arrest

Protein Interactions

Oskar protein directs the deployment of Nanos, the posterior body-patterning morphogen in Drosophila. To avoid inappropriate activation of nos, osk activity must appear only at the posterior pole of the oocyte, where the OSK mRNA becomes localized during oogenesis. Translation of OSK mRNA is, and must be, repressed prior to its localization; absence of repression allows Osk protein to accumulate throughout the oocyte, specifying posterior body patterning throughout the embryo. Translational repression is mediated by an ovarian protein, Bruno, that binds specifically to Bruno response elements (BREs), present in multiple copies in the OSK mRNA 3'UTR. Addition of BREs to a heterologous mRNA renders it sensitive to translational repression in the ovary (Kim-Ha, 1995).

Bruno physically interacts with Vasa. Repression of translation by Bruno is alleviated once OSK mRNA is localized to the posterior pole of the oocyte. The mechanism of this process is unknown; however, it seems likely that the RNA helicase Vasa is involved, since it is localized to the posterior pole of the oocyte and is required for efficient activation of OSK translation. Immune sera, reactive to Bruno protein, cause a shift in the electrophoretic mobility of Osk protein (Webster, 1997).

The precise restriction of proteins to specific domains within a cell plays an important role in early development and differentiation. An efficient way to localize and concentrate proteins is by localization of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination. A central issue is how localized mRNAs are derepressed. Regulatory elements for both RNA localization and translational repression are situated in the 3' UTR of OSK mRNA, as they are in NOS. In the case of OSK, premature translation is prevented by Bruno, a 68-kD protein encoded by the arrest (aret) locus. Bruno recognizes a repeated conserved sequence (BRE, for Bruno response element) in the osk 3' UTR, and colocalizes with OSK mRNA to the posterior pole. In contrast to NOS, however, 3' UTR-mediated localization at the posterior pole is not sufficient for translation, as heterologous transcripts localized under the control of the full-length OSK 3' UTR are not translated. This indicates that the OSK 3' UTR, although it may participate, is not sufficient for translational activation, and that sequences elsewhere in the transcript are required for translation of OSK mRNA (Gunkel, 1998).

When OSK mRNA reaches the posterior pole of the Drosophila oocyte, its translation is derepressed by an active process that requires a specific element in the 5' region of the mRNA. This novel type of element is a translational derepressor element, whose functional interaction with the previously identified repressor region in the OSK 3' UTR is required for activation of Oskar mRNA translation at the posterior pole. The derepressor element only functions at the posterior pole, suggesting that a locally restricted interaction between trans-acting factors and the derepressor element may be the link between mRNA localization and translational activation. Specific interaction of two proteins with the OSK mRNA 5' region is shown; one of these also recognizes the 3' repressor element. p50 is a BRE binding protein that recognizes 3' repressor motifs similar to those recognized by Bruno. p50 functions as a second translational repressor independent of Bruno. The involvement of a second repressor protein in OSK translational control is not unexpected. Indeed, aubergine (aub), a gene required for efficient OSK mRNA translation, is required even when Bruno-mediated repression is alleviated by mutations in the BRE, leading to the suggestion that the aub gene product enhances translation by counteracting the action of a second repressor. It is interesting to note that the requirement for aub function in OSK translation is conferred not only by the OSK 3' UTR but also involves the 5' end of OSK mRNA. Consistent with this possible involvement of the OSK 5' end in translational repression, it is found that in transgenic flies containing an inefficient BRE, premature translation increases when the 5' end is truncated. Understanding the extent to which the 5' end of the OSKtranscript might contribute to overall translational repression will require mutations that selectively disrupt 5' repressor function without simultaneously affecting derepressor function (Gunkel, 1998).

The second protein interacting with the 5' end, p68, could act as a transcriptional activator. p68 is shown to be independent of Bruno. So far it has not been possible to define a p50-binding specificity distinct from that of p68 and to abolish selectively the binding of one or the other protein. Hence, the data do not allow the affirmation that p50 functions as a repressor, not only by binding to the BRE, but also through its interaction with the OSK 5', or that p68 is the derepressor protein. There are several mechanisms by which OSK could be activated at the posterior pole. The translation repressor proteins Bruno and p50 could be degraded by an activity localized at the posterior pole or else be displaced competitively by a derepressor protein. Alternatively, Oskar protein expression could be activated by concentration of the mRNA, resulting in the accumulation of small amounts of Oskar protein by leaky translation, thus initiating a positive feedback loop in which Oskar protein stimulates its own translation. None of these mechanisms is involved in the initial event of translational derepression. In the absence of the 5' derepressor element, OSK transcripts remain repressed, arguing against a passive, local repressor inactivation model. Therefore, the mode of action of the derepressor element is distinct from that of previously described cases, in which repression is released passively by inactivation of a repressor protein and no additional RNA elements are required. The derepressor element does not coincide with the BRE, suggesting that a competitive displacement of the repressor protein from the BRE is unlikely to be the mechanism leading to derepression. Finally, a combination of leaky translation and positive feedback of Oskar protein on its own translation as a mechanism for derepression is unlikely, as reporter transcripts can be derepressed in the absence of endogenous Oskar. Thus mechanisms by which 3' UTR-binding proteins repress translation are still not understood and it is unclear how the 5' derepressor element overcomes translational repression. The fact that transcripts lacking the derepressor element are localized but not translated demonstrates that the element plays little or no role in RNA localization and that localization does not suffice for translational derepression (Gunkel, 1998).

Translational recruitment of OSK mRNA is always accompanied by posterior localization of the mRNA, indicating that localization may trigger the release from translational repression. It is suggested that RNA localization directs osk transcripts into a cytoplasmic subcompartment containing trans-acting factors that interact specifically with the 5' element to mediate derepression. The spatial restriction of the derepression machinery could be achieved by prelocalization of at least some of the components to the posterior pole, or by the localized activation of uniformly distributed factors. During the early stages of oogenesis, OSK mRNA initially fills the entire cytoplasm of the growing oocyte and yet no Oskar protein is detected, even in the posterior region. This suggests that the derepressor proteins are expressed or activated only at certain stages of oocyte development, possibly through signals from the posterior pole. The existence of localized derepressors is supported by the observation that reporter transcripts bearing the BCD 3' UTR into which the OSK repressor element is inserted are localized to the anterior ofoocytes of embryos and not derepressed, even when they contain the derepressor element. The DEAD-box RNA helicase Vasa (whose SDS-PAGE mobility is similar to that of p68), the 120-kD double-stranded RNA-binding protein Staufen, and Aubergine, whose gene has not yet been cloned, play a role in the translation of OSK mRNA. On the basis of the data presented in this report, Staufen and Aubergine could be required to overcome p50-mediated repression, as both are necessary for osk translation, even in the absence of BRE-mediated repression (Gunkel, 1998).

The coupled regulation of Oskar mRNA localization and translation in time and space is critical for correct anteroposterior patterning of the Drosophila embryo. Localization-dependent translation of Oskar mRNA, a mechanism whereby Oskar RNA localized at the posterior of the oocyte is selectively translated and the unlocalized RNA remains in a translationally repressed state, ensures that Oskar activity is present exclusively at the posterior pole. Genetic experiments indicate that translational repression involves the binding of Bruno protein to multiple sites, the Bruno Response Elements (BRE), in the 3' untranslated region (UTR) of Oskar mRNA. A cell-free translation system, derived from Drosophila ovaries, has been established that faithfully reproduces critical features of mRNA translation in vivo, namely cap structure and poly(A) tail dependence. This ovary extract, containing endogenous Bruno, is able to recapitulate Oskar mRNA regulation in a BRE-dependent way. Thus, the assembly of a ribonucleoprotein (RNP) complex leading to the translationally repressed state occurs in vitro. Moreover, a Drosophila embryo extract lacking Bruno efficiently translates Oskar mRNA. Addition of recombinant Bruno to this extract establishes the repressed state in a BRE-dependent manner, providing a direct biochemical demonstration of the critical role of Bruno in Oskar mRNA translation. This approach opens new avenues to investigate translational regulation in Drosophila oogenesis at a biochemical level (Castagnetti, 2000).

The product of the oskar gene directs posterior patterning in the Drosophila oocyte, where it must be deployed specifically at the posterior pole. Proper expression relies on the coordinated localization and translational control of the Oskar mRNA. Translational repression prior to localization of the transcript is mediated, in part, by the Bruno protein, which binds to discrete sites in the 3' untranslated region of the Oskar mRNA. To begin to understand how Bruno acts in translational repression, a yeast two-hybrid screen was performed to identify Bruno-interacting proteins. One interactor, described here, is the product of the apontic gene. Expression occurs in both the somatic follicle cells and the germline nurse cells and oocyte. APT transcripts are detected as early as stage 2A at low levels in the germarium and at higher levels in the follicle cells. The amount of APT mRNA in the soma decreases during the remainder of oogenesis, while the level in the germline increases. APT mRNA becomes concentrated in the oocyte and also accumulates in the nurse cells at about stage 6. APT transcripts continue to be found in both the oocyte and nurse cells throughout oogenesis. To determine when and where Apt protein is expressed during oogenesis, antisera directed against a recombinant Apt protein were prepared and used for protein detection in whole-mount ovaries by confocal microscopy. Apt protein appears in both the germline and somatic cells of the ovary throughout all stages of oogenesis. In the germline, Apt protein is present in both cytoplasm and nuclei. Within the nurse cells the protein is more concentrated in the cytoplasm, while in the oocyte more protein is found in the nucleus. The protein, however, is not localized to any subdomain within the cytoplasm of either the nurse cells or the oocyte. Although Apt protein is not strictly nuclear or cytoplasmic in cells of the female germline, the protein is highly concentrated in nuclei of the ovarian follicle cells and in post-cellularization-stage embryos. The developmental differences in subcellular location suggest that Apt may have functions, perhaps different, in both nuclei and cytoplasm. Nuclear proteins expressed from maternal mRNAs are sometimes present at high levels in the cytoplasm of early embryos. Examples include the Bicoid, Caudal and Hunchback proteins, which appear in both nuclei and cytoplasm shortly after egg laying. As nuclear divisions progress and the density of nuclei increases, nuclear localization of these proteins remains strong while the fraction of protein in the cytoplasm diminishes. Thus there appears to be no early impediment to nuclear localization, simply a paucity of nuclei. In contrast, the subcellular distribution of Apt protein appears to be actively controlled in early development. Apt protein was monitored in early embryos. Even after migration of nuclei to the surface of the embryo, Apt protein remains evenly distributed between nuclei and cytoplasm, unlike any of the examples described above. This unusual persistence of Apt protein in the cytoplasm suggests the existence of a mechanism to control its distribution, reinforcing the notion of roles for Apt in both cytoplasm and nuclei (Lie, 1999a).

Apt is an RNA binding protein. Remarkably, the regions of the OSK 3' UTR bound by Apt, the AB and C regions, are precisely those bound by Bru. A test of Apt binding was performed to determine if Bru and Apt have the same RNA binding specificity: a series of RNAs was used to map the Bru binding sites, called BREs, within the OSK C region. Three of these RNAs retain the BREs and are bound by Bru, while a fourth RNA, C�4, lacks the BREs and fails to bind Bru. Apt binds all four RNAs, including C�4, indicating that Apt can bind to sites other than BREs (Lie, 1999a).

Coimmunoprecipitation experiments lend biochemical support to the idea that Bruno and Apontic proteins physically interact in Drosophila. Genetic experiments using mutants defective in apontic and bruno reveal a functional interaction between these genes. arrest (aret) mutants are defective for Bru (i.e. aret and bru are the same gene and lead to a developmental arrest early in oogenesis. Mutants in apt are zygotic lethal, and some alleles also cause arrested oogenesis. Several different apt alleles were used for all analyses, since the genetics of apt are complex and different alleles have different effects. Testing for a genetic interaction between the aret and apt mutants, dosages of both genes were reduced to see if this would provide a distinct phenotype. Females heterozygous for aret, heterozygous for any of the five apt alleles, or transheterozygous for both aret and an apt allele, were crossed to wild-type males, and the progeny embryos were then examined for cuticular defects. Females transheterozygous for aret and apt produce a fraction of embryos with head defects. Head defects can result from ectopic or excessive posterior body patterning activity, because this activity interferes with expression of the anterior body patterning morphogen, Bicoid. Consequently, the observed head defects could be explained if both Bru and Apt contribute to repression of OSK mRNA translation. Given this interaction, Apontic is likely to act together with Bruno in translational repression of Oskar mRNA (Lie, 1999a).

Earlier genetic analyses of apt have concentrated on the zygotic phenotype. To define more completely the role of apt in the female germline, females were created with apt minus germline clones using the FLP/DFS method. Ovaries containing germline clones were dissected, stained with DAPI to highlight nuclei, and examined for phenotype. Different apt mutants display dramatically different ovarian phenotypes. One allele is indistinguishable from wild type, because females with germline clones of this allele have phenotypically wild-type ovaries and lay eggs that develop into fertile adults. Females with germline clones of another allele also have phenotypically wild-type ovaries, but a small fraction of the eggs laid developed into embryos with head defects. In contrast, ovaries from females with germline clones from two other mutants have phenotypes that are similar to one another and severe: development is arrested in early oogenesis, and the oocyte fails to differentiate, with all nuclei becoming polyploid. In addition, some of the egg chambers have an abnormal number of nuclei. It is concluded that apt is necessary for oogenesis and that loss of apt activity leads to a developmental arrest during oogenesis. Just as for aret mutants, the arrest occurs too early to allow the ovaries to be examined for defects in OSK mRNA translation (Lie, 1999a).

Interestingly, Apontic, like Bruno, is an RNA-binding protein and specifically binds certain regions of the Oskar mRNA 3' untranslated region. A sequence shared by all of the bound RNAs could not be identified. Thus, despite its ability to efficiently discriminate between different parts of the OSK mRNA, Apt appears to be relatively promiscuous in its binding and may recognize many sites or perhaps a structural feature common to many RNAs (Lie, 1999a).

Translational regulation plays a prominent role in Drosophila body patterning. Progress in elucidating the underlying mechanisms has been limited by the lack of a homologous in vitro system that supports regulation. Global control over all transcripts can be achieved through changes in the activity of the translation machinery. More specific controls often rely on cis-acting regulatory elements present within mRNAs, in many cases in their 3' untranslated regions (3' UTRs). Extracts prepared from Drosophila tissues are competent for translation. Ovarian extracts, but not embryonic extracts, support the Bruno response element-dependent and Bruno-dependent repression of Oskar mRNA translation, which acts in vivo to prevent protein synthesis from transcripts not localized to the posterior pole of the oocyte. Consistent with suggestive evidence from in vivo experiments, regulation in vitro does not involve changes in poly(A) tail length. Moreover, inhibition studies strongly suggest that repression does not interfere with the process of 5' cap recognition. Translational regulation mediated through the Bruno response element is thus likely to occur via a novel mechanism (Lie, 1999b).

Two types of experiments were performed to explore the possibility that OSK translational regulation occurs by changes in the poly(A) tail. First, a direct assay was used to determine the length of the poly(A) tail on OSK ovarian transcripts. The poly(A) tail is short, suggesting that polyadenylation is unlikely to function in regulating translation of OSK mRNA. Second, the distribution of poly(A)-binding protein (PABP) in the Drosophila ovary was determined. Although PABP is present at high levels in the germline cells early in oogenesis, it is noticeably depleted from the oocyte at the stage when OSK mRNA is localized to the posterior pole and translationally activated. Although these experiments are suggestive, neither observation provides a compelling argument against a role for polyadenylation in translational activation. For example, the poly(A) tail could act in a manner not requiring PABP. Furthermore, the fraction of translationally active OSK mRNA may be small, as observed for NOS mRNA, making it difficult to detect the presence of transcripts with longer poly(A) tails. To facilitate biochemical analysis of translational control in Drosophila and definitively address the role of polyadenylation in regulation of OSK mRNA translation, in vitro translation systems were developed from Drosophila tissues. Although similar systems have been prepared from other sources, they are unlikely to contain the factors necessary for specific translational regulation of Drosophila mRNAs. Translational activity of the extracts was monitored using reporter mRNAs encoding luciferase (Lie, 1999b).

The ability of Drosophila extracts to recapitulate regulated translation was tested using the 3' UTR of the OSK mRNA, which contains the BRE control elements that mediate Bru-dependent translational repression. The luciferase (luc) reporter mRNA was modified by addition of the following sequences to its 3' end: either the wild-type OSK 3' UTR (BRE+) or a point-mutated version of the OSK 3' UTR (BRE-) that is unable to bind Bru protein in vitro and fails to support translational repression in vivo. In embryo extracts, as well as reticulocyte lysates, both mRNAs were translated with similar efficiencies, revealing no inherent differences in their abilities to be translated. In contrast, translation of the two mRNAs is markedly different in ovary extracts: the luc BRE+ message is translationally repressed approximately 9-fold, on average, in comparison to the luc BRE- message. These results indicate that the ovary extract supports specific regulation of translation (Lie, 1999b).

To determine whether the dependence of regulated translation on BREs reflects a requirement for Bru, two related experiments were performed. First, ovarian extracts were immunodepleted using anti-Bru antibodies or antibodies purified from normal rat serum and were then tested for translational activity. Notably, the control antibodies have no effect on the relative levels of BRE+ and BRE- translation, but depletion with anti-Bru antibodies largely eliminates BRE-dependent repression. Purified recombinant Bru protein was added to the immunodepleted extracts but was not sufficient to restore translational regulation activity. This result suggests that, in addition to removing Bru, immunodepletion using anti-Bru antibodies may remove other proteins required for BRE-mediated translational regulation. Indeed, a significant fraction of Bru protein is present in a large macromolecular complex. In a second type of experiment, purified Bru was added to embryonic extracts that lack Bru and do not support BRE-mediated regulation of translation. Thus BRE-dependent translational repression in vitro requires Bru. Although addition of Bru to embryo extract promotes BRE-dependent repression, the relative translation of BRE- versus BRE+ RNAs is less (average 3.2-fold) than that measured in ovary extracts (average 9.2-fold). There are at least two likely explanations for this difference: the recombinant Bru protein may not be fully active, although the protein displays RNA-binding activity indistinguishable from that of ovarian Bru; alternatively, additional protein(s) present in ovaries but not in embryos may also contribute to repression and may be required for wild-type levels of activity (Lie, 1999b).

The use of BRE+ and BRE- mRNAs reveals the importance of BREs for translational regulation, but does not address the possible role of other sequences in the OSK mRNA 3' UTR. To determine if BREs alone are sufficient, luc reporter mRNAs bearing multimerized consensus BREs (8 copies), either wild-type or containing point mutations that abrogate Bru binding, were generated and their translation measured in vitro. The BRE+ and BRE- RNAs were translated with only a modest difference in efficiency, suggesting that binding of Bru alone is insufficient for complete repression. The 8x BRE supports translational repression in vivo, but repression is not efficient, consistent with the results of the more quantitative in vitro assay. In contrast, RNAs containing the full OSK AB region (a 124 nt region of the OSK mRNA 3' UTR containing at least four consensus BREs interspersed among other sequences) were translationally regulated with a much higher efficiency. Thus it appears that other cis-acting sequences and presumably other factors contribute to Bru-mediated translational repression, a result consistent with the incomplete repression conferred by the addition of Bru to embryonic extracts (Lie, 1999b).

To begin to explore the mechanism of Bru-mediated repression, the in vitro system was used to rigorously test the role of the poly(A) tail in translational regulation, as well as the role of the 5' cap. A number of maternal mRNAs in Xenopus have been shown to be translationally regulated in a manner involving changes in the length of the poly(A) tail (e.g. cyclin mRNAs and c-mos mRNA), and RNA control elements and proteins that contribute to this process have been identified (e.g. the cytoplasmic polyadenylation element (CPE) and the CPE-binding protein (CPEB). Similar forms of regulation have been reported for maternal mRNAs from other animals such as Bicoid and Hunchback mRNAs in Drosophila and tPA and c-mos mRNAs in mouse, and for mRNAs from somatic tissues such as alpha-CaMKII mRNA. To specifically study the requirement for the poly(A) tail in OSK mRNA translational regulation, BRE+ and BRE- reporter RNAs that lacked poly(A) tails were tested in the in vitro system. The translation of these RNAs was regulated in a manner similar to that of transcripts with a poly(A) tail. Though the overall level of translation from the poly(A)-messages was slightly reduced, BRE-dependent repression remains strong. To address the possibility that reporter mRNAs might be polyadenylated during the course of the reaction, two types of experiments were performed using poly(A)- reporter RNAs bearing only the 5' portion of the OSK 3' UTR, including the AB region. This part of the OSK 3' UTR lacks all of the normal polyadenylation elements, including sequences that resemble the binding site for Xenopus CPEB, a factor involved in cytoplasmic polyadenylation. Initially, the translational regulation of these reporter mRNAs was tested and found to be similar to that of RNAs containing the intact 3' UTR. Then the lengths of reporter mRNAs were monitored over the course of incubation in the in vitro translation extract: no changes in size were observed, confirming that polyadenylation does not occur in vitro. It is concluded from these data that translational regulation of OSK mRNA in vitro is independent of the poly(A) tail and that regulation occurs via a novel mechanism (Lie, 1999b).

Another mRNA feature often implicated in control events is the 5' cap. Cap-dependent translational initiation requires recognition of the cap by the eIF4F complex (or its constituent components), a process that is modulated by several types of regulation. A simple test of the importance of the cap in Bru-mediated regulation is to monitor relative translation efficiencies of BRE+ and BRE- mRNAs lacking cap structures. However, uncapped mRNAs are quite unstable in the extracts. An alternative approach is to inhibit cap-dependent initiation by the addition of excess free cap analog (7-methyl-GpppG). In a preliminary experiment, it was shown that translation in the extracts can be substantially inhibited by free cap analog and is thus largely cap-dependent. To test for cap-dependence of Bru regulation, translation of BRE+ and BRE- mRNAs was tested in the presence of free cap. If Bru interferes with recognition or use of the cap, the levels of BRE- and BRE+ translation would be expected to equalize under conditions where cap-dependent initiation is inhibited. Notably, the ratio of BRE-/BRE+ translation remains similar under all conditions tested, ranging from 0 to 2 mM cap competitor. These results very strongly suggest that Bru interferes with a step in translation that is distinct from cap recognition. Additional evidence supporting this conclusion could come from examination of dicistronic mRNAs, in which translation of one encoded protein is initiated in a cap-independent pathway through the use of an internal ribosome entry site (IRES). However, such an experiment must await the identification of an IRES that is active in Drosophila ovaries (Lie, 1999b).

Prior analysis of OSK mRNA translational regulation has provided strong but indirect evidence that Bru acts as a repressor. Molecular genetic data reveals the essential role for BREs, while genetic evidence demonstrates that Bru acts in controlling the level of OSK activity. However, the complex phenotype of mutants defective in Bru prevented a direct demonstration that the absence of Bru leads to a derepression of OSK translation. The results of the in vitro studies now provide compelling evidence that Bru is in fact required for translational repression mediated through the BREs. How the binding of Bru to the 3' UTR of OSK mRNA leads to translational repression remains uncertain, although the availability of the in vitro system defined here is likely to prove useful in addressing that question. Indeed, a definitive demonstration that the 5' cap and changes in poly(A) tail length are not involved in regulation were only made possible through use of this system (Lie, 1999b).

The Drosophila gene squid (sqd) encodes a heterogeneous nuclear RNA binding protein (hnRNP), also known as hrp40. hnRNPs are a large family of proteins that have been implicated in the processing of nascent mRNA transcripts. Recent studies have demonstrated that a subset of hnRNPs, including human hnRNP A1 and A2, Saccharomyces cerevisiae Nplp3 and Hrp1, and Chironomus tentans Hrp36, rapidly shuttle between the cytoplasm and the nucleus. A specific motif, termed M9, has been shown to mediate this nucleocytoplasmic shuttling, and this motif is present in Sqd. Nuclear import of M9-containing hnRNPs is achieved by an association with the nuclear import protein Transportin. Studies of several of these hnRNPs have indicated that one of their major roles is the nuclear export of mRNAs, suggesting that Sqd may perform a similar function during Drosophila oogenesis (Norvell, 1999 and references).

Sqd protein is detected within the somatic follicle cells and the germ-line-derived nurse cells and oocyte. A germ-line mutation in a squid causes female sterility as a result of mislocalization of Gurken (GRK) mRNA during oogenesis. Alternative splicing produces three isoforms: SqdA, SqdB, and SqdS. These isoforms are not equivalent; SqdA and SqdS perform overlapping but nonidentical functions in GRK mRNA localization and protein accumulation, whereas SqdB cannot perform these functions. Furthermore, although all three Sqd isoforms are expressed in the germline cells of the ovary, they display distinct intracellular distributions. Both SqdB and SqdS are detected in germ-line nuclei, whereas SqdA is predominantly cytoplasmic. It is argued that SqdS is involved in the transport and localization of GRK mRNA. The ability of SqdA to prevent translation of ventrally localized GRK mRNA and its ability to provide peak levels of Grk protein required on the dorsal side of the egg chamber, strongly suggests that SqdA has a role in the accumulation of Grk protein. Moreover, the role of SqdA is both positive and negative, suggesting that SqdA may influence the association of GRK mRNA with appropriate translational regulators (Norvell, 1999).

Evidence is provided that GRK mRNA localization and translation are coupled by an interaction between Sqd and the translational repressor protein Bruno. Because Sqd protein binds GRK mRNA directly and belongs to the class of hnRNPs implicated in nuclear mRNA export, it seems likely that Sqd functions in the nuclear export of GRK mRNA. One complication to this model is that in the sqd mutant, GRK mRNA is still able to leave the nucleus and accumulate in the oocyte cytoplasm but not in the dorso-anterior corner. Therefore, the function of the Sqd protein appears to be in the regulated nuclear export of GRK mRNA, such that Sqd is responsible for delivering the GRK message to a cytoplasmic protein involved in its anchoring, possibly coupled to translation. Thus, one might expect that Sqd protein should interact with some cytoplasmic ovarian proteins (Norvell, 1999).

A number of proteins have been implicated in the translational regulation of GRK mRNA, both positively (e.g., encore and Vasa) and negatively (e.g., Bruno). Therefore, an investigation was carried out to see whether Sqd protein could directly associate with any of these candidates. Using the in vitro association assay, interactions were sought between the Sqd isoforms and Encore, Vasa, or Bruno. Although a direct interaction between Sqd and Encore or Vasa could not be found, Sqd protein associates with Bruno protein in vitro. Although the Sqd-Bruno interaction observed in vitro is not extremely strong, it is very consistently observed over multiple experiments. These data show that Sqd protein and Bruno protein can associate with one another. Moreover, these data provide evidence for a link between GRK mRNA localization and translational regulation (Norvell, 1999).

In addition to its requirement in oogenesis, Sqd is also required somatically. A number of lethal alleles of sqd were generated and the ability of the individual isoforms to restore viability of sqd null alleles was investigated. Again, as with the ability of the specific isoforms to function during oogenesis, the three Sqd isoforms differ in their ability to rescue the viability of a lethal sqd allelic combination. Both SqdS and SqdB were capable of rescuing the essential somatic Sqd function: expression of either of these transgenes allows recovery of 11% and 19% of the expected number of mutant sqd adults, respectively. In contrast, however, SqdA is incapable of restoring the essential somatic function of Sqd, since less than 0.2% of the expected number of sqd adults were recovered. These data further demonstrate that the individual Sqd isoforms are not functionally equivalent (Norvell, 1999).

At least two lines of evidence indicate that Sqd protein must be present within the oocyte nucleus for GRK mRNA to be localized properly during oogenesis. (1) Oof the three Sqd isoforms, only SqdS is detected within the oocyte nucleus; among the Sqd transgenic females, only those expressing the SqdS isoform show properly localized GRK mRNA. The differential nuclear accumulation of the SqdS protein is associated with its ability to interact with Drosophila Transportin. SqdA does not possess a Transportin interaction motif. (2) The relationship between K10 and the distribution of Sqd protein also demonstrates the importance of Sqd accumulation within the oocyte nucleus. Mutations in both fs(1)K10 and sqd, consistently cause a mislocalization of GRK mRNA along the entire anterior cortex of the oocyte and lead to the production of strongly dorsalized eggs. Although the nuclear import of SqdS protein is most likely driven by its association with Transportin, K10 function is required for the stable accumulation of Sqd in the oocyte nucleus. This places K10 function upstream of Sqd in the germ line. Accumulation of Sqd protein in the nurse cells is not affected, and in addition Sqd is detectable within the oocyte cytoplasm of K10 mutants. Since the SqdS protein is the only Sqd isoform that is normally detected within the oocyte nucleus, the major effect of K10 must be on the nuclear retention of SqdS (Norvell, 1999 and references).

At this time it is unclear how K10 performs this function mechanistically. K10 protein will physically interact with the Sqd isoforms. The only known motif within the K10 protein is a potential helix-turn-helix domain in the carboxyl terminus, but site-directed mutagenesis of this domain has revealed that this motif is unnecessary for K10 function. K10 could be responsible for the modification of Sqd protein in such a manner as to promote nuclear retention, or alternatively, K10 could form a complex with Sqd protein that stabilizes its accumulation within the oocyte nucleus. In either case, the finding that Sqd protein requires the presence of K10 to accumulate in the oocyte nucleus further suggests that the phenotype of K10 mutant eggs is attributable to an effect on Sqd (Norvell, 1999 and references).

The ability to investigate the roles of the individual Sqd isoforms in the regulation of Grk during Drosophila oogenesis has revealed that there are two key aspects of Grk regulation: GRK mRNA localization and Grk protein accumulation. Both of these critical aspects of Grk regulation are accomplished by Sqd protein; however, these functions are performed differentially by the SqdS and SqdA isoforms. The severity effects of sqd mutation, therefore, reflect the loss of function of both of these proteins within the germ line, thus causing the mislocalization of GRK mRNA and the inappropriate accumulation of ectopic Grk protein. Restoration of either of these levels of regulation allows partial rescue of the D-V patterning defects of sqd mutants, but full rescue requires the function of both SqdS and SqdA. The data suggest that Sqd protein is a key regulator of both aspects of Grk regulation. The interaction of Sqd with the translational repressor protein Bruno provides a link between GRK mRNA localization and its translational regulation. Bruno has been shown directly to have a role in the translational repression of unlocalized Oskar mRNA. The interaction between Bruno and OSK mRNA is mediated by a specific sequence within the OSK message, which is termed BRE. As of yet, the molecular mechanism of Bruno action is not fully understood. However, correctly localized OSK mRNA must somehow be relieved from the Bruno-mediated repression by specific trans-acting factors localized to the posterior of the embryo (Norvell, 1999).

The interaction between Sqd and Bruno suggests that Bruno may play a role in the translational regulation of GRK mRNA. In support of this, GRK mRNA is known to contain a BRE within its 3' UTR, and Bruno has been shown to bind the GRK message. In addition, it has been demonstrated that during late stages of oogenesis, Bruno protein is concentrated at the anterior end of the oocyte, in a position that is coincident with localized GRK mRNA. On the basis of protein interaction data, it is suggested that Bruno may serve as a translational repressor of unlocalized GRK mRNA. As is the case for localized OSK mRNA, the appropriately localized GRK message would be relieved of its Bruno-mediated repression by other localized trans-acting factors. Moreover, the physical association between Sqd protein and Bruno protein suggests an appealing model to explain the similarity of the sqd and K10 mutant phenotypes. These two female sterile mutations represent the only cases in which mislocalized GRK mRNA is translated consistently and efficiently in all egg chambers. It is proposed that the role of Sqd protein is to take GRK mRNA from the oocyte nucleus, recruit Bruno in the cytoplasm, and deliver GRK mRNA to an anchor. In the absence of nuclear Sqd, in either sqd or K10 egg chambers, GRK RNA exits the nucleus by a generalized export mechanism, but does not associate efficiently with the repressor Bruno nor with the anchor. Because the interaction between GRK mRNA and Bruno does not occur, even the unlocalized GRK mRNA is translated efficiently. Using this model, Sqd protein provides the physical link between GRK mRNA transport, localization, and its appropriately regulated translation (Norvell, 1999).

Bruno regulates gurken during Drosophila oogenesis

Translational regulation of localized transcripts is a powerful mechanism to control the precise timing and localization of protein expression within a cell. In the Drosophila germline, oskar transcript must be translationally repressed until its localization at the posterior pole of the oocyte, since ectopic production of Oskar causes severe patterning defects. Translational repression of oskar mRNA is mediated by the RNA-binding protein Bruno, which binds to specific motifs in the oskar 3'UTR. Bruno over-expression is shown to cause defects in antero-posterior and dorso-ventral patterning, consistent with a role of Bruno in both oskar and gurken mRNA regulation. Bruno and gurken interact genetically. Finally, Bruno is shown to bind specifically to the gurken 3'UTR; the dorso-ventral defects caused by Bruno over-expression are due to a reduction of Gurken levels in the oocyte. It is concluded that Bruno plays similar roles in translational regulation of gurken and oskar (Filardo, 2003).

Bru has mainly been studied with regard to its role in translational repression of the posterior determinant Osk. However, the most obvious effect of aret mutations in females is premature arrest of oogenesis, a phenotype unrelated to translational misregulation of osk mRNA. During early oogenesis, the cystoblast fails to develop into a 16-cell cyst in the presence of strong aret mutant alleles. In contrast, weak aret alleles produce apparently normal egg chambers, which then undergo degeneration at stage 9. Hence, Bru affects a number of cellular processes that take place in the germline, including osk translational regulation. By analogy to osk regulation, the aret phenotype might therefore be caused by misregulation of target RNAs which, in the wild-type, are tightly regulated by Bru. Another not mutually exclusive possibility is that lack of functional Bru impairs other processes in which the protein is involved and that are unrelated to its RNA-binding activity. The fact that Bru over-expression causes phenotypes similar to Bru loss-of-function, and the fact that these defects can be modulated by simultaneous over-expression of BRE-containing RNA, supports the hypothesis that at least some of the aret early oogenesis phenotypes are indeed the result of RNA mis-regulation (Filardo, 2003).

Bru over-production, like Bru loss-of-function, impairs ovarian development. Most remarkably, Bru-over-expressing egg chambers that develop beyond the earliest stages undergo an extra round of division with incomplete cytokinesis, suggesting a role of Bru in regulation of the cystocyte divisions. Another gene, encore (enc), has also been shown to be involved both in regulation of germline mitoses and in establishment of oocyte polarity, the latter due to its role in grk mRNA localization and translation. enc encodes a 210 kDa protein with one conserved R3H domain, a single-stranded nucleic acid-binding domain. Thus, Bruno is not the only RNA-binding protein to be involved in regulation of the cystoblast divisions and in establishment of polarity. Given the nature of the proteins, it is likely that both Enc and Bru mediate their oogenesis effects through RNA binding. In contrast to Enc, which is required for Grk accumulation, Bru appears to negatively regulate Grk levels, most likely at the level of translation (Filardo, 2003).

Bru also provides a new example of genes whose activity affects establishment of both the A/P and the D/V axis. Bru has previously been shown to repress osk mRNA translation and new results show that Bru negatively regulates grk as well. The grk 3'UTR contains a single BRE. The interaction between Bruno and grk is most likely responsible for the observed reduction in Grk signal in egg chambers in which Bru is over-expressed. Another protein involved in regulation of both osk and grk is the DEAD-box RNA helicase Vasa, although in this case mutant alleles show a reduction in Osk and Grk levels, suggesting a positive role for Vas in osk and grk mRNA translation. oo18 RNA binding (orb), encoding the Drosophila cytoplasmic polyadenylation element binding protein (CPEB) is also required for both osk and grk mRNA localization and translation. Thus, regulation of the mRNAs encoding the embryonic polarity determinants Osk and Grk appears to be intimately related, involving many of the same RNA regulatory proteins (Filardo, 2003).

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

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

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

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).

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

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

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

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

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

The results show that Cup is an eIF4E binding protein that is involved in translational repression of osk RNA during oogenesis. The conserved YxxxxLφ motif in Cup is important for eIF4E binding and Cup and eIF4G are likely to bind the same surface of eIF4E. These results suggest that Cup competes with eIF4G for eIF4E binding, and hence inhibits translation initiation. 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).

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

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

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

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

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

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

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

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

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

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

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).