-BPs and Maskin suggest that derepression may target the Cup-eIF4E interaction. In the case of 4E-BPs, extracellular growth signals lead to the hyperphosphorylation of 4E-BPs. Phosphorylation of a critical set of residues in 4E-BPs abolishes the interaction with eIF4E, such that global translation becomes efficient (Gingras, 1999; 2001). The Cup-eIF4E interaction may be regulated similarly, although potential target residues that correspond with 4E-BP phosphorylation sites cannot be identified in Cup by a simple sequence comparison (Nakamura, 2004).

A second possibility is that Cup may out-compete eIF4G for eIF4E binding. In the Xenopus oocyte, progesterone-induced oocyte maturation promotes the cytoplasmic polyadenylation of cyclin-B1 RNA. The newly elongated poly(A) tail recruits the poly(A) binding protein (PABP) to the RNA. PABP in turn binds eIF4G to stabilize the eIF4G-eIF4E interaction, dissociating the Maskin-eIF4E interaction (Stebbins-Boaz, 1999; Cao, 2002). Since Bru-mediated translational repression is effective even for a long poly(A)-tailed RNA (Castagnetti, 2003), derepression by cytoplasmic polyadenylation would not be the case for the eIF4E-Cup-Bru complex. However, it is still possible that as yet unknown factor(s) interact with eIF4G and promote the dissociation of the eIF4E-Cup interaction. It is also possible that the eIF4E-Cup-Bru interactions are regulated by a novel mechanism (Nakamura, 2004).

Derepression of osk translation requires a specific cis-element on the 5' side of the RNA (Gunkel, 1998). This element is required to overcome BRE-dependent translational repression and functions only at the posterior pole of the oocyte. Thus, p68, an unidentified ovarian protein that binds the element (Gunkel, 1998), is a good candidate for interfering with the eIF4E-Cup-Bru interactions to promote osk translation. (Nakamura, 2004).

Females with strong cup mutations have defects in early oogenesis, but cup^Δ212 females, in which a truncated Cup that fails to bind eIF4E is expressed, do not. Thus, Cup has additional functions that are independent of the eIF4E interaction. Cup interacts with Nanos in a yeast two-hybrid assay (Verrotti, 2000). Since this interaction appears to be required in early oogenesis (Verrotti, 2000), the Cup-Nanos-mediated process may be independent of the interaction with eIF4E. Cup also interacts genetically with ovarian tumor (otu) and fs(2)B (Keyes, 1997). Otu functions, together with Cup, to organize nurse cell chromosome structure. Since nurse cell chromosomes have normal morphology in cup^Δ212 ovaries, Cup's cooperative function with Otu is, again, likely to be independent of its interaction with eIF4E (Nakamura, 2004).

Finally, Cup and eIF4E associate with Me31B, forming a multiprotein-RNA complex that contains many maternal RNAs (Wilhelm, 2000; Nakamura, 2001). Me31B is also required to repress osk translation in oogenesis (Nakamura, 2001). Considering that Me31B belongs to the DEAD-box RNA helicase family, whose members modulate RNA-RNA and protein-RNA interactions, Me31B may organize the assembly of the RNP complex. In this scenario, loss of Me31B causes the Cup-eIF4E complex to fail to associate properly with osk RNA and results in the premature expression of the Osk protein in early oogenesis (Nakamura, 2004).

Alternatively, Me31B may act independent of the Cup-eIF4E-mediated process. Although Cup is essential for the repression of osk translation, it is possible that the Cup-mediated process is not the only repression mechanism in oogenesis. Additional factors involved in osk RNA translation and localization should also be enriched in the complex, because the complex contains the osk RNA (Wilhelm, 2000; Nakamura, 2001). Identification and characterization of the additional components of this complex and analysis of the interactions among them will provide further insight into the mechanism underlying localization-coupled translational control of the osk RNA (Nakamura, 2004).

GENE STRUCTURE

cDNA clone length - 4041 bases

Bases in 5' UTR - 157

Exons - 7

Bases in 3' UTR - 485

PROTEIN STRUCTURE

Amino Acids - 1132

Structural Domains

Cup consists of 1132 amino acids and contains clusters of glutamine residues in the 300 amino acids proximal to the carboxyl terminal, referred to as the 'Q-rich region'. A BLAST search revealed no clear homolog in other species. However, careful inspection of the Cup sequence shows that it has a short sequence that matches the consensus eIF4E binding motif, YxxxxLφ. This sequence has been shown by X-ray crystallography (Marcotrigiano, 1999) to be directly involved in eIF4E binding (Nakamura, 2004).

date revised: 3 April 2004

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