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).
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 (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 nosRC 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 nosRC. Each deletion derivative also rescues the abdominal segmentation defects associated with nosBN 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 nosRC mutant females. Cystoblasts that give rise to the germline components of the egg chamber do not develop normally in nosRC mutant ovaries, and germline stem cells that give rise to cystoblasts are not maintained. As a result, nosRC mutant ovaries contain only rare mature egg chambers. In contrast, in cup-/+; nosRC/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-/+; nosRC /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 nosRC allele, but not the embryonic defects. Nine different cup alleles tested suppress the defects associated with nosRC, 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 nosRD 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 nosRC, 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 nosRC (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 nosRC and cup. However, nosRC, 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 nosRC? To address this question, it was asked whether nosRC actually encodes a very low level of functional protein. Semi-quantitative RT-PCR was used to determine whether nosRC 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 nosRC 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 nosRC-encoded protein (Verrotti, 2000).
In an attempt to detect protein encoded by nosRC directly, transgenic flies were prepared bearing a nosRC-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 nosRC-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 nosRC is in the order of 1%-2% of wild type. By crossing the nos+-myc and nosRC-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 nosRC 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 nosRC ovaries. This finding suggests that Cup acts to inhibit the residual protein encoded by nosRC 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 nosRC 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).
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).
Translational regulation plays an essential role in development and often involves factors that interact with sequences in the 3' untranslated region (UTR) of specific mRNAs. For example, Nanos protein at the posterior of the Drosophila embryo directs posterior development, and this localization requires selective translation of posteriorly localized nanos mRNA. Spatial regulation of nanos translation requires Smaug protein bound to the nanos 3' UTR; binding represses the translation of unlocalized nanos transcripts. While the function of 3' UTR-bound translational regulators is, in general, poorly understood, they presumably interact with the basic translation machinery. Smaug is shown to interact with the Cup protein and Cup is an eIF4E-binding protein that blocks the binding of eIF4G to eIF4E. Cup mediates an indirect interaction between Smaug and eIF4E, and Smaug function in vivo requires Cup. Thus, Smaug represses translation via a Cup-dependent block in eIF4G recruitment (Nelson, 2004),
To understand the mechanisms that underlie Smg's ability to repress translation, attempts were made to identify Smg-binding proteins. Initial work focused on proteins that would interact with amino acids 583-763. This region contains the Smg SAM domain, which is the protein's RNA-binding domain. An affinity resin carrying covalently coupled GST-Smg583-763 was mixed with early embryo extracts. After extensive washing, bound proteins were eluted and detected via silver staining following SDS-PAGE. Several proteins were eluted from both the GST-Smg583-763 resin and a resin carrying covalently coupled GST-Smg179-307. However, an `80 kDa protein and an `140 kDa protein were specifically eluted from the Smg583-763 resin. Both proteins were subjected to MALDI-TOF mass spectrometry, and while the smaller protein was not identified the larger was identified as Cup, which plays an essential but ill-defined role during oogenesis and early embryogenesis. To confirm that Cup interacts with Smg583-763, Cup was generated via in vitro translation in rabbit reticulocyte lysate. This protein interacted with GST-Smg583-763, as assayed by capture of Cup on glutathione agarose in the presence of GST-Smg583-763 (Nelson, 2004),
Biochemical and genetic evidence is presented that is consistent with Cup functioning as an eIF4E-binding protein that mediates an interaction between Smg and eIF4E. Cup blocks the eIF4E/eIF4G interaction, suggesting that Smg-dependent translational repression of SRE-containing mRNAs results from a Cup-mediated block in the recruitment of eIF4G. Cup's role in Smg function is therefore similar to that played by Maskin in translational repression mediated by CPEB. Given that Maskin and Cup are not homologous, this suggests that other undiscovered adaptor eIF4E-binding protein/3' UTR-binding protein pairs will employ this mechanism to regulate translation (Nelson, 2004),
Cup interacts with eIF4E using both an eIF4E-binding motif and a second site that interacts with eIF4E through a distinct mechanism. Despite this difference, the second site is still able to inhibit the eIF4E/eIF4G interaction in vitro. Further work will be required to assess the significance of this site to Cup function in vivo (Nelson, 2004),
This model for Cup suggests that Smg represses translation at the level of initiation. However, the association of repressed nos mRNA with polysomes indicates that translational repression is achieved at a step after initiation. This apparent contradiction may reflect the fact that repression of nos translation is mediated by at least two trans-acting factors: Smg and a yet to be identified factor that functions through sequences in the nos 3' UTR that are distinct from the SREs. Thus, while Smg regulates translation at the level of initiation, additional factors may function at other levels. Similarly, Smg itself may utilize multiple mechanisms to repress nos expression, only one of which is Cup dependent (Nelson, 2004),
Regulation of translation during development often involves both translational repression and translational activation. The combination of these controls can spatially or temporally restrict the expression of an mRNA, thereby directing the proper development of a cell type or tissue. For example, nos translation is spatially regulated allowing for the proper development of the posterior of the Drosophila embryo. Smg plays an essential role in this process by repressing the translation of unlocalized nos mRNA, while nos mRNA localized to the posterior escapes this repression allowing for the accumulation of Nos protein specifically at the posterior. Given that Smg protein is distributed throughout the embryo, this suggests that Smg function must be over-ridden at the posterior. Cup is also distributed throughout the embryo, suggesting that spatial regulation of nos translation may involve disrupting Cup and/or Smg function specifically at the posterior. Osk protein, which is localized to the posterior, is required for nos translation and Osk interacts with Smg. Thus translational activation could involve Osk binding to Smg thereby blocking Smg function. Interestingly, Cup and Osk interact with the same region of the Smg protein. This might imply that Osk's interaction with Smg could disrupt the Cup/Smg complex and in so doing play a role in activating nos translation at the posterior (Nelson, 2004),
In Xenopus, temporal regulation of translation involves Maskin-mediated repression of target mRNAs in immature oocytes. Upon oocyte maturation, this repression is disrupted resulting in the activation of translation . This activation of translation involves a CPEB-mediated increase in the length of the transcript's poly(A) tail and subsequent recruitment of poly(A)-binding protein (PABP) to the message. PABP brings eIF4G to the mRNA, which in turn disrupts the Maskin/eIF4E complex resulting in translational activation. Measurement of the length of the nos poly(A) tail suggests that regulation of nos translation does not involve changes in poly(A) tail length. Thus, activation of nos translation does not likely involve disruption of the Cup/eIF4E complex through poly(A)-dependent eIF4G recruitment. Taken together, these results also suggest that the use of adaptor proteins such as Cup in translational regulation mediated by sequence-specific RNA-binding proteins is not restricted to mRNAs whose translation is regulated through their poly(A) tail (Nelson, 2004),
The data demonstrate that the same region of Smg that has previously been shown to function in sequence-specific RNA binding also interacts with Cup. The model therefore suggests that this region of the protein would be sufficient to repress translation. However, a transgene that expresses the Smg RNA-binding domain plus a short carboxy-terminal extension fails to rescue the smg mutant phenotype. These results would suggest that Smg has other essential functions in the early embryo in addition to Cup-dependent translational repression. Smg has been suggested to induce the degradation of target mRNAs in a process that may be distinct from its ability to repress translation. Perhaps this ability to induce mRNA degradation is essential and requires regions of Smg outside of amino acids 583-763 (Nelson, 2004),
Phenotypic analysis of several cup mutant alleles highlights Cup's involvement in a number of different biological processes during oogenesis and early embryogenesis, including oocyte growth, maintenance of chromosome morphology, and the establishment of egg chamber polarity. However, the molecular mechanisms that underlie Cup function have not been characterized. The demonstration that Cup is an eIF4E-binding protein suggests that at least some of the defects associated with mutations in the cup gene result from misregulation of translation. Consistent with this possibility is the fact that Cup has been previously shown to interact with Nos protein, which is itself a translational repressor. Genetic experiments suggest that Cup negatively regulates Nos activity during oogenesis, but the molecular mechanisms are not understood. This contrasts with Cup's positive effect on Smg-mediated translational repression. Thus Cup might utilize different molecular mechanisms to influence different translational repressors. The pleiotropic nature of the cup mutant phenotype suggests that Cup may serve as an adaptor protein that is utilized by multiple translational repressors to interact with eIF4E (Nelson, 2004),
Cup is homologous to 4E-T, a human nucleocytoplasmic shuttling protein that employs an eIF4E-binding motif to transport eIF4E into the nucleus (Dostie, 2000). The similarity between these proteins may suggest that Cup also functions to transport eIF4E into the nucleus. Thus some of the phenotypes associated with cup mutants may be related to a defect in eIF4E shuttling during oogenesis. The similarity between Cup and 4E-T also suggests that 4E-T might function in translational repression as an adaptor protein that mediates interactions between eIF4E- and 3' UTR-binding proteins. Specifically, 4E-T could function in translational repression mediated by the human Smg homolog. Similarly, additional RNA-binding proteins that interact with other eIF4E-binding proteins could function to regulate translation spatially or temporally. These protein pairs could control the translation of different mRNAs in various cell types throughout development (Nelson, 2004),
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).
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 cupΔ212 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).
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).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.