InteractiveFly: GeneBrief

cup: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

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).

The E2 Marie Kondo and the CTLH E3 ligase clear deposited RNA binding proteins during the maternal-to-zygotic transition

The maternal-to-zygotic transition (MZT) is a conserved step in animal development, where control is passed from the maternal to the zygotic genome. Although the MZT is typically considered from its impact on the transcriptome, previous work found that three maternally deposited Drosophila RNA-binding proteins (ME31B, Trailer Hitch [TRAL], and Cup) are also cleared during the MZT by unknown mechanisms. This study shows that these proteins are degraded by the ubiquitin-proteasome system. Marie Kondo, an E2 conjugating enzyme (FlyBase: Ubiquitin conjugating enzyme E2H), and the E3 CTLH ligase are required for the destruction of ME31B, TRAL, and Cup. Structure modeling of the Drosophila CTLH complex suggests that substrate recognition is different than orthologous complexes. Despite occurring hours earlier, egg activation mediates clearance of these proteins through the Pan Gu kinase, which stimulates translation of Kdo mRNA. Clearance of the maternal protein dowry thus appears to be a coordinated, but as-yet underappreciated, aspect of the MZT (Zavortink, 2020).

Proper embryogenesis is critical for animal development. Many of the earliest events occur prior to the onset of zygotic transcription, and they are instead directed by maternally deposited proteins and messenger RNAs (mRNAs). During the maternal-to-zygotic transition (MZT), genetic control of developmental events changes from these maternally loaded gene products to newly made zygotic ones. Thus, the MZT requires both the activation of zygotic transcription and clearance of maternal transcripts. Failure to mediate either of these processes is lethal for the embryo (Zavortink, 2020).

In contrast to understanding of the transcriptome during the MZT, much less is known about changes in the proteome. Despite the fact that the maternal dowry of proteins plays key roles in embryogenesis, there are only a handful of examples of cleared maternal proteins. Recently, three RNA-binding proteins (ME31B, Trailer Hitch [TRAL], and Cup) were found to be rapidly degraded during the MZT in Drosophila melanogaster, at a time point coinciding with the major wave of zygotic transcription. ME31B, TRAL, and Cup form a complex that blocks translation initiation. All three proteins are required for oogenesis, and they appear to bind and repress thousands of deposited maternal mRNAs. The degradation of ME31B, TRAL, and Cup coincides with many of the hallmarks of the MZT, but explorations into this issue have been hindered by a lack of understanding of how their destruction is controlled (Zavortink, 2020).

An intriguing observation has been made that genetically linked the clearance of ME31B, TRAL, and Cup, to the Pan Gu (PNG) kinase (Wang, 2017). Composed of three subunits (PNG, Giant Nuclei [GNU], and Plutonium [PLU]), the PNG kinase is central to the oocyte-to-egg transition and mediates key aspects of embryogenesis, including resumption of the cell cycle, zygotic transcription, and maternal mRNA clearance. Unlike many animals, the oocyte-to-egg transition in Drosophila does not require fertilization but is instead triggered by egg activation. The PNG kinase is activated by mechanical stress as the oocyte passes through the oviduct, and then phosphorylation and degradation of the GNU subunit quickly inactivates the kinase, restricting its activity to the first half hour after egg activation (Hara, 2017). One way that PNG mediates the oocyte-to-embryo transition is by rewiring post-transcriptional gene regulation. Possibly by phosphorylating key RNA-binding proteins such as Pumilio, PNG activity leads to changes in the poly(A)-tail length and translation of thousands of transcripts during egg activation (Hara, 2018). Importantly, two targets induced by PNG activity are the pioneer transcription factor Zelda, which is responsible for initial zygotic transcription, and the RNA-binding protein Smaug, which is responsible for clearance of many maternal transcripts. The PNG kinase also phosphorylates ME31B, Cup, and TRAL (Hara, 2018), but it is unclear what effect phosphorylation has on these proteins. One possibility has been that PNG phosphorylation could lead to the degradation of ME31B, TRAL, and Cup, but this model has been thus far unexplored (Zavortink, 2020).

The ubiquitin-proteasome system is a major protein degradation pathway. A series of ubiquitin activating enzymes, conjugating enzymes, and ligases (E1, E2, and E3, respectively) lead to the post-translational addition of a polyubiquitin chain on a target protein, which then serves as a molecular beacon for degradation by the proteasome. E3 ligases are typically thought to recognize target proteins, while E2 conjugating enzymes provide the activated ubiquitin and in turn recognize the E3 ligase. There are hundreds of different E3 ligases and 29 annotated E2 conjugating enzymes in Drosophila, but most of the client substrates are unknown, and few have been implicated in the MZT (Zavortink, 2020).

Given the key roles of ME31B, Cup, and TRAL in oogenesis and embryogenesis, it was of interest to understand the mechanisms controlling their degradation. In particular, this study sought to answer how PNG activity at egg activation leads to the degradation of these three RNA-binding proteins several hours later, and how their degradation is coordinated with other elements of the MZT, including zygotic transcription and maternal mRNA clearance. To answer these questions, a selective RNAi screen was performed in Drosophila, and the E2 conjugating enzyme was identified as UBC-E2H/Marie Kondo (Kdo) and the E3 ligase as the CTLH complex. Interestingly, structural models based on the S. cerevisiae complex (Qiao, 2020) suggest that the Drosophila version is organized differently than its orthologous complexes. The CTLH complex recognized and bound ME31B and Cup even in the absence of PNG activity, strongly suggesting that phosphorylation is not required for the destruction of these proteins. In contrast, Kdo mRNA is translationally upregulated by more than 20-fold upon egg activation in a PNG-dependent manner. Thus, egg activation through PNG mediates translation upregulation of Kdo and so leads to ME31B, Cup, and TRAL destruction (Zavortink, 2020).

Kdo is conserved from yeast to humans and is known to work through the CTLH E3 ligase, a multicomponent complex. (Note that the S. cerevisiae complex is called the Gid complex.) Using BLAST for the human CTLH components, it was easy to identify putative D. melanogaster homologs: RanBPM (homologous to Hs RanBP9), Muskelin, CG3295 (homologous to Hs RMND5A/GID2), CG7611 (homologous to Hs WDR26), CG6617 (homologous to Hs TWA1/GID8), and CG31357 (homologous to Hs MAEA). Putative homologs for Hs GID5/ARMC8 or Hs GID4 were not found. Notably, none of these genes were annotated as putative E3 components in FlyBase, and thus none were included in an original screen (Zavortink, 2020).

ME31B, Cup, and TRAL are RNA-binding proteins that are degraded during the MZT. Despite occurring several hours after egg laying, degradation of these proteins is triggered by egg activation through the activity of the PNG kinase and appears to be mediated by the ubiquitin-proteasome system. Through a medium-scale RNAi screen, the E2 conjugating enzyme Kdo was identified as being required for the clearance of ME31B, TRAL, and Cup. Kdo is conserved from yeast to humans and, as in those systems, appears to work with the CTLH complex, which acts as the E3 ligase. Components of the CTLH complex physically interact with ME31B and Cup, and the CTLH complex is also required for the degradation of ME31B, TRAL, and Cup during early embryogenesis. Structure-based homology suggests that, despite its conservation from yeast to humans, the Drosophila CTLH complex has an unusual architecture, and it remains unclear how it recognizes its substrate. The association of CTLH with ME31B occurs in the absence of PNG activity, suggesting that, although ME31B (as well as TRAL and Cup) are phosphorylated by the kinase, phosphorylation may not be required for their destruction. Instead, translation of Kdo appears to be suppressed during oogenesis by its short poly(A) tail length and binding of ME31B. Its translation is dramatically upregulated at the oocyte-to-embryo transition, in a process that depends on PNG activity. Together, these data suggest a model that egg activation via the PNG kinase leads to translational activation and production of Kdo, which then allows the CTLH complex to ubiquitinate ME31B, TRAL, and Cup and ultimately leads to their destruction. Interestingly, based on RNA-seq data from FlyBase, Muskelin shows exquisite tissue-specificity and is only strongly expressed in the ovaries. This observation, together with the translational control of Kdo, may partly explain how ME31B, a ubiquitous protein, is specifically destabilized in the early embryo (Zavortink, 2020).

Although the CTLH complex is conserved, it has not yet been studied in Drosophila. The data point to this complex being composed of multiple components (Muskelin, RanBPM, Houki, Souji, and Katazuke), as in other organisms. However, due to a lack of available reagents, the stoichiometry of these components is unknown, and it remains possible that there are additional, Drosophila-specific components. Nonetheless, so far, the CTLH complex in Drosophila appears different than the human and yeast complexes. Although Gid7 and WDR26 are important in the yeast and human versions, respectively, and a Drosophila ortholog (CG7611) was identified, no evidence was found of its association with ME31B or requirement for ME31B degradation; the role of CG7611 in the Drosophila CTLH complex warrants further investigation. Orthologs of Gid4 and Gid5, which are critical for substrate recognition in S. cerevisiae, were not found. Intriguingly, the residues and domains important for the Gid1-Gid4 and Gid8-Gid5 interactions in budding yeast appear absent to be in RanBPM and Hou, raising the fundamental question of how the Drosophila CTLH complex recognizes and positions its substrate proteins. Answering this question will require future investigation and may shed light on other proteins targeted by the Drosophila CTLH complex and the extent to which ME31B, a ubiquitously expressed protein, is targeted outside of the MZT (Zavortink, 2020).

One unexpected result is the role of PNG in mediating the destruction of ME31B, TRAL, and Cup. PNG phosphorylates all three proteins, and so the initial hypothesis was that this modification also stimulated their destruction. However, contrary to expectations, ME31B and Cup interacted with the CTLH complex even in png50 embryos, demonstrating that phosphorylation by PNG was not required for binding of ME31B and Cup by the E3 ligase. An unresolved question, then, is how PNG phosphorylation affects the activities of ME31B, TRAL, and Cup. Intriguing observations from the Orr-Weaver lab suggest that the modification can impact the ability of these proteins to repress gene expression (Hara, 2018). It is tempting to speculate that phosphorylation may then contribute to the MZT by modulating the activities of ME31B, TRAL, and Cup, rather than their stability (Zavortink, 2020).

The link between PNG and the destruction of ME31B, TRAL, and Cup instead appears to be mediated through the translational upregulation of Kdo. Although PNG may contribute through other, as-yet undiscovered, mechanisms as well (such as phosphorylating unknown CTLH adaptor proteins), this link is sufficient to explain the PNG requirement for ME31B degradation: in the absence of Kdo, ME31B is stable during the MZT, and in the absence of PNG, Kdo is not detectably expressed. An important question for the future will be to understand what elements in the Kdo mRNA are responsible for its translational repression during oogenesis. One hint may be that the 3'UTR of Kdo contains several putative Pumilio-binding sites, and translation of Kdo is upregulated in ovaries where Pumilio has been knocked down. Pumilio is also a target of PNG (Hara, 2018), and so a possible model is that translational repressors, such as Pumilio, are phosphorylated and inactivated at egg activation, leading to the production of Kdo (Zavortink, 2020).

PNG also mediates the translational upregulation of key MZT effectors: Zelda, the pioneer transcription factor, and Smaug, an RNA-binding protein that targets nearly two-thirds of the maternal transcriptome for degradation. Together with the current results, a picture is emerging that egg activation stimulates the production of multiple key factors that are important for clearing the maternal RNA and protein dowry and for producing zygotic gene products (Zavortink, 2020).

Although the MZT has typically been considered from the perspective of RNA, a role for maternal protein clearance is becoming clearer. Over the past few years, the list of proteins degraded during the Drosophila MZT has grown and now includes GNU, Matrimony, Cort, Smaug, ME31B, TRAL, and Cup. Unbiased mass spectrometry experiments also suggest that Wispy and Dhd are also robustly degraded. As this list of proteins in Drosophila and other developmental systems increases, a new question is emerging: how many maternally deposited proteins are degraded during the MZT? Understanding the mechanisms controlling protein degradation during the MZT as well as the impact of removing the maternal protein dowry will be key issues to explore in the future (Zavortink, 2020).

Precise temporal regulation of post-transcriptional repressors is required for an orderly Drosophila maternal-to-zygotic transition

In animal embryos, the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. The maternal proteome represents more than half of the protein-coding capacity of Drosophila melanogaster's genome, and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). Although the ubiquitin-proteasome system is necessary for clearance of these repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL, and ME31B for degradation early in the MZT and the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG abrogates F-box protein interaction and confers immunity to degradation. Persistent SMG downregulates zygotic re-expression of mRNAs whose maternal contribution is degraded by SMG. Thus, clearance of SMG permits an orderly MZT (Cao, 2020).

This study has shown that, in Drosophila, an extremely small subset of its maternal proteome is cleared during the MZT. This contrasts with the massive degradation of the maternal mRNA transcriptome that occurs during the MZT of all animals. Previous studies in other animals have suggested that the maternal proteome may behave very differently from the maternal transcriptome during the MZT. For example, in C. elegans, a quarter of the transcriptome is downregulated, whereas only 5% of the proteome shows a similar decrease. In frog embryos, there is also a discordance between the temporal patterns of protein and mRNA (Cao, 2020).

The set of proteins cleared during the Drosophila MZT is enriched for RNP granule components. This is consistent with the importance of post-transcriptional processes during the first ('maternal') phase of the MZT and the possible need to downregulate these processes upon ZGA and the switch to zygotic control of development. By focusing on a subset of these RNP components, which function as post-transcriptional repressors, this study has uncovered precise temporal control of their clearance by two distinct E3 ubiquitin ligase complexes: the SCF E3 ligase governs the degradation of SMG, whereas the CTLH E3 ligase is responsible for the degradation of Cup, TRAL, and ME31B. Intriguingly, SMG is degraded later during the MZT compared with its co-repressors Cup, TRAL, and ME31B. This study also showed that clearance of SMG is essential for appropriate levels of re-expression of a subset of its targets during ZGA. The results raise questions about how temporal specificity of protein degradation is regulated, as well as why at least two temporally distinct mechanisms of protein degradation exist during the MZT (Cao, 2020).

Expression data support the hypothesis that timing of E3 ligase function might, at least in part, be determined by the timing of expression of one or more of their component subunits, notably Muskelin for CTLH and CG14317 for SCF. During the Drosophila MZT, most components of the CTLH complex display constant expression levels, but Muskelin protein is degraded with a similar profile to its target repressors. Mammalian Muskelin has been shown to be auto-ubiquitinated and targeted for degradation. Detection of a ubiquitinated peptide in Muskelin supports the possibility that the Drosophila CTLH complex may be negatively autoregulated through its Muskelin subunit during the MZT. In contrast, activation of CTLH function at the beginning of the MZT may not depend on changes in complex composition: previous studies have shown that going from stage 14 oocytes to activated eggs or early (0-1 h) embryos, there are no significant changes in either the levels of CTLH subunit proteins (including Muskelin) or the ribosome association of their cognate transcripts. Thus, it is speculated that post-translational modification of one or more CTLH subunits may activate CTLH function (Cao, 2020).

Modification of substrates may also play a role: the degradation of Cup, TRAL, and ME31B depends on the PNG kinase, which itself has temporally restricted activity coinciding with degradation of these repressors. PNG-dependent phosphorylation of Cup, TRAL, and ME31B may make them ubiquitination substrates. Furthermore, evidence suggests that temporal regulation of the E2 ubiquitin-conjugating enzyme, UBC-E2H, at this stage depends on the PNG kinase and may also contribute to the timing of ubiquitin ligase complex function during the MZT. Concomitant PNG-dependent activation of the CTLH complex, its cognate E2, and its substrates, coupled with subsequent self-inactivation of the complex through Muskelin degradation, would provide a precise time window for CTLH function and, therefore, for degradation of Cup, TRAL, and ME31B early in the MZT (Cao, 2020).

In contrast with these three co-repressors, degradation of SMG occurs near the end of the MZT and depends on zygotic gene expression. Although the levels of most SCF complex subunits are constant during the MZT, the F-box protein CG14317 displays a unique expression pattern: CG14317 protein and mRNA are absent at the beginning of the MZT, are zygotically synthesized, peak in NC14 embryos, and sharply decline shortly thereafter. Thus, CG14317 expression coincides with the timing of SMG protein degradation and, coupled with the zygotic nature of its accumulation, makes it a strong candidate to be a timer for SCF function. The fact that knockdown of SLMB stabilizes SMG protein suggests that both F-box proteins may be necessary for SMG degradation, with CG14317 serving as the timer. At present there are no forward or reverse genetic reagents available to test this hypothesis. Additionally, the function of SLMB in directing SMG-protein ubiquitination may itself be temporally restricted. Both Drosophila SLMB and its mammalian homolog are known to bind phosphorylated motifs. Phosphorylated residues have been detected in SMG in the embryo, including residues within its C terminus; one of these, S967, resides close to a ubiquitinated lysine, K965. In summary, despite the stable expression of SLMB during the MZT, temporal regulation of phosphorylation of its target proteins, including SMG, through yet uncharacterized mechanisms, may also contribute to temporal control of SMG protein degradation (Cao, 2020).

Because Cup, TRAL, and ME31B are known to function as co-repressors in a complex with SMG, why are the timing of degradation of Cup-TRAL-ME31B and SMG differentially regulated? Although the SMG-Cup-TRAL-ME31B-mRNA complex has been characterized to be extremely stable in vitro, it would be disrupted in vivo by the degradation of Cup, TRAL, and ME31B (or by the degradation of nos and other SMG-target mRNAs). SMG directs translational repression both through AGO1 and through Cup-TRAL-ME31B, as well as transcript degradation through recruitment of the CCR4-NOT deadenylase. CTLH-driven degradation of Cup, TRAL, and ME31B would abrogate SMG-Cup-TRAL-ME31B-dependent translational repression, but not AGO1-dependent repression, because AGO1 levels increase during the MZT. However, the relative contributions of AGO1 versus Cup-TRAL-ME31B to translational repression by SMG are unknown. That said, the CCR4-NOT deadenylase is present both during and after the MZT (Temme et al., 2004); thus, SMG-dependent transcript degradation would occur both before and after clearance of Cup, TRAL, and ME31B. 12% of SMG-associated transcripts are degraded, but not repressed, by SMG. Perhaps this subset is bound and degraded by SMG late in the MZT, after the drop in Cup, TRAL, and ME31B levels (Cao, 2020).

Another possible role for clearance of ME31B and TRAL derives from studies in budding yeast, where it has been shown that their orthologs, respectively, Dhh1p and Scd6p, have a potent inhibitory effect on 'general' translation. If this is also true in Drosophila, then degradation of ME31B and TRAL, which are present at exceedingly high concentrations in embryos, might also serve to permit high-level translation during the second phase of the MZT (Cao, 2020).

Previous work has shown that SMG has both direct and indirect roles in the MZT. SMG's direct role is to bind to a large number of maternal mRNA species and target them for repression and/or degradation. Two indirect effects have been shown in smg mutants. First, if maternal transcripts fail to be degraded and/or repressed, ZGA fails or is significantly delayed, likely because mRNAs encoding transcriptional repressors persist. Second, because zygotically synthesized microRNAs direct a second wave of maternal mRNA decay during the late MZT, in smg mutants, failure to produce those microRNAs results in failure to eliminate a second set of maternal transcripts late in the MZT (Cao, 2020).

This study has uncovered a role for rapid clearance of the SMG protein itself late in the MZT: to permit normal levels of zygotic re-expression of a subset of it targets. Notably, stabilized SMG (SMG767Δ999) rescues both clearance of its maternal targets and ZGA, excluding the possibility that lower-than-normal levels of re-expressed targets are a result of defective SMG function upon deletion of its C terminus. Indeed, in control experiments, SMG's exclusively maternal targets actually dropped to lower levels than normal, likely because SMG767Δ999 continues to direct their decay beyond when SMG normally disappears from embryos. Furthermore, in another control, strictly zygotic transcripts that lack SMG binding sites were expressed at higher levels in SMG767Δ999-rescued mutants than in full-length SMG-rescued mutants. This result is consistent with the hypothesis that clearance of transcriptional repressors by SMG permits ZGA; persistent SMG would clear these repressors to lower levels than normal, hence resulting in higher zygotic expression. The higher-than-normal expression of zygotic transcripts that lack SMG binding sites makes the lower-than-normal levels of SMG's zygotically re-expressed target transcripts by SMG767Δ999 even more striking. Together, these data support a model in which the timing of both SMG synthesis and clearance are important for orderly progression of the MZT (Cao, 2020).

Comparative Proteomics Reveal Me31B's Interactome Dynamics, Expression Regulation, and Assembly Mechanism into Germ Granules during Drosophila Germline Development

Me31B is a protein component of Drosophila germ granules and plays an important role in germline development by interacting with other proteins and RNAs. To understand the dynamic changes that the Me31B interactome undergoes from oogenesis to early embryogenesis, this study characterized the early embryo Me31B interactome and compared it to the known ovary interactome. The two interactomes shared RNA regulation proteins, glycolytic enzymes, and cytoskeleton/motor proteins, but the core germ plasm proteins Vas, Tud, and Aub were significantly decreased in the embryo interactome. Follow-up on two RNA regulations proteins present in both interactomes, Tral and Cup, revealed that they colocalize with Me31B in nuage granules, P-bodies/sponge bodies, and possibly in germ plasm granules. It was further shown that Tral and Cup are both needed for maintaining Me31B protein level and mRNA stability, with Tral's effect being more specific. In addition, evidence is provided that Me31B likely colocalizes and interacts with germ plasm marker Vas in the ovaries and early embryo germ granules. Finally, it was shown that Me31B's localization in germ plasm is likely independent of the Osk-Vas-Tud-Aub germ plasm assembly pathway although its proper enrichment in the germ plasm may still rely on certain conserved germ plasm proteins (McCambridge, 2020).

To summarize, although Me31B's localization to the posterior of an oocyte is likely independent of Osk, Aub, and Dart5, its proper enrichment at the site may still rely on Aub. Together with a previous report that Me31B's localization pattern is not affected in vas and tud mutants, it is speculated that Me31B's localization in a developing oocyte may be independent of the Osk-Vas-Tud-Aub assembly pathway, but its proper enrichment at the posterior germ plasm may still depend on certain conserved germ plasm proteins like Aub. (McCambridge, 2020).

This speculation, together with earlier conclusions in this study, led to the proposal of a hypothetical model for Me31B localization and enrichment process in the germline cells (see Hypothetical model of Me31B localization and enrichment into germ plasm). In this model, Me31B and conserved germ plasm proteins, Osk-Vas-Tud-Aub, exist in distinct granules in the germ plasm, Osk-Vas-Tud-Aub in germ plasm granules and Me31B (possibly associated with Tral and Cup) in separate granules but in close proximity. Me31B granules use an Osk-Vas-Tud-Aub-independent mechanism to localize to the cortex and the posterior of a developing oocyte, then the posteriorly localized Me31B granules interact with the germ plasm granules, which is necessary for proper Me31B granule enrichment in the germ plasm. In the early embryos, Me31B proteins begin to degrade rapidly and become dispersed in the cytoplasm (McCambridge, 2020).

REGULATION

Protein Interactions

Cup is an eIF4E binding protein

Cup 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. To find out if Cup binds eIF4E through this sequence, the interactions were examined of GST-eIF4E with mutant Cup proteins, in which the conserved residues were replaced with Ala. Mutations in the conserved residues result in a severe reduction of the eIF4E-Cup interaction. From these results, it is concluded that Cup binds eIF4E directly through the conserved eIF4E binding site (Nakamura, 2004).

It has been reported that Trp-73 of mammalian eIF4E contacts Leu and φ residues in the conserved eIF4E binding sequence, and that the eIF4E-W73A mutant cannot bind its interacting partners that contain the conserved binding sequence (Marcotrigiano, 1999; Pyronnet, 1999). Thus Trp-117 of Drosophila eIF4E (equivalent to Trp-73 in mammalian eIF4E) was mutated into Ala (W117A) and its interaction with Cup was examined. GST-eIF4E-W117A fail to pull down Cup, suggesting that Cup binds eIF4E in the same manner as other eIF4E binding proteins, including eIF4G (Nakamura, 2004).

Drosophila Cup is known to be crucial for diverse aspects of female germ-line development. Its functions at the molecular level, however, have remained mainly unexplored. Cup was found to directly associate with eukaryotic translation initiation factor 4E (eIF4E). In this report, Cup is shown to be a nucleocytoplasmic shuttling protein, and the interaction with eIF4E promotes retention of the Cup protein in the cytoplasm. Cup is required for the correct accumulation and localization of eIF4E within the posterior cytoplasm of developing oocytes. cup and eIF4E interact genetically, because a reduction in the level of eIF4E activity deteriorates the development and growth of ovaries bearing homozygous cup mutant alleles. These results reveal a crucial role for the Cup-eIF4E complex in ovary-specific developmental programs (Zappavigna, 2004).

Cup is a Bruno binding protein

To better understand how Cup-mediated repression of osk translation is achieved, Cup-interacting proteins were sought using a yeast two-hybrid screen with full-length Cup as the bait. Sequence analysis of positive clones identified the translational repressor of osk RNA, Bruno, as a potential interacting partner of Cup. To determine which portion of Cup interacts with Bru, several deletion derivatives of the Cup bait construct were made and their interaction with Bru in yeast cells was analyzed. The C-terminal Q-rich region (residues 821-1132) of Cup is sufficient for the Bru interaction. This region contains at least two domains that could interact with Bru as two nonoverlapping fragments, 821-920 and 921-1132, interact with Bru with similar affinity. The Cup-interacting region of Bru was determined; residues 320-520 are sufficient to interact with Cup. Finally, Cup821-1132 and Bru320-520 are sufficient to interact with each other. Notably, the Bru-interacting domain of Cup does not contain the conserved eIF4E binding motif, and the RNA binding domains of Bru are dispensable for the interaction with Cup. These results suggest that Cup specifically interacts with Bru under physiological conditions, and that the interaction requires neither eIF4E nor RNA (Nakamura, 2004).

To examine if Cup associates with Bru in vivo, ovarian extracts were immunoprecipitated with α-Cup, α-eIF4E, and α-Me31B, and the precipitates were analyzed by Western blotting. Bru is coprecipitated by α-Cup, α-eIF4E, and α-Me31B, indicating that Bru is a component of the complex. Furthermore, although RNase treatment of the extracts disrupts the interaction of Me31B with Bru, it does not interfere with the coimmunoprecipitation of Bru by α-Cup and α-eIF4E. These results indicate that Bru associates with the Cup-eIF4E complex in vivo, and that the interactions between these three proteins are RNA independent (Nakamura, 2004).

Nanos interacts with Cup in the female germline of Drosophila

Nanos (Nos) is a translational regulator that governs abdominal segmentation of the Drosophila embryo in collaboration with Pumilio (Pum). In the embryo, the mode of Nos and Pum action is clear: they form a ternary complex with critical sequences in the 3'UTR of Hunchback mRNA to regulate its translation. Nos also regulates germ cell development and survival in the ovary. While this aspect of its biological activity appears to be evolutionarily conserved, the mode of Nos action in this process is not yet well understood. In this report it is shown that Nos interacts with Cup, which is required for normal development of the ovarian germline cells. nos and cup also interact genetically: reducing the level of cup activity specifically suppresses the oogenesis defects associated with the nos^RC allele. This allele encodes a very low level of mRNA and protein that, evidently, is just below the threshold for normal ovarian Nos function. Taken together, these findings are consistent with the idea that Nos and Cup interact to promote normal development of the ovarian germline. They further suggest that Nos and Pum are likely to collaborate during oogenesis, as they do during embryogenesis (Verrotti, 2000).

To identify proteins that interact with Nos, a yeast two-hybrid screen was performed using full-length Nos fused to the GAL4 DNA-binding domain as the bait. Two of the interactors proved to be fragments of Cup, a novel cytoplasmic protein of unknown biochemical function that is required for normal oogenesis (Keyes, 1997). In the ovaries of cup mutant females, egg chamber maturation arrests between stages 5 and 14, and the nurse cells have aberrant nuclear morphology. However, all of the extant cup alleles encode detectable protein, and thus the null phenotype may be stronger. The interaction with Nos appears to be specific, since Cup fails to interact with a variety of other baits in yeast. In particular, Cup does not interact with the RNA-binding domain of Pum (Verrotti, 2000).

To test the interaction between Nos and Cup in vitro, the minimum region of Cup required for interaction in yeast was defined by deletion analysis. Residues 593-963 constituted the smallest fragment of Cup tested that interacted with Nos in yeast. A GST fusion protein bearing these Cup residues was prepared in bacteria and was incubated with embryonic extracts from either wild-type or transgenic flies that produced a Myc epitope-tagged Nos that was fully functional and rescued the defects in otherwise nos^- embryos and ovaries. Approximately 10% of the Nos-Myc from the extract is retained by GST-Cup under the reaction conditions, whereas a negligible amount of Nos-Myc is retained in a control reaction with GST. In summary, Nos appears to interact specifically with Cup in yeast and in vitro (Verrotti, 2000).

Which portion of Nos mediates the interaction with Cup? Nos contains two regions -- a well-conserved C-terminal Zn²⁺-binding domain that mediates the interaction with Pum and HB mRNA, and a poorly conserved N-terminal region. The N-terminal region of Nos mediates interaction with Cup. No biochemical function has previously been ascribed to this portion of Nos, which is very poorly conserved even among closely related Dipteran homologs. Further deletion analysis of the N-terminal region reveals that it contains at least two redundant sub-domains that can interact with Cup (Verrotti, 2000).

The function of the N-terminal region of Nos in vivo is not clear. A recent analysis of 60 nos^- alleles reveals that mis-sense mutations that eliminate both ovarian and embryonic function alter residues in the conserved C-terminal domain, consistent with the idea that this domain is required for function in both tissues (Arrizabalaga, 1999). In contrast, no such mis-sense mutations are found in the coding region for the poorly conserved N-terminal domain. Microinjection of mRNAs encoding deletion derivatives of Nos into nos^- embryos suggests that no single part of the N-terminal region is essential for regulation of HB mRNA. No comparable analysis of Nos residues required for ovarian function has been reported (Verrotti, 2000).

To identify residues of Nos essential for its activity in the ovary, nos transgenes were prepared that encode deletion derivatives. No part of the N-terminal region of Nos is essential for Nos to function in either the embryo or the ovary. Maternal expression from a single transgene encoding each deletion derivative rescues the ovarian morphology and egg-laying defects associated with nos^RC. Each deletion derivative also rescues the abdominal segmentation defects associated with nos^BN either completely (a full complement of 8 abdominal segments) or nearly completely (6-8 abdominal segments). One derivative, deltaG, appears to have somewhat less activity than the others; however, expression from two maternal copies of the deltaG transgene completely rescues abdominal segmentation in 100% of embryos. Thus, it is concluded that the N-terminal region of Nos contains no unique sequence that is essential for its activity in either the embryo or the ovary. This latter observation is consistent with the finding that interaction with Cup is mediated by redundant elements in the N-terminal region of Nos (Verrotti, 2000).

To determine whether the interaction between Nos and Cup is functionally significant, it was asked whether lowering the level of Cup modified any of the ovarian or embryonic phenotypes associated with altered Nos function. In one case, a strong genetic interaction was observed: introduction of a single cup allele substantially suppresses the oogenesis defects in hemizygous nos^RC mutant females. Cystoblasts that give rise to the germline components of the egg chamber do not develop normally in nos^RC mutant ovaries, and germline stem cells that give rise to cystoblasts are not maintained. As a result, nos^RC mutant ovaries contain only rare mature egg chambers. In contrast, in cup^-/+; nos^RC/Df(nos) ovaries, many of the egg chambers appear normal and mature into oocytes that are fertilized and oviposited. (The resulting embryos develop no abdominal segments, presumably because they lack sufficient Nos activity to repress HB translation.) The cup^-/+; nos^RC /Df(nos) females lay eggs for at least 3 weeks, suggesting that germline stem cells are maintained and function normally. Thus, reducing the level of Cup appears to specifically suppress the oogenesis defects associated with the nos^RC allele, but not the embryonic defects. Nine different cup alleles tested suppress the defects associated with nos^RC, suggesting that it is simply a reduction of Cup activity that suppresses the oogenesis phenotype. In contrast, the genetic interaction appears to be specific to the RC allele; the nos^RD mutant encodes an unstable protein bearing a substitution at one of the conserved Cys residues in the C-terminal domain. This allele exhibits oogenesis defects similar to nos^RC, but these defects are not ameliorated by lowering the level of Cup, presumably because the level of active Nos protein is insufficient. In addition, reducing the level of Cup has no effect on the oogenesis defects associated with two different allelic combinations of pum. Thus, reduction of Cup activity does not appear to globally suppress oogenesis defects resulting from alterations in Nos or Pum activity, but specifically suppresses the defects associated with nos^RC (Verrotti, 2000).

The genetic interaction between nos and cup suggests that expression of the protein encoded by each gene coincides, and previous reports support this idea (Keyes, 1997). However, it was of interest to visualize the distributions of Nos and Cup simultaneously to determine whether the spatiotemporal distribution of the proteins is consistent with the observed genetic interaction. Using anti-Nos antibodies, Nos protein could not be reliably detected in the germarium. Therefore, the localization of Cup and Myc-tagged Nos was examined in the transgenic flies that carry a fully functional nos⁺ transgene altered to encode a Myc epitope tag at the C terminus of the protein. Cup is present throughout the cytoplasm of all the germ cells in the germarium, the terminal region of the ovary that contains the stem cells, cystoblasts and most immature egg chambers. In contrast, the Nos-Myc distribution is not uniform. It is present in the germline stem cells and cystoblasts in region 1 of the germarium, falls beneath the level of detection during the early cystoblast cleavages in regions 1 and 2, rises to relatively high levels in the germline cysts in region 2, and falls to somewhat lower levels in the maturing cysts of region 3. The significant finding is that Nos and Cup co-localize to the cytoplasm of the stem cells, the cystoblasts and the cysts, consistent with the genetic interaction described above (Verrotti, 2000).

The data reported above support the idea that Cup interacts with the N-terminal region of Nos and thereby lowers its activity, perhaps titrating it away from regulatory targets. This follows from the observed physical interaction and the genetic interaction between nos^RC and cup. However, nos^RC, which bears a mutation in the splice donor of intron 1 in the pre-mRNA, has been described as a null allele, and no mature mRNA is detectable by either in situ hybridization or Northern blot. How then can a physical interaction between Cup and Nos account for the genetic interaction between cup and nos^RC? To address this question, it was asked whether nos^RC actually encodes a very low level of functional protein. Semi-quantitative RT-PCR was used to determine whether nos^RC flies contain low levels of mRNA. Using primers that flank intron 1, a low level of NOS mRNA was detected in extracts prepared from whole flies. Since the major site of transcription in adult females is the ovary, it is assumed that most of this mRNA is derived from the rudimentary nos^RC ovaries. Two major cDNA species were detected by ethidium bromide staining of the PCR product following electrophoresis. To further characterize these cDNAs, the PCR products were subcloned and six individual clones were sequenced. The six clones appear to represent mRNAs generated by processing from cryptic splice sites; the open reading frame is preserved in three different clones, and one of these encodes a Nos derivative that is four amino acids larger than wild type. This cDNA clone plausibly represents the mRNA species that gives rise to the nos^RC-encoded protein (Verrotti, 2000).

In an attempt to detect protein encoded by nos^RC directly, transgenic flies were prepared bearing a nos^RC-myc gene that encodes an epitope-tagged protein that is otherwise identical to the nos⁺-myc gene described above. Using Western blots, a very low level of nearly full-length protein was detectable in nos^RC-myc ovaries from five different transgenic lines. Comparison with dilutions of extracts prepared from nos⁺-myc transgenic ovaries suggests that the level of protein encoded by nos^RC is in the order of 1%-2% of wild type. By crossing the nos⁺-myc and nos^RC-myc transgenes into cup^-/+ backgrounds and comparing the level of Nos protein in ovarian extracts, it was found that reducing the level of Cup does not significantly affect the level of protein encoded by either transgene. Thus, low levels of Cup do not appear to suppress the nos^RC ovarian phenotype by stabilizing Nos. It is concluded that, in the presence of reduced levels of Cup, 1%-2% of the wild-type level of Nos is sufficient to promote normal maintenance of the germline stem cells and differentiation of the cysts (Verrotti, 2000).

It is concluded that reducing the level of functional Cup suppresses the oogenesis defects in hemizygous nos^RC ovaries. This finding suggests that Cup acts to inhibit the residual protein encoded by nos^RC and prevent it from acting on potential regulatory targets. The identities of such targets are not currently known. In addition, the sequence of Cup sheds no light on its function. A search of the current database reveals no significant homologies to proteins of known function, neither does it possess recognizable motifs using programs such as Prosite, although Keyes (1997) has suggested that Cup may be a microtubule-associated protein. A fragment of human sequence bears high homology to a part of the fly protein, and thus it seems likely that one or more of the Cup functions are evolutionarily conserved. Further analysis of the significance of the Nos-Cup interaction awaits definition of the biochemical activities of Cup and the identification of Nos-regulated genes in the ovary (Verrotti, 2000).

While physiological levels of Cup are capable of inhibiting the low level of Nos activity in hemizygous nos^RC flies, it is not understood what role the Cup-Nos interaction plays in the ovaries of wild-type flies. Over-expression of Nos is deleterious in many different tissues -- the embryo, the eye imaginal disc and the male germline -- suggesting that Nos is a potent regulator of gene expression. Consistent with this idea, it has been found that extremely low levels of Nos, in the order of a few percent of the wild-type amount, suffice for biological activity in the ovary. Thus, it seems possible that the interaction with Cup helps restrict Nos activity, which otherwise might interfere with normal ovarian development. Alternatively, Nos and Cup may act together to govern some aspect of germ cell development. Cup appears to play a role in early germline development, since cup and ovarian tumor interact genetically in the ovary, leading to over-proliferation of germline cells (Keyes, 1997; Verrotti, 2000).

Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz

In Drosophila oocytes, precise localization of the posterior determinant, Oskar, is required for posterior patterning. This precision is accomplished by a localization-dependent translational control mechanism that ensures translation of only correctly localized oskar transcripts. Although progress has been made in identifying localization factors and translational repressors of oskar, none of the known components of the Oskar complex is required for both processes. Cup has been identified as a novel component of the oskar RNP complex. cup is required for oskar mRNA localization and is necessary to recruit the plus end-directed microtubule transport factor Barentsz to the complex. Surprisingly, Cup is also required to repress the translation of oskar. Furthermore, eukaryotic initiation factor 4E (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).

To identify novel components that play a role in either localization or translational regulation of oskar mRNA, an oskar RNP complex was purified that contains Exuperantia (Exu), Ypsilon Schachtel (Yps), and six unidentified proteins (Wilhelm, 2000). Using mass spectrometry, the 147-kD protein of this complex has been identified as Cup. To confirm that Cup is a bona fide component of the oskar RNP complex, both GFP-Exu and Yps were immunoprecipitated and immunoblotted with anti-Cup antibody. Cup specifically coimmunoprecipitates with both GFP-Exu and Yps, demonstrating that Cup is a component of the complex (Wilhelm, 2003).

cup was originally identified as a female sterile mutation that forms eggs that are open at the anterior due to a failure in chorion deposition at the anterior of the oocyte (Schupbach, 1991; Keyes, 1997). This previous work established that Cup is a cytoplasmic protein that is localized early to the oocyte (Keyes, 1997). Since Cup copurifies with components of an oskar RNP complex, the distribution of Cup during oogenesis was examined in more detail. Immunostaining of different stage egg chambers reveals that Cup accumulates at the posterior of the oocyte during stages 1-6, consistent with previously published results. At stages 7 and 8, Cup localizes to the anterior of the oocyte, followed by redistribution to the posterior of the oocyte during stages 9 and 10. Thus, Cup copurifies with components of the oskar RNP complex and is localized within the oocyte in a temporal-spatial pattern identical to that of oskar mRNA (Wilhelm, 2003).

One of the rationales for using GFP-Exu as a biochemical handle for the purification of localization complexes is that GFP-Exu forms particles in nurse cells that move in a microtubule-dependent manner. Yps, which binds directly to Exu, localizes to these motile particles (Wilhelm, 2000). To determine if Cup is also a component of these particles, egg chambers were immunostained for both Cup and Yps. The particulate staining observed for both Cup and Yps in the nurse cells show a high degree of overlap, indicating that Yps and Cup are part of the same particles in vivo. Recently, a novel component of the oskar mRNA localization machinery, Btz, was identified that has a staining pattern that is strikingly similar to that of Cup (van Eeden, 2001). Egg chambers were immunostained for both Cup and Btz to determine if they were also present in the same nurse cell particles. Most cytoplasmic particles contained both Cup and Btz. Interestingly, Btz protein that localizes tightly to the nuclear rim does not display a large amount of overlap with Cup, indicating that this pool of Btz might be part of a separate complex. Thus, Cup is present in motile RNP particles that contain Btz, a known component of the oskar mRNA localization machinery (Wilhelm, 2003).

Since Cup colocalizes and copurifies with components of the oskar RNP complex, it was next asked if Cup plays a role in oskar mRNA localization. For this and subsequent experiments, attention was focussed on the heteroallelic combination of cup¹/cup⁴⁵⁰⁶ since the combination of the strong cup⁴⁵⁰⁶ allele with the intermediate strength cup¹ allele allowed oogenesis to proceed far enough to assay oskar mRNA localization. This allelic combination yielded results that were representative of other heteroallelic combinations and also allowed minimization of the effects of secondary mutations since cup¹ and cup⁴⁵⁰⁶ were isolated in separate screens. In situ hybridization of oskar mRNA in cup¹/cup⁴⁵⁰⁶ egg chambers revealed that although oskar mRNA localization is normal in stages 1-7 of oogenesis, during stages 8-10, oskar mRNA is predominantly cortical with some enrichment at the posterior pole. This dispersed localization pattern is similar to that observed in weak alleles of btz (van Eeden, 2001) where low levels of oskar mRNA are localized to the posterior pole (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 (Dostie, 2000), 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 (Mader, 1995). 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 (Dostie, 2000; Macchi, 2003). 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 (van Eeden, 2001), 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 (Wilhelm, 2003; van Eeden, 2001), 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 (Stebbins-Boaz, 1999), 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).

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-Smg^583-763 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-Smg^583-763 resin and a resin carrying covalently coupled GST-Smg^179-307. However, an `80 kDa protein and an `140 kDa protein were specifically eluted from the Smg^583-763 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 Smg^583-763, Cup was generated via in vitro translation in rabbit reticulocyte lysate. This protein interacted with GST-Smg^583-763, as assayed by capture of Cup on glutathione agarose in the presence of GST-Smg^583-763 (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),

Efficient Gurken protein trafficking requires trailer hitch, a component of a ribonucleoprotein complex that includes Cup and Yps

Translational control of localized messenger mRNAs (mRNAs) is critical for cell polarity, synaptic plasticity, and embryonic patterning. While progress has been made in identifying localization factors and translational regulators, it is unclear how broad a role they play in regulating basic cellular processes. Drosophila trailer hitch (tral) has been identified as required for the proper secretion of the dorsal-ventral patterning factor Gurken, as well as the vitellogenin receptor Yolkless. Surprisingly, biochemical purification of Tral reveals that it is part of a large RNA-protein complex that includes the translation/localization factors Me31B and Cup as well as the mRNAs for endoplasmic reticulum (ER) exit site components, that regulate exit of proteins from the ER. This complex is localized to subdomains of the ER that border ER exit sites. Furthermore, tral is required for normal ER exit site formation. These findings raise exciting new possibilities for how the mRNA localization machinery could interface with the classical secretory pathway to promote efficient protein trafficking in the cell (Wilhelm, 2005).

In order to better understand the role of Tral in regulating membrane trafficking, the identification of Tral-associated proteins was attempted by immunoprecipitating Tral from Drosophila embryo extract using Tral antibody. By colloidal blue staining, three major bands were found that specifically coimmunoprecipitated with Tral: p147, p70, and p50. Using mass spectrometry, p147 was identified as the eIF4E binding protein Cup, and p70 as poly(A) binding protein (PABP). p50 was found to be a mixture of the RNA binding protein Ypsilon Schactel (Yps) and the RNA helicase Me31B. To confirm the identities of the Tral-associated proteins, Tral was immunoprecipitated from ovarian extracts and immunoblotted for Cup, Yps, and Me31B. Me31B, Yps, and Cup all specifically coimmunoprecipitate with Tral, indicating that these proteins are bona fide components of the complex. Because Me31B, Yps, and Cup have been previously shown to be part of an RNA-protein complex, the ability of each protein to coimmunoprecipitate with Tral was tested in RNase-treated ovarian extracts. It was found that while the association of Tral with Me31B, Yps, and Cup is RNase resistant, the association of Yps with Cup is sensitive to RNase treatment, indicating the presence of RNA in the complex (Wilhelm, 2005).

Previous work has shown that Me31B, Cup, and Yps colocalize in vivo. In order to demonstrate that Tral is part of the Me31B-Cup-Yps complex in vivo, egg chambers were immunoprecipitated for Tral and Me31B as well as Tral and Cup. The particulate staining in nurse cells showed a high degree of overlap for both the Tral/Cup and Tral/Me31B double-labeled egg chambers. Furthermore, the temporal-spatial pattern of Tral localization within the oocyte is identical to that previously described for Cup, Me31B, and Yps. These results, together with the previously demonstrated colocalization of Me31B, Cup, and Yps, indicate that Tral, Cup, Me31B, and Yps all exist as a complex in vivo (Wilhelm, 2005).

Because Tral is present on discrete domains of the ER, it was next asked whether other components of the complex were also present on the ER. Colocalization studies of GFP-KDEL with either Me31B or Cup showed that Me31B and Cup are both present on discrete ER subdomains. This observation, together with the biochemical analysis of the Tral complex, demonstrates that Tral is part of an RNA-protein complex that is associated with the ER (Wilhelm, 2005).

Because mutations in tral have such striking effects on morphology of COPII foci, attempts were made to define the relationship between these foci and components of the Tral complex. Using GFP-Sar1 as a marker for COPII complex formation, it was found that while some COPII sites are not associated with the Tral complex, a number of sites either colocalize with or are bordered by the Tral complex. These observations are highly suggestive of a direct role in regulating exit site function, as recent work has implicated the regions around COPII sites in exit from the ER (Wilhelm, 2005).

Cup interacts with Bruno and inhibits oskar translation: Bruno acts as a dual repressor of oskar translation, promoting mRNA oligomerization and formation of silencing particles

Prior to reaching the posterior pole of the Drosophila oocyte, oskar mRNA is translationally silenced by Bruno binding to BREs in the 3' untranslated region. The eIF4E binding protein Cup interacts with Bruno and inhibits oskar translation. Validating current models, the mechanism proposed for Cup-mediated repression has been directly demonstrated: inhibition of small ribosomal subunit recruitment to oskar mRNA. However, 43S complex recruitment remains inhibited in the absence of functional Cup, uncovering a second Bruno-dependent silencing mechanism. This mechanism involves mRNA oligomerization and formation of large (50S-80S) silencing particles that cannot be accessed by ribosomes. Bruno-dependent mRNA oligomerization into silencing particles emerges as a mode of translational control that may be particularly suited to coupling with mRNA transport (Chekulaeva, 2006).

Tight restriction of Oskar protein to the posterior pole of the Drosophila oocyte is crucial for development of the future embryo and is largely achieved by posterior localization of oskar mRNA and its translational inhibition prior to localization. This molecular analysis of oskar mRNA translational repression and of the relative roles of Bruno and Cup in this process has demonstrated the existence of two distinct modes of repression by Bruno and their mechanistic basis. This study has demonstrated directly the mechanism hypothesized for Bruno/Cup function, whereby cap-dependent 43S complex recruitment is inhibited. It has also been discovered that Bruno exerts its function through a second mechanism that does not require functional Cup and its interaction with eIF4E. This mode of repression involves Bruno-dependent oskar mRNA oligomerization and assembly into silencing particles, unusually large RNPs in which oskar remains inaccessible to the translation machinery (Chekulaeva, 2006).

This analysis of ribosomal complexes assembled on oskar reporter mRNA in vitro revealed that 48S initiation complex formation is inhibited both in the presence and in the absence of Cup-eIF4E interaction. This result is compatible with either of two possible mechanisms: (1) inhibition of small ribosomal subunit recruitment and (2) blocking of the following step: scanning of the 5'UTR by the small ribosomal subunit. Indeed, such scanning complexes in which the 43S subunit moves along the mRNA searching for the initiation codon are not stable and can easily dissociate during centrifugation in the sucrose density gradient. Therefore, as with a failure in recruitment of the small ribosomal subunit, interfering with scanning would also result in a reduction of the 48S peak (Chekulaeva, 2006).

The first of the two oskar repression mechanisms requires the interaction of Cup and eIF4E. This Cup-dependent repression process also requires a m⁷GpppN cap on the mRNA. Since binding of the small ribosomal subunit represents the cap-dependent step in translation initiation, these results provide a direct demonstration of the hypothesized mechanism for Cup regulation of oskar mRNA: a block of cap-dependent 43S recruitment mediated by a functional interaction between Cup-eIF4E and Bruno. Interestingly, it was observed that Cup recruits eIF4E to the mRNA in a cap-independent manner, suggesting an unexpected role for Cup, over and beyond its role in translational repression. Recruitment of eIF4E to oskar mRNA complexes by Cup might ensure colocalization and local enrichment of this otherwise limiting translation factor at the posterior pole, where oskar mRNA is translationally activated (Chekulaeva, 2006).

The second mechanism of oskar regulation revealed by this analysis also involves Bruno but requires neither Cup-eIF4E interaction nor a m⁷GpppN cap. It is therefore unlikely that this mechanism directly interferes with cap-dependent recruitment of the 43S complex (Chekulaeva, 2006).

This analysis shows that repressed oskar reporter mRNA forms unusually heavy complexes sedimenting between the 48S and 80S peaks. Importantly, these complexes form in the absence of the Cup-eIF4E interaction and of ribosomal subunit binding, as revealed by their persistence upon addition of cap analog. It is therefore proposed that oskar mRNA is sequestered in such large RNP complexes and hence inaccessible to the 43S preinitiation complex. Consistent with such a sequestration hypothesis, the repressed mRNA is selectively protected from the degradation machinery. Interestingly, a model of 'masked' (translationally inactive, stable) mRNAs was put forward 40 years ago. Masking factors were proposed to bind to mRNA and promote aggregation into higher-order condensed particles, protected from any processive events, including translation, degradation and polyadenylation/deadenylation (Chekulaeva, 2006).

The current experiments reveal that assembly of oskar mRNA into RNP complexes as large as monoribosomes can occur without any involvement of the RNA with the ribosomal subunits. These findings shed an unexpected light on the published literature, where complexes of 80S and larger can be intuitively taken as an indication of ribosomal association and translation elongation. Based on the co-sedimentation of oskar mRNA with polysomes and experiments involving the polysome-disrupting agent puromycin, it has been concluded that in the ovary, repressed oskar mRNA is associated with translating ribosomes (Braat, 2004). The current data challenge this conclusion, because it is shown directly that heavy RNPs (up to 80S in vitro) can form on oskar reporter mRNA without ribosomal subunit binding. The Braat study employed experimental conditions in which more than one variable was simultaneously changed. Specifically, the Mg²⁺ concentration, which can affect both polysome and RNP stability, differed by an order of magnitude between the puromycin-treated samples (2.5 mM Mg²⁺) and the cycloheximide control (25 mM Mg²⁺). This experiment was repeated, altering only one variable (puromycin). When the Mg²⁺ concentration is kept constant, puromycin does not affect the heavy RNPs that were previously interpreted as being 'polysomal'. It is suggested that oskar mRNA is engaged in puromycin-insensitive, heavy silencing particles that are sequestered from ribosomal engagement and that cosediment with polysomes (Chekulaeva, 2006).

Remarkably, oskar silencing particles comprise not single mRNA molecules but mRNA oligomers, whose formation is dependent on the specific association of Bruno with the BREs. The fact that the same components, Bruno and BREs, are responsible for both translational repression and mRNA oligomerization into silencing particles suggests a causal relationship between oligomerization and translational silencing (Chekulaeva, 2006).

The interesting finding that Cup is present in the heavy but not in the light RNP peak highlights the role of silencing particles in oskar repression. The sucrose gradient analysis of repressed complexes in cup^Δ212 extract demonstrates that Cup-4E interaction is not required for silencing particle formation. However, the fact that Cup is exclusively associated with the silencing particles but not with the light RNP peak of repressed mRNA suggests that particle formation may contribute not only to Cup-independent repression but also to Cup-dependent repression (Chekulaeva, 2006).

Consistent with the in vitro demonstration of oskar mRNA multimerization in silencing particles, it has been demonstrated that oskar mRNA molecules can self-associate through the 3′UTR for localization to the posterior pole of the oocyte. Since oskar mRNA is translationally repressed prior to posterior localization, it is tempting to speculate that the large silencing complexes that have been identified as containing oskar mRNA multimers are related to oskar mRNA localization complexes. It should be noted, however, that at present, there is no evidence for a role of the translational repressor Bruno in oskar mRNA localization. It is also possible that direct intermolecular RNA-RNA interactions might contribute to oskar oligomerization, as in the case of bicoid mRNA (Chekulaeva, 2006).

The current work suggests that silencing particles in Drosophila ovary extracts form by Bruno-mediated mRNA oligomerization from lower complexity precursors. Recent reports have described the presence of large particles, P bodies, in both yeast and mammalian cells. From these P bodies, silenced mRNAs may either return to the translating pool or be targeted for degradation. Furthermore, this work has suggested that RNP particles may aggregate from precursors into higher-order structures. In this regard, it is notable that both Cup and Me31B [Me31B has been implicated in translational regulation of Oskar mRNA during early oogenesis (Nakamura, 2001) and is a homolog of the S. cerevisiae P body component and translational repressor Dhh1p (Coller, 2005)] are present in silencing particles -- it has recently been shown that the mammalian eIF4E binding protein, 4E-T, and Dhh1p, the S. cerevisiae homolog of Me31B, are P body components. While the factors that promote P body aggregation in mammals and yeast are currently unknown, Bruno has been identified as a critical factor for silencing particle formation. Interestingly, this analysis shows that while Bruno is associated with the repressed mRNA both in silencing particles and lighter RNPs, Cup associates only with the mRNA in silencing particles. The fact that Bruno does not recruit Cup in the light RNP peak suggests that effectors may exist that regulate this interaction and cause RNP transition to silencing particles by addition/modification of factors and/or conformational change. It will be interesting to further explore the relationship between silencing particles and P bodies (Chekulaeva, 2006).

The exciting finding that oskar silencing particles comprise not single mRNA molecules, but mRNA multimers, suggests a mode of mRNA translational control that seems particularly suited to coupling of translational repression with mRNA transport within the cell. Such a repression mechanism would also allow coordinate repression of multiple oskar mRNAs, as well as coordinate derepression of the mRNAs within the silencing mRNP, upon its localization at the oocyte posterior pole. The particles could in principle contain other RNAs regulated and assembled into RNPs by common components. It will be interesting to determine if gurken mRNA, which is translationally repressed by Bruno (but not Cup) and colocalizes with oskar mRNA during the early stages of oogenesis, is coassembled with oskar mRNA in silencing particles (Chekulaeva, 2006).

Squid, Cup, and PABP55B function together to regulate gurken translation in Drosophila

During Drosophila oogenesis, the proper localization of gurken (grk) mRNA and protein is required for the establishment of the dorsal–ventral axis of the egg and future embryo. Squid (Sqd) is an RNA-binding protein that is required for the correct localization and translational regulation of the grk message. Cup and polyA-binding protein (PABP) interact physically with Sqd and with each other in ovaries. cup mutants lay dorsalized eggs, enhance dorsalization of weak sqd alleles, and display defects in grk mRNA localization and Grk protein accumulation. In contrast, pAbp mutants lay ventralized eggs and enhance grk haploinsufficiency. PABP also interacts genetically and biochemically with Encore. These data predict a model in which Cup and Sqd mediate translational repression of unlocalized grk mRNA, and PABP and Enc facilitate translational activation of the message once it is fully localized to the dorsal–anterior region of the oocyte. These data also provide the first evidence of a link between the complex of commonly used trans-acting factors and Enc, a factor that is required for grk translation (Clouse, 2008).

This study has taken a direct approach to identify proteins that interact with Sqd protein in ovaries. Using an Sqd antibody, immunoprecipitations out of ovarian extracts were performed, proteins were isolated that specifically interacted with Sqd, and those proteins were identified by mass spectrometry. Four of the Sqd-interacting proteins were positively identified in the mass spectrometry analysis: Cup, PABP55B, Imp, and Hrb27C/Hrp48. The remaining bands were not identified with certainty. Imp and Hrb27C/Hrp48 are two factors that have previously been shown to be involved in RNA localization, and both Hrb27C/Hrp48 and Imp bind to grk mRNA. The identification of these two factors confirmed that the immunoprecipitation method could successfully identify functional Sqd interactors (Clouse, 2008).

One of the Sqd interactors identified in the mass spectrometry analysis was the novel 150-kDa protein Cup. cup mutants display egg chambers with nurse cell nuclear morphology defects and eggs with open chorions. Cup interacts with several factors known to be required for osk localization and translation, such as Exu, Yps, eIF4E, Me31B, and Bruno and independent studies have shown that osk mRNA is prematurely translated in cup mutants. Cup co-localizes with the cap-binding protein, eIF4E, and eIF4E is not properly localized to the oocyte posterior pole in cup mutants. Cup competes away eIF4G, another translation initiation factor, for binding to eIF4E, thereby repressing translation. Together, these data are consistent with the following model for Cup-mediated translational repression; Cup represses the translation of RNAs containing BREs through interactions with Bruno. In this complex, Cup binds directly to eIF4E and interferes with eIF4G binding to eIF4E. Because eIF4G binding to eIF4E is a prerequisite for translation initiation, Cup represses translation by blocking this interaction. Direct biochemical data supporting this model have recently been obtained (Chekulaeva, 2006). It is proposed that Cup represses grk translation by a similar mechanism prior to its localization to the dorsal–anterior of the oocyte (Clouse, 2008).

Cup activity is used by several transcript-specific factors to mediate translational repression of that RNA in a developmentally appropriate context. For instance, Cup is required to mediate the translational repression of the nanos (nos) transcript. Cup has been shown to interact with Nos protein and co-localizes with Nos in the germarium. cup and nos also interact genetically, as heterozygosity for cup suppresses nos-induced phenotypes in early oogenesis. Later in development, Cup binds to Smaug, a factor that specifically binds to nos RNA and is required for its translational repression in embryos. In this example, Cup is required for Smaug to interact with eIF4E and mediate nos repression. Consistent with this biochemical model, Smaug-mediated translational repression is less efficient in cup mutants (Clouse, 2008).

This study as shown that Cup is also required for grk translational repression. This contrasts with previous reports that grk expression is normal in cup mutants, but these earlier reports used relatively weak cup alleles and monitored Grk levels by immunofluorescence. In contrast, in this study alleles were used that allowed assessment of the eggshell phenotype in cup mutants, providing the most sensitive assay for defects in Grk levels. These analyses showed that the different cup alleles vary greatly in phenotypic strength and range of phenotypes (Clouse, 2008).

Using two different alleles of cup from two distinct genetic backgrounds, it was shown that cup mutants lay dorsalized eggs, display defects in Grk protein accumulation, and display less efficient grk mRNA localization. Furthermore, Cup interacts biochemically with Sqd and Hrb27C/Hrp48 in ovarian extracts. Finally, heterozygosity for cup is able to enhance the moderate dorsalization observed in weak allelic combinations of sqd. Together, these data strongly support a model in which Cup functions with Sqd and Hrb27C/Hrp48 to mediate the translational repression of the grk message (Clouse, 2008).

Once grk mRNA is properly localized to the future dorsal/anterior of the oocyte, translational control must be switched from repressive to promoting. In many cellular situations, this activation is accomplished by binding of PABP to polyA tails of transcripts. In fact, PABP55B contains four RNA-recognition motifs (RRMs) that directly bind to polyA tails. PABP55B also has a C-terminal polyA domain that is used for oligomerization of PABP55B on polyA tails. Once PABP55B is bound to RNA, it binds to eIF4G, and this interaction helps to increase the affinity of eIF4G for eIF4E. With this increased affinity, eIF4G is able to effectively compete with Cup for binding to eIF4E, and translation is able to begin (Clouse, 2008).

There are at least three polyA-binding proteins in the Drosophila genome (CG5119 at 55B, CG4612 at 60D, and CG2163 at 44B), which are predicted to function as general translation factors, so it is conceivable that PABP55B could regulate a subset of RNAs. CG2163 has also been designated as PABP2 and has been shown to have essential roles in germ line development and in early embryogenesis (Benoit, 2005). This study has shown that PABP55B mediates the translational activation of fully localized grk mRNA. Specifically, heterozygous pAbp55B mutants lay ventralized eggs in certain genetic combinations, and heterozygosity for pAbp55B also enhances the weakly ventralized phenotype of grk heterozygotes, consistent with a role in translational activation of grk (Clouse, 2008).

PABP55B binds to Enc in ovarian extracts, and that this interaction may be direct and not bridged by an RNA molecule. Furthermore, heterozygosity for pAbp55B is able to enhance the weakly ventralized phenotype of enc mutants raised at 25 °C. Taken together, the biochemical and genetic interactions suggest that PABP55B and Enc function together to mediate the translational activation of grk mRNA once it is localized to the dorsal–anterior of the oocyte (Clouse, 2008).

Previously, Enc has been shown to be required for activation of grk translation in mid-oogenesis. An effect on osk mRNA localization has also been previously observed in enc mutants, but it is unclear at what level this process is affected, or whether this effect is direct. In addition, Enc has been shown to interact with subunits of the proteasome early in oogenesis. Because of its large size and its ability to interact with several different proteins, Enc may play multiple roles during oogenesis. Considering the function of Enc in grk translational activation and its localization to the dorsal–anterior region of the oocyte, It is hypothesized that Enc could function as a scaffolding protein that helps to mediate the transition from translational repression to activation of grk mRNA (Clouse, 2008).

Cup functions with Sqd in a protein complex that mediates the translational repression of grk mRNA before it is properly localized. It is clear from the analysis of mutants such as spn-F and encore, in which mislocalized grk mRNA is translationally silent, that these two steps can be uncoupled. It is proposed that once the RNA has reached the future dorsal–anterior region of the oocyte, PABP, Sqd, and Enc facilitate the translational activation of grk mRNA, PABP is shown associating with the complex once it is fully localized; however, it is possible that PABP associates with the grk transport complex in an inactive form that is remodeled following its anchorage at the dorsal–anterior of the oocyte (Clouse, 2008).

Previous studies have shown that Bruno (Bru) binds directly to Cup protein and is required for the translational repression of osk. Bru binds to specific sequence elements in the osk 3′ UTR called Bruno Response Elements (BREs), and mutations in these BREs have been shown to reduce Bru binding and result in ectopic Osk accumulation in the oocyte. Similarly, Bru has also been shown to bind to grk mRNA and to Sqd protein. Overexpression of bru cDNA leads to ventralization of the eggshell, consistent with reduced Grk protein expression in the oocyte. Furthermore, disrupting bru expression in certain genetic contexts has been shown to result in excess Grk protein in the oocyte, consistent with Bru being required to mediate grk translational repression. In light of the results presented in this study, it is proposed that this phenotype is the result of Bru-mediated repression of grk translation by Cup (Clouse, 2008).

The mechanism of grk translation and the trans-acting factors required for translational control largely parallel the mechanism employed by osk RNA, so an important question to be answered is how these two different RNAs are differentially transported and translationally regulated in distinct parts of the oocyte at the appropriate stage in oogenesis. Since the same group of trans-acting factors is involved in the expression of both RNAs, the specificity could be provided by cis-acting sequences within the RNA molecules themselves that affect the activity of common trans-acting factors. Alternatively, RNA-specificity could be generated by as-yet unidentified trans-acting factors. Given that Enc functions in grk translational activation, but is not required for osk translational activation, it is possible that Enc is providing some degree of specificity to the commonly used machinery that mediates translational control of multiple, unrelated transcripts. Currently, Enc is the only factor known to function uniquely in the translational activation of grk mRNA, and these results provide the first evidence of a link between this factor and the general translational control machinery that is used by multiple RNAs in oogenesis (Clouse, 2008).

Similar modes of interaction enable Trailer Hitch and EDC3 to associate with DCP1 and Me31B in distinct protein complexes

Trailer Hitch (Tral or LSm15) and enhancer of decapping-3 (EDC3 or LSm16) are conserved eukaryotic members of the (L)Sm (Sm and Like-Sm) protein family. They have a similar domain organization, characterized by an N-terminal LSm domain and a central FDF motif; however, in Tral, the FDF motif is flanked by regions rich in charged residues, whereas in EDC3 the FDF motif is followed by a YjeF_N domain. This study shows that in Drosophila cells, Tral and EDC3 specifically interact with the decapping activator DCP1 and the DEAD-box helicase Me31B. Nevertheless, only Tral associates with the translational repressor CUP, whereas EDC3 associates with the decapping enzyme DCP2. Like EDC3, Tral interacts with DCP1 and localizes to mRNA processing bodies (P bodies) via the LSm domain. This domain remains monomeric in solution and adopts a divergent Sm fold that lacks the characteristic N-terminal alpha-helix, as determined by nuclear magnetic resonance analyses. Mutational analysis revealed that the structural integrity of the LSm domain is required for Tral both to interact with DCP1 and CUP and to localize to P-bodies. Furthermore, both Tral and EDC3 interact with the C-terminal RecA-like domain of Me31B through their FDF motifs. Together with previous studies, these results show that Tral and EDC3 are structurally related and use a similar mode to associate with common partners in distinct protein complexes (Tritschler, 2008).

This study shows that Tral and EDC3 are structurally related proteins that associate with common partners in distinct protein complexes. The N-terminal domains of Tral and EDC3 adopt a divergent Sm fold that mediates their interaction with the decapping activator DCP1 and is sufficient for P-body targeting. In Tral, this domain also mediates the interaction with the translational regulator CUP. Tral and EDC3 share an additional common partner, the RNA helicase Me31B. Both proteins interact with the C-terminal RecA-like domain of Me31B via their FDF motifs, suggesting that their binding is mutually exclusive. This agrees with the conclusion that Tral and EDC3 function in distinct protein complexes. EDC3-containing complexes are known to play a role in mRNA decapping. The localization of Tral in P bodies, its association with decapping activators, and translational repressors suggest that this protein functions in translational repression and/or mRNA degradation (Tritschler, 2008).

P bodies are cytoplasmic domains that accumulate a variety of proteins involved in mRNA degradation, translational repression, mRNA surveillance, and RNA-mediated gene silencing, together with their mRNA targets. The mechanisms leading to P-body assembly are not fully understood, but several P-body components, including RNA, are required for P-body integrity. In yeast cells, P-body assembly occurs through parallel redundant pathways, requiring either Edc3 or LSm4. In multicellular organisms, several P-body components are required for P-body formation, since depleting them disperses the remaining P-body components throughout the cytoplasm (Tritschler, 2008).

In vertebrates the Tral ortholog RAP55 is among the essential P-body components. In contrast, in Drosophila S2 cells, depleting either Tral or EDC3 does not affect P bodies , suggesting that these proteins are not essential for P-body assembly or that multiple redundant pathways lead to P-body formation. Similarly, CAR-1 is not required for P-granule formation in C. elegans. Still, Tral and EDC3 likely contribute to the assembly of RNP particles. Both proteins have a modular domain organization that allows them to interact with additional P-body components and probably with multiple RNPs, bringing them in close proximity and thereby facilitating the nucleation of P bodies (Tritschler, 2008).

Over the past few years, the number of proteins shown to localize to P bodies has increased dramatically. A question that remains open is what causes these proteins to accumulate into P bodies. Some proteins may passively accumulate in P bodies as components of mRNP complexes. In this case, RNA-binding domains or protein-protein interaction domains likely mediate their localization to P bodies. However, the aggregation of individual mRNPs into large granules detectable by light microscopy requires either (1) multidomain proteins that can bridge more than one mRNP or (2) specific proteins or protein domains that self-aggregate (Tritschler, 2008).

One such domain has been described in S. cerevisiae LSm4. LSm4 consists of a canonical LSm domain followed by a C-terminal extension rich in glutamine and arginine residues (Q/N-rich domain), which is not part of the Sm fold. This extension is required to localize LSm4 to P bodies). LSm4 is partially redundant with Edc3 for P-body assembly in yeast cells, but in cells lacking Edc3, P-body assembly relies on the Q/N-rich extension of LSm4. Indeed, a truncated version of LSm4 lacking the Q/N-rich region cannot sustain P-body assembly in the absence of Edc3 . It was proposed that the Q/N-rich extension of LSm4 has prion-like properties and promotes P-body formation by aggregating with itself or with additional Q/N-rich domains, similar to the assembly mechanisms for Q/N-rich domains in prions (Tritschler, 2008).

In contrast to LSm4, the LSm domains of Tral and EDC3 are sufficient to localize them to P bodies. These domains lack Q/N prion-like features and remain monomeric in solution, indicating that they are not self-aggregating domains. Furthermore, these domains do not bind RNA, suggesting that they accumulate in P bodies through protein-protein interactions. Nevertheless, in EDC3, mutations that disrupt DCP1-binding do not affect P-body localization, suggesting that interactions with additional P-body component(s) drive this protein into P bodies. In Tral, no mutations were identified that reduced its accumulation in P bodies without affecting the folding of the LSm domain (Tritschler, 2008).

RAP55 is the vertebrate ortholog of Tral and was originally identified in the salamander Pleurodeles waltl as a component of cytoplasmic RNP particles containing translationally repressed maternal mRNAs. Orthologous proteins have been described in several eukaryotic species, including C. elegans (CAR-1), X. laevis (xRAP55), and mammals (RAP55). Like Tral, these proteins localize in diverse cytoplasmic RNP granules that share components with P-bodies and serve as storage sites for translationally inactive mRNAs in germ cells. For instance, in young oocytes Tral, xRAP55 and murine RAP55 localize to the Balbiani body, a large organelle aggregate that includes mitochondria, endoplasmic reticulum, germinal granule proteins, and RNAs that become incorporated into germ cells in the developing embryos (Tritschler, 2008).

RNP granules containing Tral, CAR-1, or xRAP55 comprise additional proteins with roles in translational repression and/or mRNA decapping. These include, Me31B and its orthologs (C. elegans CGH-1 and X. laevis Xp54), the Y-box domain-containing proteins (D. melanogaster YPS, C. elegans CEY-2-4, and X. laevis FRGY2), which are major components of maternal RNP granules, as well as the eIF4E-binding proteins CUP and 4E-T. Moreover, the Drosophila fragile-X mental retardation protein (dFMRP), which is involved in translational repression, colocalizes with Tral in RNP-containing granules in both embryos and neuronal cells. Tral and CAR-1-containing granules in germ cells also include DCP1 and additional components of somatic P-bodies (Tritschler, 2008).

The localization of Tral and its orthologs in RNP granules in germ cells and young embryos and their association with proteins involved in translational repression, together with the conservation of these interactions in eukaryotes, suggest that these proteins play a fundamental role in regulating translation of maternal mRNAs during oogenesis and early embryogenesis. In agreement with this, xRAP55 represses translation both in vivo and in vitro. Nevertheless, the precise molecular mechanism by which Tral orthologs exert their regulatory functions remains to be established (Tritschler, 2008).

Depleting or mutating Scd6p, CAR-1, or Tral alters endoplasmic reticulum morphology and causes diverse developmental phenotypes. These phenotypes are likely due to the misregulation of specific mRNAs. However, only few mRNA targets of these proteins are known. It is also not yet clear whether these targets are conserved and whether Tral orthologs recognize specific cis-acting sequence elements on regulated mRNAs (Tritschler, 2008).

CAR-1 and xRAP55 expression is confined to the germ line and early embryos. In contrast, human RAP55 is ubiquitously expressed and localizes to P bodies in somatic cells at rest and in stress granules in cells exposed to heat shock or oxidative stress. Similarly, Tral expression is not confined to the germ line, the protein is detected in S2 cells and in neurons, where it is also a component of neuronal RNP granules and participates in neuronal translation regulation. The localization of human RAP55 and Tral in somatic P bodies and neuronal granules suggests that, in addition to their role in regulating maternal mRNAs, these proteins have acquired more general roles in mRNA metabolism (Tritschler, 2008).

Based on the structural similarity between Tral and EDC3 and their association with common partners, one might anticipate that these proteins represent alternative subunits in the assembly of DCP1- and Me31B-containing complexes. These complexes may perform dual and partially overlapping functions; they may repress translation (e.g., in oocytes) or enhance decapping (e.g., in somatic cells), depending on the additional partners with which they associate (Tritschler, 2008).

CUP promotes deadenylation and inhibits decapping of mRNA target

Cup is an eIF4E-binding protein (4E-BP) that represses the expression of specific maternal mRNAs prior to their posterior localization. This study shows that Cup employs multiple mechanisms to repress the expression of target mRNAs. In addition to inducing translational repression, Cup maintains mRNA targets in a repressed state by promoting their deadenylation and protects deadenylated mRNAs from further degradation. Translational repression and deadenylation are independent of eIF4E binding and require both the middle and C-terminal regions of Cup, which collectively is termed the effector domain. This domain associates with the deadenylase complex CAF1-CCR4-NOT and decapping activators. Accordingly, in isolation, the effector domain is a potent trigger of mRNA degradation and promotes deadenylation, decapping and decay. However, in the context of the full-length Cup protein, the decapping and decay mediated by the effector domain are inhibited, and target mRNAs are maintained in a deadenylated, repressed form. Remarkably, an N-terminal regulatory domain containing a noncanonical eIF4E-binding motif is required to protect Cup-associated mRNAs from decapping and further degradation, suggesting that this domain counteracts the activity of the effector domain. These findings indicate that the mode of action of Cup is more complex than previously thought and provide mechanistic insight into the regulation of mRNA expression by 4E-BPs (Igreja, 2011).

Therefore, binding to eIF4E is not required for Cup-mediated translational repression and deadenylation of target mRNAs. Instead, the Mid and Q-rich regions, which are collectively termed the Cup effector domain, are essential for these functions. The isolated effector domain is a potent trigger of mRNA degradation and promotes deadenylation-dependent decapping and subsequent decay of the mRNA. In the context of full-length Cup, however, the N-terminal domain counteracts the activity of the effector domain, allowing deadenylation to occur but inhibiting mRNA decapping and subsequent degradation. The protective function of the N-terminal domain requires the noncanonical 4E-BM2 motif, which contributes to, but is not essential for, eIF4E-binding. Taken together, these results show that Cup uses multiple mechanisms to regulate protein output of its targets and further reveal unexpected functions for the noncanonical eIF4E-binding motif (4E-BM2) and the Mid and Q-rich regions in this regulation (Igreja, 2011).

This study shows that Cup consists of two functional domains: a regulatory N-terminal domain and a C-terminal effector domain. The effector domain consists of the Mid and Q-rich regions and elicits mRNA degradation. Although the isolated Mid and Q-rich regions are also able to trigger mRNA degradation, in the context of full-length Cup, these regions are both required for translational repression and mRNA deadenylation; a protein containing the N-terminal region but lacking either the Mid or Q-rich region does not repress translation or promote deadenylation. Thus, the Mid and Q-rich regions act together as a single effector domain. This domain serves as a binding platform for deadenylating and decapping factors. Further work will be required to determine whether the effector domain interacts with these factors directly, and whether these interactions occur simultaneously or consecutively (Igreja, 2011).

One important observation from these studies is that the effector domain can repress translation in the absence of deadenylation, suggesting that deadenylation is required not to establish repression, but rather to sustain the repressed state. This observation is consistent with previous studies showing that deadenylation contributes to, but is not essential for, the repression of nanos and oskar mRNAs (Igreja, 2011).

A second important finding is that Cup can promote deadenylation independent of Smaug. This finding provides one explanation for the observation that the repression of oskar mRNA involves deadenylation, even though Smaug is not involved in the repression of this mRNA. The results suggest that Cup could be responsible for the observed deadenylation of unlocalized oskar mRNA, maintaining it in a repressed state. In agreement with this possibility, the deadenylation of oskar mRNA is catalyzed by CCR4. Furthermore, the translation activation of oskar mRNA at the posterior pole requires polyadenylation mediated by Orb, the Drosophila homolog of cytoplasmic polyadenylation element-binding protein (CPEB) and Drosophila poly(A) polymerase (PAP), reinforcing the hypothesis that oskar mRNA repression involves deadenylation (Igreja, 2011).

The model that Cup inhibits translation by competing with eIF4G for binding to eIF4E is supported in vivo by the observation that a Cup mutant (Cupδ212) that lacks N-terminal residues, including the canonical eIF4E-binding motif, fails to repress the expression of unlocalized oskar mRNA in Drosophila oocytes. Thus, the observation that the 4E-BM1 motif is not required for Cup-mediated repression is contrary to the current model for Cup function. However, the results obtained with Cupδ212 mutant (which is thought to initiate translation at Met348) are difficult to interpret because this mutant lacks a large portion of the N-terminal region of the protein, the function of which remains unknown. Moreover, it is important to note that direct evidence that Cup binding to eIF4E is required for translational repression is lacking; for example, it has not been directly shown that point mutations in the canonical 4E-BM1 relieve Cup-mediated translational repression (Igreja, 2011).

Furthermore, in agreement with current findings, several lines of evidence indicate that the mRNA 5' cap structure, and thus eIF4E binding, is not essential for Cup-mediated translational repression. First, the δ212 mutation does not impair Cup function during oogenesis and only affects oskar mRNA expression in the embryo, whereas other Cup alleles have strong effects on oocyte maturation and ovary development, leading to female sterility. Second, oskar mRNA was repressed in Drosophila embryo extracts isolated from flies carrying the δ212 mutation, although it is not clear whether this repression was mediated by the N-terminal-truncated form of Cup. Third, the repression of oskar and nanos mRNA reporters was independent of the presence of a 5' cap structure in cell-free extracts. Fourth, mRNA reporters containing a nanos 3' UTR, the translation of which was initiated in a cap-independent manner (by the cricket paralysis virus IRES), were repressed in vitro. Finally, the results show that the 4E-BM1 motif is dispensable for the repression of a reporter containing the oskar 3' UTR to which Cup was recruited through interactions with Bruno, indicating that binding to eIF4E may contribute to, but is neither necessary nor sufficient for, Cup repression of its targets (Igreja, 2011).

Thus, it is possible that the 4E-BM1 motif has other functions. For example, through its binding to eIF4E, this motif may stabilize eIF4E binding to localized mRNAs so that translation can resume immediately after Cup-mediated repression is relieved. Consistent with this interpretation, the posterior localization of eIF4E has been shown to depend on Cup. Thus, the effects of the δ212 mutation may be partially related to a defect in eIF4E localization and/or on eIF4E phosphorylation (Cup also controls the eIF4E phosphorylation status in the ovary). A role for the canonical 4E-BM1 motif in recruiting eIF4E to mRNA targets is also supported by the observation that Cup can recruit eIF4E to mRNAs that lack an m7G cap structure. Thus, the 4E-BM1 motif may enable eIF4E to piggyback on transported mRNAs (Igreja, 2011).

The noncanonical 4E-BM2 motif also contributes to eIF4E binding, although to a lesser extent than the canonical 4E-BM1. This observation raised the question of whether this motif plays a role in translational repression. A major finding in the present study is that this motif is required to inhibit the potent decapping and degradative activity of the Cup effector domain in tethering assays. However, additional sequences within the N-terminal regulatory domain also contribute to protect Cup-associated mRNAs from degradation, as the isolated effector domain exhibits a stronger degradative activity than Cup Mut2 and Mut1+2 (Igreja, 2011).

It is unclear how the regulatory domain and the 4E-BM2 motif can counteract the activity of the effector domain. One possible mechanism could be that the regulatory domain and 4E-BM2 block decapping indirectly by making the cap structure less accessible to DCP2. This protection could be achieved by increasing eIF4E affinity for the cap structure or, alternatively, competing with unknown proteins that facilitate eIF4E dissociation. However, the interaction of 4E-BM2 with eIF4E is weak, suggesting that the inhibitory activity of 4E-BM2 may be independent of eIF4E binding. Another possible mechanism could be that the regulatory domain and/or 4E-BM2 directly antagonizes DCP2 recruitment and/or activation at mRNAs associated with Cup. This possibility is consistent with the observation that when the regulatory domain is deleted or 4E-BM2 is mutated, Cup promotes the decapping of deadenylated targets, indicating that DCP2 is recruited to and activated at Cup-associated mRNAs in the absence of the regulatory domain or the 4E-BM2 motif. It is also possible that the regulatory domain or 4E-BM2 directly interferes with the activity of the effector domain. However, this possibility is considered unlikely because the regulatory domain of Cup is sufficient to stabilize bound mRNAs. Finally, neither wild-type Cup nor a 4E-BM2 mutant (Mut2) interacted with DCP2, EDC4, or XRN1, indicating that 4E-BM2 does not inhibit decapping by directly preventing the interaction between Cup and decapping enzymes (Igreja, 2011).

Regardless of the specific mechanism, the observation that the regulatory domain and the 4E-BM2 motif inhibit decapping of associated mRNAs suggest that their activity could be subject to regulation, possibly via post-translational modifications or binding partners, so that mRNAs associated with Cup could be either fully degraded (e.g., if unlocalized) or stored in a repressed deadenylated form (Igreja, 2011).

More generally, the finding that the regulatory domain and the 4E-BM2 motif can block mRNA decapping in cis is both striking and unprecedented. This result opens up the exciting possibility that similar domains and motifs are present in other proteins, where they could specify alternative fates for bound mRNAs: complete degradation or storage in a deadenylated, repressed form for translation at a later time point. Therefore, this study has important implications for the understanding of translational regulation by 4EBPs. This topic warrants further investigation, as the repression of 4EBP targets may also be achieved through diverse, potentially 4E-independent mechanisms (Igreja, 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).

DEVELOPMENTAL BIOLOGY

Oocyte

To learn where cup is transcribed during oogenesis, the 4.1 kb cDNA was hybridized in situ to wild-type ovaries. cup RNA is present in germ-line cells throughout pre-vitellogenic development, but is not detected in the somatic follicle cells. The RNA is present in region 1 of the germarium, where it is detectable in stem cells, cystoblasts and dividing cysts. Steady state RNA levels decrease in region 2a, rise again in region 2b, and peak around stage 3 or 4. Subsequently, RNA levels decline and reach undetectable levels by stage 8. A second round of expression begins during stage 10 and continues through stage 14. The transcript is not differentially localized within the germ line at any stage. None of the strong alleles abolish all cup RNA expression (Keyes, 1997).

Polyclonal antibodies were raised against two independent domains of the Cup protein. Antisera against both domains produce similar results when used to analyze ovarian extracts on Western blots. Both sera detect a single predominant band of approximately 150 kDa that is abundant in wild-type ovaries but greatly reduced in cup mutant females. This is similar in size to the 130 kDa product predicted by the 4.1 kb cDNA. A second smaller band of 75 kDa reacts more weakly and variably with all Cup sera tested, but this protein is unaffected by mutants and is presumed to be encoded by a separate locus. Testes contain very little of the 150 kDa protein; the anti-Cup antibodies instead detect bands of approximately 180, 110 and 45 kDa. This suggests that the testis-specific cup RNAs encode non-identical but antigenically related Cup proteins (Keyes, 1997).

The protein products of the EMS- and P-derived cup alleles were examined by Western analysis. All but one of the cup alleles affect the quantity, rather than size of the 150 kDa protein, indicating that these alleles are not the products of premature termination. None of the strong cup alleles completely eliminates all immunoreactive protein, suggesting that null alleles have not been recovered. The level of Cup protein detected in these experiments correlates well with the genetic strength of the allele in question: weak alleles produce much more of the 150 kDa Cup protein than strong alleles such. One allele produced a smaller Cup protein of about 135 kDa, further confirming that the gene analyzed corresponds to cup (Keyes, 1997).

Immunofluorescent antibody staining was used to determine the expression pattern and subcellular localization of the Cup protein. Beginning with the stem cells, Cup protein is found in the cytoplasm of all germ-line cells, but was never detected inside either nurse cell or oocyte nuclei. Staining of early embryos confirms that Cup protein is maternally deposited in the egg. The protein is abundant and uniformly distributed in the cytoplasm of all cleavage stage embryos through stage 9, after which signal intensity declines. Staining is effectively absent by gastrulation, and remains so throughout the remainder of embryogenesis (Keyes, 1997).

The subcellular location and distribution of Cup protein within 16-cell cysts undergoes marked changes during the course of oogenesis. Cup protein accumulates preferentially in the future oocyte within 16-cell cysts of the germarium. This enrichment begins very early: localization to a single cell is detectable in region 2a, prior to overt differentiation of the oocyte. Unlike most oocyte-enriched proteins such as Bic-D or Oskar, no corresponding enrichment of cup mRNA was observed. Cup protein may be selectively transported from the nurse cells and/or differentially translated or stabilized within the oocyte. The retention of relatively high levels of Cup protein in the nurse cells suggests that the protein may function in both cell types (Keyes, 1997).

Cup protein continues to be selectively enriched in the oocyte until approximately stage 8, a time when egg chamber microtubules are extensively reorganized. During stage 9 the nurse cells and oocyte contain similar amounts of Cup protein, and by stage 10 most of the protein lies in the nurse cells. Prior to stage 8 Cup forms a cap at the posterior of the oocyte; later much smaller amounts of protein are found at the oocyte surface and also persist in a small cap at the posterior (Keyes, 1997).

The behavior of Cup protein in the nurse cells is particularly interesting. Cup accumulates almost exclusively in large aggregates, whose location varies as egg chambers develop. In early chambers, and especially around stage 4, Cup aggregates are found predominantly around the periphery of the nurse cell nuclei. After stage 4, Cup leaves the nuclear membrane and becomes dispersed throughout the nurse cell cytoplasm in large aggregates that eventually move toward the cellular periphery. By stage 10, Cup protein is localized almost exclusively in particulate structures along the subcortical surface of the nurse cells. The movement of Cup protein away from the nucleus corresponded in time to the sharp reduction in cup mRNA levels after stage 4, suggesting that a decrease in the rate of Cup synthesis might play a role in these changes (Keyes, 1997).

EFFECTS OF MUTATION

The ovarian tumor gene is required in germ-line cells for cyst formation, nurse cell chromosome structure and egg maturation. A gene has been analyzed, fs(2)cup, that participates in many of the same processes and interacts with otu genetically. Both nurse cell and oocyte chromosomes require cup to attain a normal morphology. The cup gene is needed for the oocyte to grow normally by taking up materials transported from the nurse cells. cup encodes a 1132-amino-acid protein containing a putative membrane-spanning domain. Cup protein (but not CUP mRNA) is transported selectively into the oocyte in germarial cysts, like the p104 Otu protein. It is strongly associated with large structures in the cytoplasm and the perinuclear region of nurse cells; like Otu, Cup moves to the periphery of these cells in stages 9-10. Moreover, cup mutations dominantly disrupt meiotic chromosome segregation. It is proposed that cup, otu and another interacting gene, fs(2)B, take part in a common cytoplasmic pathway with multiple functions during oogenesis (Keyes, 1997).

The finding that Cup can bind eIF4E implies that Cup may be involved in the translational control of maternal RNAs. The distribution of the Osk protein was examined in cup mutants, because the Cup-associated protein Me31B has been found to be involved in the repression of osk translation in early oogenesis (Nakamura, 2001). Although the translation of osk RNA is repressed until stage 8 of oogenesis in wild-type ovaries, it is prematurely translated in stage 4-7 egg chambers of several cup mutants, including cup²¹ and cup³². Premature Osk expression was also observed in cup¹/cup¹³⁵⁵ ovaries. These results show that Cup is required for the repression of osk translation during early oogenesis. However, since the egg chambers of these cup mutants start to degenerate at mid-oogenesis, it was not possible to analyze the effects in later oogenesis. Furthermore, the molecular nature of all EMS-induced cup alleles (Keyes, 1997) remains uncharacterized (Nakamura, 2004).

To isolate a molecularly definitive cup mutation, a series of derivative lines were generated by mobilizing the P element in cup⁴⁵⁰⁶. Imprecise excision of the P element causes a small deletion within the cup locus. This line, cup^Δ212, produces a truncated Cup, in which the amino-terminal third of the protein is deleted and the conserved eIF4E binding sequence is disrupted. Immunoprecipitation-Western analyses reveal that Cup^Δ212 protein fails to interact with eIF4E in vivo. Females homozygous for the cup^Δ212 mutation produce eggs, although the eggs are fragile and were not analyzed further. Thus, the cup^Δ212 allele is sufficiently weak to investigate, in detail, the role of Cup in later oogenesis (Nakamura, 2004).

Immunostaining cup^Δ212 ovaries for Osk reveals that osk is prematurely translated starting at early oogenesis, as in the EMS-induced cup mutants. The protein is concentrated in the posterior of the oocyte in stage 4-6 egg chambers. In the stage 8 egg chamber, Osk protein is ectopically concentrated at the anterior of the oocyte. The signal was frequently highest at the anterior-dorsal corner of the oocyte. As oogenesis proceeds, Osk becomes concentrated at the posterior pole of the oocyte. However, large Osk particles remained in the cytoplasm of the oocyte. This type of signal was never observed in wild-type egg chambers. These results show that osk translation is not repressed in the cup^Δ212 oocyte (Nakamura, 2004).

osk RNA distribution was examined by fluorescence in situ hybridization. osk RNA forms cytoplasmic particles, which are especially obvious in early oogenesis. In wild-type ovaries, osk RNA signals are concentrated in the oocyte in early egg chambers, transiently accumulate in the anterior side of the oocyte during stage 7-8, and localize to the posterior pole of the oocyte from stage 8 onward. In cup^Δ212 egg chambers, osk RNA signals show larger particles in the cytoplasm, suggesting that the assembly of osk RNA particles is also affected in the cup^Δ212 egg chambers. However, in spite of the abnormally large osk RNA particles in the cup^Δ212 egg chambers, osk RNA is concentrated in the oocyte in early egg chambers, and at the posterior pole of the oocyte from stage 8 onward. At stage 10, osk RNA is accumulated at the posterior pole of the cup^Δ212 oocyte, although some signal remains in the cytoplasm. Microtubule polarity is normal in cup^Δ212 ovaries. In addition, no defect was found in grk RNA, Grk protein or Bicaudal-D distribution in cup^Δ212 ovaries. Thus, the defects observed in cup^Δ212 ovaries were most striking in their effects on osk translational regulation (Nakamura, 2004).

The spinal muscular atrophy protein SMN affects Drosophila germline nuclear organization through the U body-P body pathway

Survival motor neuron protein (SMN) is the determining factor for the human neurodegenerative disease spinal muscular atrophy (SMA). SMN is critical for small nuclear ribonucleoprotein (snRNP) assembly. Using Drosophila oogenesis as a model system, this study shows that mutations in smn cause abnormal nuclear organization in nurse cells and oocytes. Germline and mitotic clonal analysis reveals that both nurse cells and oocytes require SMN to maintain normal organization of nuclear compartments including chromosomes, nucleoli, Cajal bodies and histone locus bodies. Previous studies found that SMN-containing U bodies invariably associate with P bodies. U bodies are cytoplasmic structures that contain uridine-rich small nuclear ribonucleoproteins and associate with P bodies. Multiple lines of evidence implicate SMN in the regulation of germline nuclear organization through the connection of U bodies and P bodies. Firstly, smn germline clones phenocopy mutations for two P body components, Cup and Ovarian tumour (Otu). Secondly, P body mutations disrupt SMN distribution and the organization of U bodies. Finally, mutations in smn disrupt the function and organization of U bodies and P bodies. Taken together, these results suggest that SMN is required for the functional integrity of the U body-P body pathway, which in turn is important for maintaining proper nuclear architecture (Lee, 2009).

The current findings demonstrate that mutation of the U body component SMN causes disruption of P bodies and exhibits very similar phenotypes to those of P body mutants during Drosophila oogenesis. SMN has been shown to be ubiquitously expressed in all cell types, which correlates with its essential role in fundamental cell processes such as snRNP biogenesis and RNA splicing. However, the expression level of SMN is not uniform among different cell types. This study observe various levels of SMN within Drosophila ovaries. Although SMN is detectable in somatic cells, germline cells in egg chambers show much higher expression of SMN. The differential expression of SMN in somatic cells and germline cells may reflect the different activities of RNA metabolism in these cells. Alternatively, the high levels of SMN in germline cells may act as a store for subsequent use during embryogenesis (Lee, 2009).

Consistent with previous studies, it was found that SMN is mostly distributed in the cytoplasm of nurse cells and oocytes. The concentration of SMN in the ooplasm is similar to that in the nurse cell cytoplasm. However, the distribution of SMN is not uniform subcellularly. SMN is undetectable in the nucleoplasm with one exception -- Cajal bodies are enriched with SMN. In the cytoplasm, concentrated spherical structures known as U bodies can be detected above the bright cytoplasmic background (Lee, 2009).

The enrichment of SMN in the U body and the Cajal body is likely related to snRNP biogenesis since both organelles contain high levels of snRNPs. However, the pattern of SMN is not identical to the pattern of snRNPs. In the nucleus, snRNPs are enriched at sites other than the Cajal body, namely on the chromosomes and in structures that are believed to be sites of splicing, known as speckle. In the cytoplasm, snRNPs are only detectable in U bodies, whereas SMN staining is bright in both the cytoplasm and in U bodies. Overall, the levels of snRNPs in the nucleus are much higher than those in the cytoplasm, while the distribution of SMN is the reverse; higher in the cytoplasm than in the nucleus. Why do snRNP and SMN distributions differ from one another? It is possible that snRNP distribution reflects both snRNP assembly and snRNP activity, whereas SMN reflects only snRNP assembly. Alternatively, SMN may have a function in the cytoplasm that is independent of snRNP assembly (Lee, 2009).

There are four lines of evidence that support the hypothesis that U bodies and P bodies interact with each other. This study, consistent with previous observations, demonstrates that U bodies invariably associate with P bodies, although the absolute number of bodies may vary from cell to cell. However, P bodies are not always found to be associated with U bodies. This may be due to the significantly larger number of P bodies than U bodies in a cell. It is not known whether U body-free P bodies are functionally different from U body-associated P bodies. In some cases, many U bodies are clustered together surrounding one or more P bodies. The association of the U body with the P body is not disrupted even when the size and number of U bodies and/or P bodies change, as observed in many mutants. This suggests that there is a mechanism to maintain the connection between these two specific cytoplasmic domains that is not disrupted in mutants of P body components (Lee, 2009).

Secondly, smnA germline clones display similar nuclear morphology phenotypes to mutants in P body components. Analysis of combined mutations in U bodies and P bodies are underway to address the possible genetic interaction among the components in these two related cytoplasmic structures (Lee, 2009).

Next, it was previously shown that disruption of P body components such as Trailer-hitch or Ago2 leads to changes in U body organization. This study demonstrated that disruption of either one of two other P body components, Cup and Otu, leads to abnormal U body distribution and size. Collectively, these results indicate that P bodies are important for normal organization of U bodies (Lee, 2009).

Finally, using multiple P body markers, this study has shown that P body patterns are altered in cells in which the U body component SMN is disrupted. This suggests that factors in the U body can influence the structure of P bodies (Lee, 2009).

How does SMN regulate nuclear structure and function? It is hypothesized that SMN regulates nuclear organization through the U body-P body (Ub-Pb) pathway, where the U body and the P body, two specialized cytoplasmic domains, work together to regulate a series of downstream events including nuclear organization (Lee, 2009).

U bodies and P bodies associate and interact with each other, but remain physically and characteristically distinct from one another. The results from these experiments suggest it is likely that U bodies and P bodies are interdependent, and that components required by one may be regulated by machinery in the other, or vice versa. Moreover, the normal organization of the U body-P body association may simply reflect the balance between U bodies and P bodies. Any interference with this balance could impair the Ub-Pb pathway, which in turn, may lead to abnormal organization of U bodies and/or P bodies (Lee, 2009).

Both U bodies and P bodies are conserved structures in many cell types in multiple organisms. It would be particularly interesting to see how the Ub-Pb pathway works in other cell types such as neurons. Some human neuronal diseases are determined by factors in U bodies and P bodies. Factors that influence egg chamber development have also been shown to play key roles in the neuron, making the egg chamber an appropriate system in which to investigate the role of SMN. For example, low expression of SMN causes SMA, while Fragile X Syndrome is mainly determined by Fragile X Mental Retardation Protein (FMRP), a P body component. Indeed, a recent study has shown that SMN associates with FMRP in vitro and in the cell (Piazzon, 2008). It is hoped that these more detailed studies of U bodies and P bodies will give new insights into the subcellular and molecular mechanism of human diseases such as Fragile X Syndrome and SMA (Lee, 2009).

The translational repressor Cup is required for germ cell development in Drosophila

In Drosophila, germ cell formation depends on inherited maternal factors localized in the posterior pole region of oocytes and early embryos, known as germ plasm. This study reports that heterozygous cup mutant ovaries and embryos have reduced levels of Staufen (Stau), Oskar (Osk) and Vasa (Vas) proteins at the posterior pole. Moreover, Cup interacts with Osk and Vas to ensure anchoring and/or maintenance of germ plasm particles at the posterior pole of oocytes and early embryos. Homozygous cup mutant embryos have a reduced number of germ cells, compared to heterozygous cup mutants, which, in turn, have fewer germ cells than wild-type embryos. In addition, cup and osk interact genetically, because reducing cup copy number further decreases the total number of germ cells observed in heterozygous osk mutant embryos. Finally, cup mRNA and protein were detected within both early and late embryonic germ cells, suggesting a novel role of Cup during germ cell development in Drosophila (Ottone, 2012).

Germ plasm assembly is a stepwise process occurring during oogenesis. Accumulation of osk mRNA at the posterior of egg chambers is necessary for correct germ plasm assembly, which requires a polarized microtubule network, the plus-end motor kinesin I, and the activity of several genes (cappuccino, spire, par-1. mago nashi, barentz, stau, tsunagi, rab11, and valois). Localization of osk mRNA is strictly linked to the control of its translation, as unlocalized osk mRNA is silent. Upon localization at the posterior pole, the relieve of osk translational repression involves several factors, including Orb, Stau, and Aubergine. Localized Osk protein, in turn, triggers a cascade of events that result in the recruitment of all factors, such as Vas, Tud, and Stau proteins and nanos, germ less mRNAs, necessary for the establishment of functional germline structures (Ottone, 2012).

Posterior anchoring of Osk requires the functions of Vas, as well as Osk itself, to direct proper germ plasm assembly. Misexpression of Osk at the anterior pole of oocytes causes ectopic pole plasm formation, indicating that Osk is the key organizer of pole plasm assembly. Moreover, it has been demonstrated that endocytic pathways acting downstream of Osk regulate F-actin dynamics, which in turn are necessary to attach pole plasm components to the oocyte cortex. As far as Cup is concerned, it has been demonstrated that Cup is engaged in translational repression of unlocalized mRNAs, such as osk, gurken, and cyclinA, during early oogenesis (Ottone, 2012).

The current results establish that Cup is also a novel germ plasm component. First, Cup colocalizes with Osk, Stau, and Vas at the posterior pole of stage 10B oocytes. Second, biochemical evidence indicates that Cup interacts with Stau, Osk, and Vas. Vas localization occurs not through its association with localized RNAs, but rather through the interaction with the Osk protein, which represents an essential step in polar granule assembly (Ottone, 2012).

As a consequence of these interactions, Cup protein is mislocalized in osk and vas mutant stage 10 oocytes, demonstrating that Osk and Vas are essential to achieve a correct localization of Cup at the posterior cortex of stage 10 oocytes. This study suggests that the presence of Cup, Osk, Stau, and Vas are required for a correct germ plasm assembly. Moreover, several immuno-precipitation experiments, using anti- Tud and anti-Vas antibodies, identified numerous P-body related proteins, including Cup, as novel polar granule components (Ottone, 2012).

All the results suggest that Cup plays at least an additional role at stage 10 of oogenesis. Cup, besides repressing translation of unlocalized osk mRNA, is necessary to anchor and/or maintain Stau, Osk, and Vas at the posterior cortex. This novel function of Cup is supported by the findings that, when cup gene dosage is reduced, Stau, Osk, and Vas are partially anchored and/or maintained at the posterior pole, even if these proteins are not degraded. Consequently, pole plasm assembly is disturbed and cup mutant females lay embryos with a reduced number of germ cells. Since the role of Cup, a known multi-functional protein during the different stages of egg chamber development, cannot be easily studied in homozygous cup ovaries, it is not surprising that the involvement of Cup in pole plasm assembly remained undiscovered until now (Ottone, 2012).

During embryogenesis, Cup exerts similar functions. In particular, Osk, Stau, and Vas proteins and osk mRNA are not properly maintained and/or anchored at the posterior pole of embryos laid by heterozygous cup mutant mothers. Surprisingly, osk mRNA is increased in heterozygous cup mutant embryos. Since osk mRNA requires sufficient Osk protein to remain tightly linked at the posterior cortex, the reduced amount of Osk protein observed in heterozygous cup embryos, should be not sufficient to maintain all osk mRNA at the embryonic pole and could stimulate, by positive feedback, de novo osk mRNA synthesis. Also, a direct/indirect involvement of Cup in osk mRNA degradation and/or deadenylation cannot be excluded. The findings that Cup has been found together with Osk, when Osk is ectopically localized to the anterior pole of the embryos, and that reducing cup copy number further decreases the total number of germ cells, observed in heterozygous osk mutant embryos, strengthen the idea that Cup is involved in germ cell formation and/or in maintenance of their identity (Ottone, 2012).

Unlike Osk protein, both cup mRNA and protein were detected within germ cells until the end of embryogenesis. These observations suggest that zygotic cup functions, during germ cell formation and maintenance, are not limited to those carried out in combination with Osk. The finding that homozygous cup mutant embryos display a further decrease of germ cell number, in comparison with heterozygous embryos, supports this hypothesis. Whether or not cup zygotic function is involved in the translational repression of specific mRNAs, different from osk, remains to be explored (Ottone, 2012).

REFERENCES

Search PubMed for articles about Drosophila cup

Arrizabalaga, G. and Lehmann, R. (1999). A selective screen reveals discrete functional domains in Drosophila Nanos. Genetics 153: 1825-1838. PubMed Citation: 10581288

Braat, A. K., et al. (2004). Localization-dependent Oskar protein accumulation control after the initiation of translation. Dev. Cell 7: 125-131. 15239960

Cao, Q. and Richter, J. D. (2002). Dissolution of the maskin-eIF4E complex by cytoplasmic polyadenylation and poly(A)-binding protein controls cyclin B1 mRNA translation and oocyte maturation. EMBO J. 21: 3852-3862. 12110596

Cao, W. X., Kabelitz, S., Gupta, M., Yeung, E., Lin, S., Rammelt, C., Ihling, C., Pekovic, F., Low, T. C. H., Siddiqui, N. U., Cheng, M. H. K., Angers, S., Smibert, C. A., Wuhr, M., Wahle, E. and Lipshitz, H. D. (2020). Precise temporal regulation of post-transcriptional repressors is required for an orderly Drosophila maternal-to-zygotic transition. Cell Rep 31(12): 107783. PubMed ID: 32579915

Castagnetti, S. and Ephrussi, A. (2003). Orb and a long poly(A) tail are required for efficient oskar translation at the posterior pole of the Drosophila oocyte. Development 130: 835-843. 12538512

Chekulaeva, M., Hentze, M. W. and Ephrussi, A. (2006). Bruno acts as a dual repressor of oskar translation, promoting mRNA oligomerization and formation of silencing particles. Cell 124(3): 521-33. 16469699

Clouse, K. N., Ferguson, S. B. and Schüpbach, T. (2008). Squid, Cup, and PABP55B function together to regulate gurken translation in Drosophila. Dev. Biol. 313(2): 713-24. PubMed citation: 18082158

Coller, J. and Parker, R. (2005). General translational repression by activators of mRNA decapping. Cell 122: 875-886. 16179257

Dostie, J., et al. (2000). A novel shuttling protein, 4E-T, mediates the nuclear import of the mRNA 5' cap-binding protein, eIF4E. EMBO J. 19: 3142-3156. 10856257

Dostie, J., Ferraiuolo, M., Pause, A., Adam, S. A., Sonenberg, N. (2000). A novel shuttling protein, 4E-T, mediates the nuclear import of the mRNA 5' cap-binding protein, eIF4E. EMBO J. 19: 3142-3156. 10856257

Gingras, A. C., Raught, B. and Sonenberg, N. (1999). eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68: 913-963. 10872469

Gingras, A. C., Raught, B. and Sonenberg, N. (2001). Regulation of translation initiation by FRAP/mTOR. Genes Dev. 15: 807-826. 11297505

Gunkel, N., Yano, T., Markussen, F. H., Olsen, L. C., and Ephrussi, A. (1998). Localization-dependent translation requires a functional interaction between the 5' and 3' ends of oskar mRNA. Genes Dev. 12: 1652-1664. 9620852

Hara, M., Lourido, S., Petrova, B., Lou, H. J., Von Stetina, J. R., Kashevsky, H., Turk, B. E. and Orr-Weaver, T. L. (2018). Identification of PNG kinase substrates uncovers interactions with the translational repressor TRAL in the oocyte-to-embryo transition. Elife 7. PubMed ID: 29480805

Keyes, L. N. and Spradling, A. C. (1997). The Drosophila gene fs(2)cup interacts with otu to define a cytoplasmic pathway required for the structure and function of germ-line chromosomes. Development 124: 1419-1431. 9118812

Kinkelin, K., Veith, K., Grunwald, M. and Bono, F. (2012). Crystal structure of a minimal eIF4E-Cup complex reveals a general mechanism of eIF4E regulation in translational repression. RNA 18(9): 1624-34. PubMed Citation: 22832024

Igreja, C. and Izaurralde, E. (2011). CUP promotes deadenylation and inhibits decapping of mRNA targets. Genes Dev. 25(18): 1955-67. PubMed Citation: 21937713

Lee, L., Davies, S. E. and Liu, J. L. (2009). The spinal muscular atrophy protein SMN affects Drosophila germline nuclear organization through the U body-P body pathway. Dev Biol 332: 142-155. PubMed ID: 19464282

Mader, S., et al. (1995). The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. Mol. Cell. Biol. 15: 4990-4997. 7651417

Macchi, P., et al. (2003). Barentsz, a new component of the Staufen-containing ribonucleoprotein particles in mammalian cells, interacts with Staufen in an RNA-dependent manner. J. Neurosci. 23: 5778-5788. 12843282

Marcotrigiano, J., Gingras, A. C., Sonenberg, N. and Burley, S. K. (1999). Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G. Mol. Cell 3: 707-716. 10394359

McCambridge, A., Solanki, D., Olchawa, N., Govani, N., Trinidad, J. C. and Gao, M. (2020). Comparative Proteomics Reveal Me31B's Interactome Dynamics, Expression Regulation, and Assembly Mechanism into Germ Granules during Drosophila Germline Development. Sci Rep 10(1): 564. PubMed ID: 31953495

Nakamura, A., Amikura, R., Hanyu, K. and Kobayashi, S. (2001). Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. Development 128: 3233-3242. 11546740

Nakamura, A., Sato, K. and Hanyu-Nakamura, K. (2004). Drosophila Cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis. Dev. Cell 6: 69-78. 14723848

Nelson, M. R., Leidal, A. M. and Smibert, C. A. (2004). Drosophila Cup is an eIF4E-binding protein that functions in Smaug-mediated translational repression. EMBO J. 23: 150-159 . 14685270

Niessing, D., Blanke, S. and Jäckle, H. (2002). Bicoid associates with the 5'-cap-bound complex of caudal mRNA and represses translation. Genes Dev. 16: 2576-2582. 12368268

Ottone, C., et al (2012). The translational repressor Cup is required for germ cell development in Drosophila. J. Cell Sci. 125(Pt 13): 3114-23. PubMed Citation: 22454519

Piazzon, N., Rage, F., Schlotter, F., Moine, H., Branlant, C. and Massenet, S. (2008). In vitro and in cellulo evidences for association of the survival of motor neuron complex with the fragile X mental retardation protein. J Biol Chem 283: 5598-5610. PubMed ID: 18093976

Pyronnet, S., Imataka, H., Gingras, A.C., Fukunaga, R., Hunter, T. and Sonenberg, N. (1999). Human eukaryotic translation initiation factor 4G (eIF4G) recruits Mink1 to phosphorylate eIF4E. EMBO J. 18: 270-279. 9878069

Pyronnet, S., Dostie, J. and Sonenberg, N. (2001). Suppression of cap-dependent translation in mitosis. Genes Dev. 15: 2083-2093. 11511540

Qiao, S., Langlois, C. R., Chrustowicz, J., Sherpa, D., Karayel, O., Hansen, F. M., Beier, V., von Gronau, S., Bollschweiler, D., Schafer, T., Alpi, A. F., Mann, M., Prabu, J. R. and Schulman, B. A. (2020). Interconversion between anticipatory and active GID E3 ubiquitin ligase conformations via metabolically driven substrate receptor assembly. Mol Cell 77(1): 150-163 e159. PubMed ID: 31708416

Schüpbach, T. and Wieschaus, E. (1991). Female sterile mutations on the second chromosome of Drosophila melanogaster. II. Mutations blocking oogenesis or altering egg morphology. Genetics 129: 1119-1136. 1783295

Stebbins-Boaz, B., Cao, Q., de Moor, C. H., Mendez, R. and Richter, J. D. (1999). Maskin is a CPEB-associated factor that transiently interacts with eIF4E. Mol. Cell 4: 1017-1027. 10635326

Tritschler, F., et al. (2008). Similar modes of interaction enable Trailer Hitch and EDC3 to associate with DCP1 and Me31B in distinct protein complexes. Mol. Cell Biol. 28(21): 6695-708. PubMed Citation: 18765641

van Eeden, F.J., et al. (2001). Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole. J. Cell Biol. 154: 511-523. 11481346

Verrotti, A. C. and Wharton, R. P. (2000). Nanos interacts with Cup in the female germline of Drosophila. Development 127: 5225-5232. 11060247

Wang, M., Ly, M., Lugowski, A., Laver, J. D., Lipshitz, H. D., Smibert, C. A. and Rissland, O. S. (2017). ME31B globally represses maternal mRNAs by two distinct mechanisms during the Drosophila maternal-to-zygotic transition. Elife 6. PubMed ID: 28875934

Wilhelm, J. E., et al. (2000). Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes. J. Cell Biol. 148: 427-440. 10662770

Wilhelm, J. E., Hilton, M., Amos, Q. and Henzel, W. J. (2003). Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz. J. Cell Biol. 163: 1197-1204. 14691132

Wilhelm, J. E., Buszczak, M. and Sayles S. (2005). Efficient protein trafficking requires trailer hitch, a component of a ribonucleoprotein complex localized to the ER in Drosophila. Dev. Cell 9(5): 675-85. 16256742

Zappavigna, V., Piccioni, F., Villaescusa, J. C. and Verrotti, A. C. (2004). Cup is a nucleocytoplasmic shuttling protein that interacts with the eukaryotic translation initiation factor 4E to modulate Drosophila ovary development. Proc. Natl. Acad. Sci. 101(41): 14800-5. 15465908

Zavortink, M., Rutt, L. N., Dzitoyeva, S., Henriksen, J. C., Barrington, C., Bilodeau, D. Y., Wang, M., Chen, X. X. L. and Rissland, O. S. (2020). The E2 Marie Kondo and the CTLH E3 ligase clear deposited RNA binding proteins during the maternal-to-zygotic transition. Elife 9. PubMed ID: 32573431

date revised: 25 September 2023

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