oskar


REGULATION

Factors effecting Oskar mRNA localization and translation

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).

Genetic analysis of the role of bruno in oogenesis is made difficult by the lack of ovaries in bruno mutants. Flies sensitized to changes in the level of Osk protein were examined for the affects of reducing wild-type Bru protein levels. If Bru acts to repress OSK translation, a reduction in Bru protein might lead to a partial derepression of OSK translation and, subsequently, to elevated Osk activity. A transgene was used which encodes a form of OSK mRNA that retains Bruno response sequences but is mislocalized to the anterior of the oocyte. Flies bearing this genotype produce embryos with modest head defects caused by the misexpressed OSK mRNA. Reduction in Bru level enhances this phenotype, resulting in progeny with extensive anterior deletions, often accompanied by duplication of posterior pattern elements (Webster, 1997).

Apontic binds the translational repressor Bruno and is implicated in regulation of oskar mRNA translation

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, CÆ4, lacks the BREs and fails to bind Bru. Apt binds all four RNAs, including CÆ4, 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 of oskar mRNA occurs independent of the cap and poly(A) tail in Drosophila ovarian extracts

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).

Premature translation of oskar in oocytes lacking the RNA-binding protein Bicaudal-C

Bicaudal-C (Bic-C) is required during Drosophila melanogaster oogenesis for several processes, including anterior-posterior patterning. The gene encodes a protein with five copies of the KH domain, a motif found in a number of RNA-binding proteins. Using antibodies raised against the Bic-C protein, it is shown that multiple isoforms of the protein exist in ovaries and that the protein, like the RNA, accumulates in the developing oocyte early in oogenesis. The pattern of Bic-C expression is similar to that of the Bic-C RNA, except that the RNA is detectable in a single cell (the presumptive oocyte) as early as germarium region 2A. In contrast, specific accumulation of BIC-C protein in the oocyte is first detectable at stages 3 and 4 of oogenesis but remains very faint until stage 5, when the protein level increases substantially. During stages 4 to 6, Bic-C protein is visible throughout the oocyte cytoplasm but is enriched at the posterior pole of the oocyte. During stages 7 to 9, Bic-C protein is abundant in the oocyte cytoplasm, with some enrichment at the anterior of the oocyte and around the oocyte cortex. In stage 10 and in later stages, the protein is expressed at high levels in the nurse cells. Bic-C protein expressed in mammalian cells can bind RNA in vitro, and a point mutation in one of the KH domains that causes a strong Bic-C phenotype weakens this binding. In addition, Oskar translation commences prior to posterior localization of Oskar RNA in Bic-C- oocytes, indicating that Bic-C may regulate Oskar translation during oogenesis (Saffman, 1998).

OSK mRNA is ectopically localized in part to the anterior of eggs produced by Bic-C/+ or Bic-C/Bic-C females. However, a substantial amount of OSK mRNA still localizes normally to the oocyte posterior, even in homozygous Bic-C oocytes. To determine whether Osk protein expression is affected in Bic-C mutants, immunohistochemistry was used to analyze Osk protein expression in ovaries from Bic-C/Bic-C females. In wild-type oocytes, translation of OSK is repressed until posterior localization of the RNA at stage 9, resulting in restriction of Osk protein to the posterior tip of the oocyte. In contrast, in Bic-C ovaries, OSK mRNA is prematurely translated, beginning in stages 7 and 8. Through stages 7 to 10, Osk protein remains diffuse and is most concentrated near the center of the oocyte. Surprisingly, despite its precocious translation in Bic-C mutants and the substantial posterior concentration of its RNA, Osk does not accumulate at the posterior pole as late as stage 10. It is possible that pole plasm-specific activation of OSK translation is also compromised by Bic-C mutations. However, as many developmental defects become apparent in Bic-C egg chambers beyond stage 9, and oogenesis fails to progress beyond stage 10, it cannot be ascertained that this failure to activate OSK translation is a specific consequence of Bic-C mutations. These results indicate that the RNA-binding activity of Bic-C is necessary for the correct repression of OSK translation. These results suggest that Bic-C may function directly to regulate the translation of target RNAs such as OSK (Saffman, 1998).

Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila

Smaug acts as a translational repressor; it binds NOS mRNA at a stem loop structure found within the Nos translational control element. Signals that mediate regulation of NOS mRNA reside in its 3' UTR. In particular, the 184 nt translational control element (TCE) contains all of the 3' UTR signals that are necessary and sufficient for NOS function. The key components of the TCE consist of a pair of redundant hairpins, each bearing the loop sequence CUGGC. These mediate both repression of NOS mRNA in the bulk cytoplasm as well as Oskar-dependent activation in the pole plasm. Thus, the TCE hairpins constitute the essential cis-acting elements of a translational switch responsible for generating a polarized distribution of Nos protein in the early embryo (Dahanukar, 1999 and references).

Using a set of mutant TCE hairpins, it was asked whether binding to Smaug in vitro correlates with TCE-mediated repression of NOS mRNA in vivo. Binding was monitored in gel mobility shift experiments, and TCE activity was monitored using transgenic flies that express appropriately altered NOS mRNAs. In brief, derepression of NOS mRNA in the bulk cytoplasm, where NOS mRNA is usually held in a translationally silent repressive complex, results in a reduction in the levels of anterior Bcd and Hb proteins, which in turn results in the development of lethal head defects. Binding of Smaug to the G12U mutant TCE, which is strongly defective in vivo, is reduced by a factor of at least 50 relative to wild type; binding to the moderately defective A15G mutant TCE is reduced by a factor of at least 5; and binding to the U18C mutant TCE, which regulates nos normally, is indistinguishable from binding to the wild-type hairpin. Thus, the variation in degree of binding of these mutant hairpins to Smaug indeed correlates with the mutant's capacity to repress translation of NOS mRNA in the bulk cytoplasm of the embryo (Dahanukar, 1999).

Overproduction of Smaug represses NOS mRNA in the pole plasm. Smaug protein is distributed throughout the preblastoderm embryo, with no detectable difference between its concentration in the bulk cytoplasm and the pole plasm. Why, then, does Smaug not repress the translation of NOS mRNA in the pole plasm? One explanation is that, in fact, Smaug-dependent repression competes with Osk-dependent activation, with Osk prevailing in wild-type embryos. To investigate this idea, Smaug was overproduced by introducing up to four extra copies of a smg+ transgene, thereby generating '6× smg+' embryos. The extent of Smaug overproduction appears to be approximately proportional to the gene dose. Otherwise, wild-type 6× smg+ embryos are completely viable and exhibit no segmentation defects. Moreover, the distribution of Nos protein during early development appears normal in such embryos, suggesting that this level of Smaug does not significantly interfere with Osk-dependent activation of wild-type NOS mRNA. However, excess Smaug clearly interferes with NOS mRNA translation in two different sensitized genetic backgrounds. While it is not understood why overproduction of Smaug is without apparent consequence in the pole plasm of wild-type embryos, one simple possibility is that a 3-fold increase in Smaug concentration is insufficient to repress translation in wild-type embryos, but that higher levels of Smaug would do so (Dahanukar, 1999).

Oskar interacts with the RNA-binding domain of Smaug. Smaug and Osk compete in the pole plasm, the former repressing and the latter activating translation of NOS mRNA. Smaug evidently acts by binding to the TCE hairpins of NOS mRNA. The molecular mechanisms by which Osk acts are not yet clear, although it plays a central role in both pole plasm assembly and activation of NOS translation. In particular, two lines of evidence suggest that Osk is the limiting component in the embryo for translational activation of NOS: (1) unlike other gene products required for pole plasm assembly, which are also present throughout the bulk cytoplasm, Osk is found only in the pole plasm; (2) overexpression of Osk is sufficient to activate NOS translation throughout the embryo. The mutually antagonistic activities of Osk and Smaug might be the result of a direct interaction between the two. To test this possibility, plasmids that direct the synthesis of various fragments of Osk and Smaug in yeast were constructed, and protein-protein interactions were assessed using the two-hybrid technique. Smaug interacts specifically with Osk in yeast. The region of Smaug that mediates this interaction corresponds to a 31 kDa fragment that contains the minimal RNA-binding domain. Further mutational analysis of this domain suggests that its TCE- and Osk-binding activities are not readily separable (Dahanukar, 1999). The region of Osk that mediates binding to Smaug consists of residues 290-418 (Dahanukar, 1999), a domain of the protein that may also mediate interactions with the pole plasm constituents Vas and Staufen (Breitwieser, 1996).

Taken with earlier work, these results support a simple model for the operation of a translational switch that governs expression from NOS mRNA. In the bulk cytoplasm, repression of NOS mRNA is dependent on the activity of Smaug, which binds to the essential targets in the 3' UTR. In the pole plasm, Smaug-mediated repression is antagonized by Osk, which interacts with the RNA-binding domain of Smaug. Currently, it is not know whether Osk interacts with Smaug bound to the TCE or whether Osk competes with the RNA for binding to Smaug. In either case, Smaug-dependent repression is overcome, and Nos protein accumulates in the posterior of the embryo. Osk also activates translation via other signals in the NOS 3' UTR. However, unlike the translational switch governed by Smaug, these signals are dispensable for NOS function in the embryo (Dahanukar, 1999 and references).

The gene aubergine is required to enhance Oskar translation. While aubergine-dependence is conferred upon Oskar mRNA by sequences in the Oskar 3' UTR, Aubergine may influence Oskar translation through an interaction with sequences upstream of the Oskar 3' UTR (Wilson, 1996).

Orb is required for efficient oskar translation at the posterior pole of the oocyte

The establishment of polarity axes in the Drosophila egg and embryo depends upon the localization and on-site expression of maternal mRNAs. The critical step in the targeting of posterior determinants is the localization of Oskar (OSK) mRNA to the pole and its on-site translation. Osk protein then recruits other posterior group gene products involved in the formation of pole plasm and in the localization and regulation of the posterior determinant, Nanos. The role of the Drosophila CPEB homolog, the orb gene, in the OSK mRNA localization pathway has been analyzed. The expression of Osk protein is dependent upon the orb gene. In strong orb mutants, Osk protein expression is undetectable, while in the hypomorphic mutant, orbmel, little or no on-site expression of Osk protein at the posterior pole is observed. The defects in Osk protein accumulation in orb mutant ovaries are correlated with a reduction in the length of the OSK poly(A) tails. OSK mRNA is found in immunoprecipitable complexes with Orb protein in ovaries. The OSK 3' UTR can be UV cross-linked to Orb protein in ovarian extracts. These data suggest that Orb is required to activate the translation of OSK mRNA and that this may be accomplished by a mechanism similar to that used by the Xenopus CPEB protein to control translation of 'masked' mRNAs (Chang, 1999).

What role does Orb protein play in the translation of OSK mRNA localized at the posterior pole in vitellogenic oocytes? One model that could potentially account for the current findings posits that translationally masked OSK mRNA would be deposited from the nurse cells into the anterior of the oocyte and then transported to the posterior pole by the Stauffen protein. Good candidates for ensuring that OSK mRNA is not translated in the nurse cells or at the anterior of the oocyte would be the Bruno protein and p50 protein. When the masked OSK mRNA arrives at the posterior pole, it would be bound by an Orb protein complex that is already anchored in the posterior cytoskeleton. Orb protein would then activate OSK mRNA translation, most probably by promoting polyadenylation (Chang, 1999 and references therein).

Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries

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).

Control of poly(A) polymerase level is essential to cytoplasmic polyadenylation and early development in Drosophila

The hiiragi (hrg) gene encodes a poly(A) polymerase (PAP), an enzyme that attaches adenylyl residues to the 3' untranslated region of mRNAs. The single Drosophila PAP is active in specific polyadenylation in vitro and is involved in both nuclear and cytoplasmic polyadenylation in vivo (Juge, 2002). Therefore, the same PAP can be responsible for both processes. In addition, in vivo overexpression of PAP during embryogenesis does not affect poly(A) tail length during nuclear polyadenylation, but leads to a dramatic elongation of poly(A) tails and a loss of specificity during cytoplasmic polyadenylation, resulting in embryonic lethality. Thus regulation of the PAP level is essential for controlled cytoplasmic polyadenylation and early development. hrg is also probably essential to cell viability since strong hrg mutant germline clones do not survive. The PAP encoded by this gene is involved in polyadenylation of oskar mRNA in oocytes. This indicates that although the reactions of nuclear and cytoplasmic polyadenylation are not identical, a single PAP is responsible for both in Drosophila (Juge, 2002).

The molecular mechanism of cytoplasmic polyadenylation has been analysed extensively in Xenopus oocytes, and some aspects of the reaction are similar to that of nuclear polyadenylation. Nuclear polyadenylation consists of endonucleolytic cleavage of pre-mRNAs followed by the synthesis of a poly(A) tail onto the upstream cleavage product. Poly(A) addition can be reconstituted in vitro from three purified mammalian factors: poly(A) polymerase (PAP), cleavage and polyadenylation specificity factor (CPSF) and poly(A)-binding protein II [PABP2, the nuclear poly(A)-binding protein]. CPSF is a complex of four proteins that binds the polyadenylation signal AAUAAA located upstream of the cleavage site. Recognition of the poly(A) site also requires cleavage stimulation factor (CstF) that binds to a GU/U-rich element downstream of the cleavage site and interacts with CPSF. PAP catalyses the polyadenylation reaction, but is also required for efficient cleavage of pre-mRNAs in vitro. PAP by itself does not recognize pre-mRNAs specifically. Specificity requires the AAUAAA element and CPSF that binds PAP through its 160 kDa subunit. Even in the presence of CPSF, PAP activity remains weak; it is again stimulated by binding of PABP2 to the poly(A) tail. Together, CPSF and PABP2 stimulate PAP activity by holding PAP on the RNA such that a full-length poly(A) tail is synthesized in a single processive event. When the poly(A) tail has reached its complete length, elongation is no longer processive and becomes slow and distributive. PABP2 is required for this poly(A) tail length control (Juge, 2002 and references therein).

In Drosophila, the role of CPEs has not been addressed, and the polyadenylation signal is dispensable in some cases, since embryonic cytoplasmic polyadenylation occurs on a bicoid engineered mRNA deleted for this element. Although genes encoding the four subunits of CPSF are present in the Drosophila genome, their role in cytoplasmic polyadenylation has not been determined. The Drosophila homolog of CPEB is the Orb protein. orb encodes germline-specific proteins different in male and female, and its function has been determined in the female germline. Strong orb mutants arrest oogenesis early, before the formation of the 16-cell cyst that would normally differentiate into nurse cells and one oocyte. Using a weaker allele, orbmel, Orb was shown to be required for anchoring of oskar mRNA at the posterior pole of the oocyte. However, this could result from a failure in oskar mRNA translation since Oskar protein is required for anchoring its own mRNA at the posterior pole. A recent study suggests that Orb could have a function analogous to that of CPEB in cytoplasmic polyadenylation. In orb mutant egg chambers, the level of Oskar protein is decreased and poly(A) tails of oskar mRNAs are shortened (Juge, 2002 and references therein).

To address a possible role for hrg in cytoplasmic polyadenylation during early development, germline clones homozygous for hrgPAP45, hrgPAP21 or hrgPAP12 were induced. No germline clones were obtained for any of these mutants, possibly as a result of a requirement of PAP for cell viability. Therefore genetic interactions were studied between hrg and orb, which is known to be involved in cytoplasmic polyadenylation. Females homozygous for the weak orbmel allele produce egg chambers at all stages and lay eggs, 30% of which show a ventralized phenotype. hrg lethal mutants act as dominant enhancers of the orbmel phenotype, since hrgPAP45/+; orbmel and hrgPAP21/+; orbmel females lay almost no eggs. In these females, oogenesis stops most frequently at stage 7/8, after which egg chambers degenerate, even though one or two stage 14 oocytes per ovary can be observed. Poly(A) tails of oskar mRNA are shortened in orb mutant ovaries. The defect of these poly(A) tails were analyzed in hrg- /+; orbmel mutants by PAT assays. oskar mRNA poly(A) tails were measured to be up to 135 residues in wild-type ovaries. These poly(A) tails are weakly reduced in orbmel, but severely reduced in hrg- /+;orbmel double mutant ovaries, their maximal length reaching 40- 50 residues. These short poly(A) tails do not result from the oogenesis defect in hrg- /+; orbmel females, since unrelated mutants that stop oogenesis early show wild-type poly(A) tails of oskar mRNA. These poly(A) tails were also found to be of wild-type length in hrgPAP21/+ and hrgPAP45/+ ovaries. This shows that the strong shortening of oskar poly(A) tails in hrg- /+; orbmel mutants does not result from an additive effect of two phenotypes, but from a synergistic effect of the two mutants due to a simultaneous decrease in PAP and Orb protein levels. This strongly suggests that hrg and orb are involved together in cytoplasmic polyadenylation. This was confirmed by measurements of poly(A) tails of a control mRNA, sop, which is thought not to be regulated by cytoplasmic polyadenylation. Poly(A) tails of sop mRNAs are unaffected in orbmel as well as in hrg- /+; orbmel mutant ovaries. It was verified that shortening of oskar mRNA poly(A) tails in hrg- /+; orbmel mutants leads to a reduction of Oskar protein level, by immunostaining of ovaries with anti-Oskar. Oskar accumulates at the posterior of the oocyte from stage 9 onwards. The amount of Oskar decreases in orbmel oocytes. This amount decreases again in hrg- /+; orbmel oocytes to a barely detectable level (Juge, 2002).

Taken together, these results show that hrg and orb cooperate in poly(A) tail lengthening during cytoplasmic polyadenylation and that alteration of this process affects protein accumulation and oogenesis (Juge, 2002).

Orb and a long poly(A) tail are required for efficient oskar translation at the posterior pole of the Drosophila oocyte

During Drosophila oogenesis, the posterior determinant, Oskar, is tightly localized at the posterior pole of the oocyte. The exclusive accumulation of Oskar at this site is ensured by localization-dependent translation of oskar mRNA: translation of oskar mRNA is repressed during transport and activated upon localization at the posterior cortex. Previous studies have suggested that oskar translation is poly(A)-independent. This study shows that a long poly(A) tail is required for efficient oskar translation, both in vivo and in vitro, but is not sufficient to overcome Bruno response element-mediated repression. Moreover, accumulation of Oskar activity requires the Drosophila homolog of Cytoplasmic Polyadenylation Element Binding protein (CPEB), Orb. Since posterior localization of oskar mRNA is an essential prerequisite for its translation, it was critical to identify an allele of orb that does localize oskar mRNA to the posterior pole of the oocyte. Flies bearing the weak mutation orbmel localize oskar transcripts with a shortened poly(A) that fails to enhance oskar translation, resulting in reduced Oskar levels and posterior patterning defects. It is concluded that Orb-mediated cytoplasmic polyadenylation stimulates oskar translation to achieve the high levels of Oskar protein necessary for posterior patterning and germline differentiation (Castagnetti, 2003).

Cytoplasmic polyadenylation of mRNA requires CPEB to recruit the enzyme poly(A) polymerase (PAP) on the regulated mRNA. Until recently, only one family of PAP, containing both a catalytic domain and an RRM-like domain, was known. A novel family of PAP has now been identified (Wang, 2002) that differs from the canonical PAP for the absence of the RRM-like domain. The prototype of this family is represented by C. elegans GLD-2 whose binding to the RNA is mediated by GLD-3, a KH domain containing protein of the BicC family. Interestingly, Drosophila BicC interacts physically with Orb in co-immunoprecipitation experiments. Since the phenotype of BicC mutants implicates BicC protein as a negative regulator of osk translation (Saffman, 1998), tests were performed to see whether Orb interacts with the translational repressor Bru. Indeed, a physical interaction between Bru and Orb has been detected, as revealed by the co-immunoprecipitation of Bru with Orb. By contrast, no direct interaction has been detected between Bru and BicC (Castagnetti, 2003).

The relevance of these interactions in vivo is further confirmed by the genetic interactions between the BicC locus and the orb and aret loci -- the latter encoding Bru protein. Females heterozygous for BicC show a number of AP patterning defects, ranging from head defects to bicaudal embryos. The BicC phenotype is suppressed when the mutation is combined with an orb allele or the strong aret allele, aretQB72. 85% of embryos produced by Bic-CYC33/+ females fail to hatch and of those 60% are bicaudal. The null allele orbF343 efficiently suppresses the Bic-C phenotype and only 13% of the embryos produced by BicCYC33/+; orbF343/+ fail to hatch, none of which show a bicaudal phenotype. The strength of the phenotype and the extent of the suppression depends on the orb allele. Embryonic viability is also improved, up to 72%, in embryos produced by BicCYC33/ aretQB72 females (Castagnetti, 2003).

Thus, combining measurement of osk poly(A) tail length in vivo with quantification of the translation activity of the corresponding mRNAs in vivo and in vitro, polyadenylation has been shown to corrolate with the translational status of the mRNA. A mutation in Orb, the Drosophila CPEB, leads to shortening of the osk poly(A) tail and to a reduction in Osk accumulation. The fact that, at least in vitro, a long poly(A) tail neither overcomes BRE-mediated repression nor is necessary for repression of osk reporter transcripts, suggests that cytoplasmic polyadenylation is not the decisive event in translational activation of osk at the posterior pole. Rather, it appears that the presence of a 200 A long tail on osk mRNA promotes its efficient translation, allowing accumulation of Osk to levels sufficient for both abdomen and germline formation to proceed (Castagnetti, 2003).

The prevailing model, which is based on studies of translational control in the Xenopus oocyte, suggests that the polyadenylation status of a transcript correlates with its translational status: a short poly(A) tail corresponding to a silenced mRNA and poly(A) tail elongation triggering translational activation. In Xenopus, upon progesterone treatment a wave of cytoplasmic polyadenylation activates translation of deadenylated and silenced maternally derived mRNAs. In Drosophila, translation of bicoid mRNA, which encodes the anterior determinant of the embryo, is repressed until egg activation, when poly(A) tail elongation triggers translation initiation. Although the correlation between adenylation and translation still holds for several transcripts, a growing body of evidence suggests that the two events may be coincidental but not directly connected. Interestingly, deadenylation and translational repression of Drosophila hunchback (hb) and mouse tPA mRNAs can occur independently of each other. The transcripts are deadenylated concomitant with translational repression, yet repression can occur in the absence of ongoing deadenylation. In arrested primary mouse oocytes, polyadenylation of the tPA mRNA is necessary to counteract the default deadenylation that affects most other oocyte mRNAs, thus preventing its degradation (Castagnetti, 2003).

Observations made in this study suggest that silencing and awakening of osk mRNA translation can occur in the absence of changes in poly(A) tail length and, in fact, osk mRNA bears a long poly(A) tail at all stages of oogenesis, including when it is unlocalized and translationally silent. However, it is still formally possible that at intermediate stages of oogenesis osk mRNA undergoes a deadenylation that goes undetected in measurements on bulk RNA, and that elongation of the poly(A) tail causes displacement of the repressor complex, leading to translational derepression. This hypothesis is supported by the fact that the repressor protein Bru shares a 50% sequence identity with the Xenopus deadenylation promoting factor EDEN-BP. However, no obvious pattern of deadenylation has been detected in vitro when Bruno is added to the embryonic extract, nor could a shortening be detected of the poly(A) tail of translationally silenced osk transcript recovered from ovarian extract. Nevertheless, the results show that BRE-mediated repression is effective independent of the length of the poly(A) tail on osk transcripts, and that a silenced mRNP can be assembled on a naked osk transcript, whether or not it bears a poly(A) tail. These results suggest that polyadenylation is not the sole determining event leading to translational derepression of osk mRNA at the posterior pole, but that the maintenance of a long poly(A) tail, by cytoplasmic polyadenylation, accounts for the enhancement of osk translation and is required for efficient osk translation, to ensure sufficient accumulation of Osk at the posterior pole of the Drosophila oocyte to promote abdominal patterning and germline differentiation (Castagnetti, 2003).

Furthermore, the physical interaction detected between Orb and Bru, and Orb and BicC suggests the existence of a multi-protein complex containing both positive and negative regulators of osk translation. In this scenario, translational silencing and polyadenylation are linked through Bru protein, offering a possible explanation as to how CPEB might be recruited to mRNAs in Drosophila, where no canonical CPE has so far been identified. Transcripts properly repressed by Bru, upon localization, could be adenylated by the recruitment of Orb by Bru itself. Loss of Bru repression would, therefore, result in loss of Orb binding with consequent deadenylation and translational silencing. In this model, modulation of the poly(A) tail would be part of the mechanism that regulates translation, ensuring a second level of control over ectopic expression while localizing all the components necessary for efficient translation. Remarkably, mutations in the BRE sites do not result in ectopic osk translation, suggesting the existence of a second layer of translational control. Moreover, during embryonic development when osk translation is no longer required, both Orb and Bru proteins are depleted in the embryo and osk mRNA undergoes complete deadenylation (Castagnetti, 2003).

Me31B regulates Oskar mRNA translation

A DEAD-box protein, Me31B, forms a cytoplasmic RNP complex with oocyte-localizing RNAs. During early oogenesis, loss of Me31B causes premature translation of oocyte-localizing RNAs within nurse cells, without affecting their transport to the oocyte. In early egg chambers that lack Me31B, at least two mRNAs in particles, OSK and Bicaudal-D mRNAs, are prematurely translated in nurse cells, though the transport of these RNAs to the oocyte is Me31B independent. These results suggest that Me31B mediates translational silencing of RNAs during their transport to the oocyte. These data provide evidence that RNA transport and translational control are linked through the assembly of RNP complex (Nakamura, 2001).

A visual screen was conducted with an ovarian GFP-cDNA library, in which fusion genes are expressed in germline cells during oogenesis. Transgenic flies were generated with this library and proteins were identified that distribute in a granular pattern during oogenesis. Screening ~3000 independent lines, one was isolated in which GFP signals were detected as cytoplasmic particles during oogenesis. The particles were dispersed in the cytoplasm of both nurse cells and oocytes but never detected within nuclei. The particles were frequently observed passing through ring canals, suggesting that the particles are assembled in nurse cell cytoplasm and transported to the oocyte (Nakamura, 2001).

The cDNA from this line was identified as me31B. In the cDNA fusion, almost the entire coding region of me31B, which lacks only the first four codons, was fused in frame with that of gfp. Me31B, a DEAD-box protein and therefore a putative ATP-dependent RNA helicase, was isolated as a gene expressed extensively during oogenesis. Me31B is a part of an evolutionally conserved DEAD-box protein group, which includes human RCK/p54 (71% identical), Xenopus Xp54 (73%), Caenorhabditis elegans C07H6.5 (76%), Schizosaccharomyces pombe Ste13 (68%) and Saccharomyces cerevisiae Dhh1 (68%). Furthermore, Me31B is phylogenetically close to two evolutionally conserved proteins, eIF4A and Dbp5/Rat8p but far from Vasa, which functions in germline development (Nakamura, 2001).

To examine distribution of the endogenous Me31B, antibodies were generated that specifically recognized Me31B. The distribution pattern of endogenous Me31B is identical to that of GFP-Me31B. No detectable signal in somatic follicle cells is observed at any stage of oogenesis. Me31B is first detected at a low level in germarium region 2B, where the signal is concentrated in the pro-oocytes. The signal remains concentrated in the oocyte until mid-oogenesis. In early egg chambers, a low level Me31B signal is detected in nurse cell cytoplasm. In both nurse cells and oocytes, the signal appears to be granular. Me31B signals in nurse cell cytoplasm become more evident from stage 5-6, when Me31B expression is drastically increased. In addition, Me31B is frequently enriched around nurse cell nuclei. Later, Me31B accumulates at the posterior pole of stage 10 oocytes. However, this posterior accumulation is transient, as revealed by uniform distribution of the signal in cleavage embryos. By cellular blastoderm stage, Me31B becomes undetectable in the entire embryonic region. No zygotic expression of Me31B was detected during embryogenesis (Nakamura, 2001).

Because Me31B is probably an RNA-binding protein that is transported to the oocyte, it was asked whether Me31B forms a complex with oocyte-localizing RNAs. Colocalization of OSK mRNA with Me31B was examined. OSK mRNA starts to accumulate in oocytes from germarium region 2B, with the concentration of OSK increasing over time. Posterior accumulation of OSK mRNA in the oocyte begins from stage 8 onwards. By fluorescent in situ hybridization, OSK mRNA exhibits particulate signals in the cytoplasm of both nurse cells and oocytes, and is frequently concentrated around nurse cell nuclei. This distribution pattern of OSK mRNA is essentially identical to that of Me31B, with colocalization present until OSK mRNA localizes to the posterior pole of stage 10 oocytes (Nakamura, 2001).

Colocalization of Me31B with other RNAs was also examined. Ovaries were doublestained for Me31B and BicD mRNA. In early egg chambers, BicD mRNA also produces particulate signals, and appears to localize in Me31B-containing particles. This colocalization becomes apparent from stage 5-6, when BicD mRNA expression is elevated. The oocyte-localizing RNAs examined [BCD, NOS, Oo18 RNA-binding (ORB), Polar granule component (PGC) and Germ cell-less (GCL)] all produce particulate signals in the cytoplasm of both nurse cells and oocytes, and colocalize with Me31B. In contrast, Vasa mRNA, which is not specifically transported to the oocyte, does not appear to be colocalized with Me31B. These results indicate that Me31B forms cytoplasmic particles that contain oocyte-localizing RNAs (Nakamura, 2001).

The complicated and redundant phenotypes observed in me31B- egg chambers in mid-oogenesis are unlikely to be the primary effect of loss of me31B function. Earlier phenotypes of me31B- egg chambers were examined using a FLP/FRT system to generate homozygous germline clones that are marked by the loss of Vas-GFP fusion protein. Based on Hoechst and phalloidin staining, me31B- egg chambers are morphologically normal until stage 4-5. From stage 6 onwards, oocytes in me31B- egg chambers fail to grow normally. At this stage, these egg chambers begin to degenerate. In early me31B- egg chambers, Exu signal is concentrated to the oocytes. Distributions of OSK and BicD mRNAs in me31B- egg chambers were examined. Both OSK and BicD mRNAs also accumulate in the oocytes of me31B- egg chambers until the chambers degenerate. Particulate signals for these RNAs are detectable in nurse cell cytoplasm in these egg chambers. These results indicate that Exu, OSK and BicD mRNAs can be transported to the oocyte even in the absence of Me31B. It is concluded that in early egg chambers, Me31B is dispensable for the transport of the molecules that form a complex with Me31B (Nakamura, 2001).

Whether loss of Me31B affects translation of OSK and BicD mRNAs was examined. Ovaries were immunostained with an anti-Osk antiserum. Although OSK mRNA is expressed during almost all stages of oogenesis, its translation is repressed to keep Osk protein level very low during early oogenesis. In me31B- egg chambers, Osk signal is significantly increased compared with that in the neighboring me31B+ egg chambers (Nakamura, 2001).

A similar increase of BicD signal in me31B- egg chambers is more evident. In wild-type egg chambers, BicD protein, like BicD mRNA, is highly concentrated in the oocytes starting from germarium region 2B. In the egg chambers lacking me31B, increased BicD signal is detected in nurse cell cytoplasm. These results suggest that loss of Me31B in germline cells causes derepression of OSK and BicD mRNA translation during their transport to the oocyte (Nakamura, 2001).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Hrp48 regulates Oskar localization and translation

The Staufen-dependent localization of oskar mRNA to the posterior of the Drosophila oocyte induces the formation of the pole plasm, which contains the abdominal and germline determinants. In a germline clone screen for mutations that disrupt the posterior localization of GFP-Staufen, three missense alleles were isolated in the hnRNPA/B homolog, Hrp48 (termed Hrb27C in FlyBase) These mutants specifically abolish osk mRNA localization, without affecting its translational control or splicing, or the localization of bicoid and gurken mRNAs and the organization of the microtubule cytoskeleton. Hrp48 colocalizes with osk mRNA throughout oogenesis, and interacts with its 5′ and 3′ regulatory regions, suggesting that it binds directly to oskar mRNA to mediate its posterior transport. The hrp48 alleles cause a different oskar mRNA localization defect from other mutants, and disrupt the formation of GFP-Staufen particles. This suggests a new step in the localization pathway, which may correspond to the assembly of Staufen/oskar mRNA transport particles (Huynh, 2004).

Previous genetic approaches have shown that Stau, Barentsz, Mago Nashi, and Y14 are required for osk mRNA localization, and colocalize with it to the posterior pole, strongly suggesting that these proteins are components of the mRNA localization complex. However, none of these proteins have been shown to bind directly to osk mRNA, although this seems likely to be the case for the RNA binding proteins, Stau and Y14. Mago, Y14, Barentsz, and eIF4AIII are part of the exon-exon junction complex (EJC) that marks where introns have been excised. This suggests that the EJC is loaded onto osk mRNA during splicing in the nucleus to control its localization in the oocyte cytoplasm. Although Stau and the EJC are thought to play a direct role in coupling osk RNA to the factors that localize it, none of these proteins has been shown to bind specifically to any sequence elements within osk mRNA, and it is unclear how the RNA is recognized (Huynh, 2004 and references therein).

Biochemical strategies have led to the identification of Bruno, Apontic, p68, and p50 as proteins that bind to specific sequences in osk RNA, all of which have been implicated in the regulation of osk mRNA translation but not in the localization of the mRNA. The results described in this study provides a link between these two approaches, by demonstrating that p50 corresponds to Hrp48 and that it is specifically required for the transport of the mRNA to the posterior of the oocyte. Germline clones of the three missense alleles of hrp48 have no effect on the polarity of the oocyte, the organization of the microtubules, or the localization of bicoid or gurken mRNAs, but abolish the posterior localization of osk mRNA (Huynh, 2004).

Since Hrp48 has been shown to regulate alternative splicing, it is possible that the oskar mRNA localization phenotype of the hrp48linha mutations is an indirect consequence of a defect in the splicing of another mRNA that encodes a factor that is directly involved in osk mRNA transport. However, this is unlikely to be the case for two reasons. (1) These missense alleles have no effect on the alternative splicing of Ubx transcripts. Since the insertion alleles of Hrp48 do disrupt the Ubx splicing pattern, the missense alleles do not appear to impair the function of Hrp48 in splicing regulation. (2) Hrp48 binds to sequences in the 5′ region and the 3′UTR of osk mRNA, and colocalizes with the mRNA at the posterior. This strongly suggests that the requirement for Hrp48 in osk mRNA localization is direct, and that it functions as an essential trans-acting factor that recognizes the RNA and plays a role in coupling it to the localization machinery (Huynh, 2004).

The phenotype of the hrp48 missense alleles differs from that of the other mutants that disrupt osk mRNA localization, suggesting that it acts at a distinct step in the localization pathway. In stau, barentsz, mago, Y14, and tropomyosinII mutants, osk mRNA also fails to reach the posterior, but most of the RNA remains at the anterior cortex. Since osk mRNA shows a transient accumulation at the anterior in wild-type before it localizes to the posterior, these proteins may be required to release osk mRNA from the anterior, and to couple it to the posterior transport pathway. In contrast, no accumulation of osk mRNA at the anterior of the oocyte is detected in the hrp48 missense mutants, and the mRNA shows a uniform distribution throughout the oocyte cytoplasm. This raises the possibility that Hrp48 is required for the transient anterior accumulation of osk mRNA, and acts upstream of the other proteins required for posterior localization, such as Stau. One argument against this interpretation is that the localization to the anterior of the oocyte is thought to be a by-product of the transport from the nurse cells into the oocyte, which occurs normally in the hrp48 missense alleles. In support of this view, all mRNAs that are transported into the oocyte also localize, at least transiently, to the anterior cortex at stage 9. Therefore an alternative model is favored in which Hrp48 acts downstream of Stau, Mago, etc. In this case, the stau class of mutants might block the release of osk mRNA from the anterior cortex, whereas the hrp48 alleles might stimulate this release, but prevent the subsequent association of the mRNA with the factors that transport it to the posterior pole (Huynh, 2004).

Many localized mRNAs appear to move as large cytoplasmic particles, leading to the suggestion that they must be packaged into transport granules in order to be localized. For example, when pair rule, wingless, or bicoid mRNAs are injected into Drosophila embryos, they assemble into particles that move in a microtubule-dependent manner, and fluorescent MBP mRNA shows a similar behavior when introduced into cultured oligodendrocytes. The formation of these transport particles has also be visualized by labeling proteins that bind to localized mRNAs. For example, injected bicoid mRNA recruits Stau into motile particles in Drosophila syncytial blastoderm embryos, while GFP-tagged mouse Stau1 has been observed to form particles that move along microtubules in neuronal processes. In this context, it is very striking that two of the hrp48 missense mutants strongly reduce the formation of GFP-Stau particles in the nurse cell and oocyte cytoplasm. This suggests that Hrp48 plays a role in the formation of Stau-containing osk mRNA transport particles, which could account for the failure to localize the mRNA to the posterior pole in these mutants. If these particles sequester the mRNA and prevent its diffusion, this may also explain why osk mRNA remains at the anterior in the stau class of mutants, but not in the hrp48 missense alleles (Huynh, 2004).

Hrp48 is one of the three most abundant HnRNPs in Drosophila, along with Hrp40 (Squid) and Hrp38, and is thought to bind most, if not all, nascent transcripts in the nucleus. It is therefore surprising to recover hrp48 mutations that have such a specific effect on the localization of osk mRNA. P element insertions in hrp48 produce a distinct phenotype. Although osk mRNA is often not localized to the posterior, it is sometimes found in the center of the oocyte, which is indicative of a defect in oocyte polarity. Consistent with this, Kin-ß-gal also localizes to the center of the oocyte in these mutants, whereas it always shows a wild-type posterior localization in the missense alleles. Thus, the P element insertions presumably disrupt the regulation of another mRNA that is required for the polarization of the oocyte microtubule network. A second important difference is that the P element alleles cause the premature translation of osk mRNA, which is consistent with the identification of Hrp48 as p50, which binds to sites in the osk BREs. In contrast, the missense alleles have no discernable effect on translational repression, since osk mRNA is completely unlocalized, but no Osk protein is produced. P alleles of Hrp48 also affect gurken mRNA localization and translation, whereas no defects are observed in the distribution of either gurken mRNA or protein in germline clones of the missense alleles. Finally, the P alleles, but not the missense alleles, disrupt the alternative splicing of Ubx RNAs. These differences presumably reflect the distinct nature of the molecular lesions in the two types of allele. All of the P element insertions fall in the promoter or an intron in the 5′UTR of the hrp48 gene, and produce reduced levels of wild-type Hrp48 protein. In contrast, each of the missense alleles expresses approximately normal levels of mutant Hrp48, in which a single amino acid has been changed (Huynh, 2004).

The comparison between the phenotypes of the two classes of hrp48 mutations indicates that the missense alleles affect regions of the protein that are specifically required for osk mRNA localization. This is particularly interesting in the case of the two Trp to Asn mutations in the Glycine-rich domain (GRD), since this domain is not involved in RNA binding, and neither mutant affects the interaction of Hrp48 with osk mRNA. Evidence from other HnRNPA/B family members suggests that this region functions as an oligomerization domain. For example, the GRD of Human HnRNP A1 has been shown to self-associate in vitro, and this interaction depends on large aromatic residues embedded with the Glycine-rich region. Thus, it is possible that Hrp48 also oligomerizes, and that this is disrupted by mutating the aromatic Tryptophans in the GRD, which could explain why both mutations impair the formation of GFP-Stau particles. These mutations may therefore abolish RNA localization because Hrp48 oligomerization is required to form high-order osk RNP complexes, which are the substrate for posterior transport (Huynh, 2004).

It is more difficult to explain why the 5B2-6 allele disrupts osk mRNA localization. Although one would expect a mutation in a conserved residue in the RNA recognition motif to disrupt RNA binding, the mutant protein binds to osk mRNA in vitro as well as the wild-type protein, in both UV-crosslinking and pull-down assays. Furthermore, it presumably also associates with osk mRNA in vivo, because it mediates normal translational repression, which requires binding to the sites in the BREs. The Hrp48 binding sites in osk mRNA that are necessary for localization have not been mapped, and these sites could be distinct from those involved in translational repression. Thus, this mutation may only disrupt Hrp48 binding to a specific subset of its target sites, including unidentified sites in osk mRNA that are required for localization. Since the glycine that is mutated is not directly involved in RNA binding, another possibility is that the mutation disrupts the interaction of Hrp48 with another protein that is required to couple osk mRNA to the localization machinery (Huynh, 2004).

Two other members of the hnRNP A/B family have also been implicated in mRNA localization. Drosophila Squid, which is most closely related to hnRNPA1, binds directly to gurken mRNA, and is required for its localization to the anterior-dorsal corner of the oocyte. Squid is also required to repress the translation of unlocalized grk mRNA, and interacts with Bruno, a translational repressor of both grk and osk mRNAs. Since Hrp48 binds to the Bruno Response elements in osk mRNA, and is required to repress its premature translation, these two Drosophila hnRNP A/B proteins perform remarkably similar functions in the regulation of osk and gurken mRNAs. Furthermore, hnRNP A2, which is one of the closest mammalian homologs of Drosophila Hrp48, plays a comparable role in the localization of MBP mRNA in oligodendrocytes. When MBP mRNA is injected into oligodendrocytes, it forms large particles that move along microtubules into the distal processes, and, like osk, the mRNA is probably transported by kinesin. This localization requires the specific binding of hnRNP A2 to a 21 nt A2RE element in the MBP 3′UTR that is necessary and sufficient for localization and efficient translation. In addition, recent work has shown that the microtubule-dependent localization into oligodendrocyte processes is mediated by the second RRM of hnRNP A2, and it is intriguing that the hrp48 5B2-6 mutation falls in the equivalent domain of the Drosophila protein. These clear parallels between the functions of Hrp48, Squid, and hnRNP A2 suggest that these hnRNP A/B proteins play a conserved role in mRNA localization and translational control in flies and mammals (Huynh, 2004 and references therein).

Hrp48 is a predominantly nuclear protein that associates with nascent transcripts, and regulates alternative pre-mRNA splicing. It therefore seems very likely that Hrp48 binds to osk mRNA in the nucleus, and is exported into the cytoplasm with the RNA, where it regulates localization and translation. This adds to the growing body of evidence that the cytoplasmic fate of mRNAs is determined in part by their nuclear history. In the case of osk mRNA, cytoplasmic localization seems to require the binding of at least four distinct proteins in the nucleus; Hrp48, which probably associates with the RNA cotranscriptionally, and Mago, Y14, and eIF4AIII, which are presumably recruited to the RNA during splicing as part of the exon-exon junction complex. The mRNA must then recruit additional essential localization factors in the cytoplasm, such as Stau and Barentsz, before it is competent to localize to the posterior pole of the oocyte. Thus, the assembly of a functional osk RNA localization complex requires the stepwise recruitment of multiple nuclear and cytoplasmic proteins, all of which are essential for posterior localization. One of the main challenges for the future will be to determine how this complex is assembled, and how these proteins act together to link the mRNA to the motor that mediates its transport to the posterior pole (Huynh, 2004).

Hrp48, a Drosophila hnRNPA/B homolog, binds and regulates translation of oskar mRNA

Establishment of the Drosophila embryonic axes provides a striking example of RNA localization as an efficient mechanism for protein targeting within a cell. oskar mRNA encodes the posterior determinant and is essential for germline and abdominal development in the embryo. Tight restriction of Oskar activity to the posterior is achieved by mRNA localization-dependent translational control, whereby unlocalized mRNA is translationally repressed and repression is overcome upon mRNA localization. The oskar RNA binding protein p50 has been identified as Hrp48, an abundant Drosophila hnRNP. Analysis of three hrp48 mutant alleles reveals that Hrp48 levels are crucial for polarization of the oocyte during mid-oogenesis. These data also show that Hrp48, which binds to the 5' and 3' regions of oskar mRNA, plays an important role in restricting Oskar activity to the posterior of the oocyte, by repressing oskar mRNA translation during transport (Yano, 2004).

HnRNPs are abundant RNA binding proteins involved in many aspects of mRNA regulation. HnRNPs associate with transcripts at their site of synthesis in the nucleus, and can remain associated with the RNA during processing and export, as well as during cytoplasmic processes such as translation and localization. Mammalian hnRNP A2 binds to specific sequences in the 3′UTR of myelin basic protein mRNA and mediates its localization and translational control in rat oligodendrocytes. In Xenopus oocytes, VgRBP60, an hnRNP I-related protein, colocalizes with and binds sequence elements in Vg1 mRNA that are critical for its localization at the vegetal pole. In the Drosophila oocyte, two isoforms of Squid/hrp40 have essential roles in localization and translational control of gurken mRNA, the dorso-ventral polarity determinant (Yano, 2004).

Drosophila Hrp48, a member of the hnRNPA/B family of proteins, is an abundant hnRNP, bearing two N-terminal RRM-type RNA binding domains and a C-terminal Glycine-rich domain. Hrp48 is expressed in somatic and germline cells of the ovary, where it is detected at low levels in the nucleus and predominantly in the cytoplasm. Mammalian hnRNP A1, a putative homolog of hrp48, has been shown to function in splice-site selection and to shuttle between the nucleus and the cytoplasm. Hrp48 is a cofactor in regulation of alternative splicing . Mutations reducing hrp48 expression cause developmental defects and lethality, indicating that hrp48 is essential in the fly (Yano, 2004).

Although hrp48 affects oskar mRNA regulation, it does not appear to regulate oskar mRNA processing, because the mRNA is accurately spliced in the mutants. The hrp48 mutants analyzed also show defects in the organization of the oocyte microtubules. These defects are due to low levels of Hrp48 in the mutants, since they are suppressed by expression of hrp48 from a transgene. Given the demonstrated involvement of Hrp48 in RNA splicing, localization, and translational control, it is likely that polarity defects in hrp48 mutants are caused by deregulation of RNAs whose products are required for oocyte polarization. The dumping defects of some hrp48 mutant egg chambers suggest that Hrp48 may also regulate the function of the actin cytoskeleton. However, the oskar-related defects observed in the mutants do not resemble anchoring defects: their onset occurs before stage 10, when cytoplasmic streaming commences, and they decrease in severity as streaming proceeds. The difference in phenotype of the mutants analyzed, in which the level of expression of wild-type Hrp48 protein is reduced, causing polarity defects, and those identified by Huynh (2004), which express mutant Hrp48 proteins at wild-type levels but show normal oocyte polarity, highlights the importance of the threshold of expression of Hrp48 and the involvement of the protein in multiple RNA regulatory events (Yano, 2004).

The only protein shown previously to bind to oskar mRNA directly and to repress its translation during transport is Bruno. Null alleles of arrest fail to develop beyond the early stages of oogenesis, and hypomorphic alleles that allow development until stage 9 show no precocious translation of oskar mRNA. The evidence that Bruno is an oskar translational repressor in vivo is extensive and comes from analysis of transgenes in which Bruno binding sites were mutated, from the cuticle phenotype of embryos in which arrest gene dosage was varied in a sensitized genetic background, and from overexpression of Bruno in the germline, which causes the development of embryos with a posterior group phenotype (Yano, 2004).

Several lines of evidence support a role of Hrp48 in oskar translational repression. (1) A substantial portion of embryos developing from hrp48K16203 germline clones show A/P patterning defects consistent with misregulation of posterior patterning activity: reduced abdominal segmentation, presumably due to defects in oskar mRNA localization, and head defects. (2) An enhancement of the head defects caused by overexpression of the oskar 3′UTR is observed when hrp48 levels are reduced, and in extreme cases embryos with a bicaudal phenotype develop. (3) Translational derepression of the lacZ-oskar translational reporter is observed in hrp48 heterozygous females. The absence of detectable Oskar protein in the cytoplasm of heterozygous or homozygous hrp48 mutant oocytes during the early stages of oogenesis suggests that the lacZ reporter is more sensitive than the antibody, and thus the extent to which oskar is derepressed in the mutants is unclear (Yano, 2004).

Bruno binds to the A, B, and C regions of the oskar 3′UTR, each of which contains a pair of U(G/A)U(A/G)U(G/A)U sequence elements defining the BRE consensus. Mutations reducing binding of Bruno to BRE region A in vitro cause precocious reporter translation in the nurse cells and in the oocyte, indicating a role of Bruno in oskar repression in both cell types. Hrp48 binds the A, B, and C regions in vitro and represses oskar translation in vivo. Sequences with homology to the characterized Hrp48 binding site in P element transposase mRNA, the F2 site (UAGGUUAAG), are located in the oskar 5′ translation regulatory region, and within 10 nucleotides of the BREs in regions A and B, and overlapping with the BRE in region C in the 3′UTR. Deletion of these F2-like sequences from an AB region probe selectively disrupts Hrp48 binding in vitro, without affecting Bruno binding. An oskar-lacZ reporter transgene bearing these deletions shows precocious translation in the oocyte, but not in the nurse cells. Hence, the F2-like elements in regions A and B of the 3′UTR may mediate the repressive effect of Hrp48 on translation of unlocalized oskar mRNA in the oocyte. Proof that the repressive effect of Hrp48 on oskar translation is mediated by binding of the protein to these elements will require a complete mutational analysis, as has been performed for Bruno, resulting in the definition of the BREs (Yano, 2004).

Interestingly, when the region to which Hrp48 binds at the 5′ end of oskar mRNA is deleted in the context of the BRE A LS5 mutation, an increase in precocious translation both in the nurse cells and in the oocyte is observed, indicating a function of the 5′ region in translational repression. The recent demonstration that mutations in Cup, an eIF4E binding protein that coprecipitates with Bruno in ovarian extracts, cause precocious oskar translation suggests that translational repression may occur at the initiation step. The fact that sequences at the 5′ and 3′ ends of oskar mRNA regulate localization-dependent translation, together with the observation that Hrp48 can homodimerize in a yeast two-hybrid assay raises the possibility that Hrp48 may promote circularization of oskar mRNA, and thus facilitate Cup-mediated repression (Yano, 2004).

Hrp48 binds oskar mRNA directly and regulates both its localization and translation, suggesting that these processes might be coupled and mediated by a bifunctional mRNA localization/repression complex comprising Hrp48. It is not known at what stage Hrp48 first associates with oskar mRNA. However, the similar distributions of Hrp48 and oskar mRNA in the oocyte and the abundance of Hrp48 in the cytoplasm suggest their association in the cytoplasm (Yano, 2004).

How mRNA localization/translation complexes are assembled and associate with the transport machinery are central questions. The involvement in oskar mRNA localization of the EJC components Mago-Nashi and Y14, which associate with RNAs at exon-exon junctions upon splicing, indicates that assembly of the oskar mRNA localization complex begins in the nucleus. The fact that all the exon-exon junctions in oskar mRNA are located in the coding region and that distinct regions of the oskar 3′UTR are required for mRNA localization indicates the involvement of a higher order complex in oskar mRNA localization. In this context, it is interesting to speculate that Hrp48 molecules bound to distinct regions of oskar mRNA may promote the association of several mRNAs and associated proteins into large transport-competent particles. The perinuclear localization of Hrp48, Barentsz, Bruno, and Cup may indicate that they associate with the mRNA at the time of its nuclear-cytoplasmic transport (Yano, 2004).

During mid-oogenesis, oskar mRNA and associated proteins are localized in a microtubule-dependent manner to the posterior pole of the oocyte, where the plus ends of the oocyte microtubule are focused. In Kinesin heavy chain mutants, oskar mRNA localization fails and the RNA remains all along the cell cortex, indicating a role of Kinesin heavy chain in polarized transport of the oskar mRNP. However, it is still unclear how the oskar mRNP associates with the motor, as Kinesin light chain is not required for oskar mRNA localization in the oocyte. Thus, future work will be required to determine the molecules and cellular mechanisms whereby nuclear-associated proteins, such as the EJC components, Barentsz, and Hrp48, assemble an oskar mRNA complex competent for transport to the posterior pole of the Drosophila oocyte (Yano, 2004).

Imp associates with Squid and Hrp48 and contributes to localized expression of Gurken and Oskar in the oocyte

Localization and translational control of Drosophila gurken and oskar mRNAs rely on the hnRNP proteins Squid and Hrp48, which are complexed with one another in the ovary. IGF-II mRNA-binding protein (Imp), the Drosophila homolog of proteins acting in localization of mRNAs in other species, is also associated with Squid and Hrp48. Notably, Imp is concentrated at sites of gurken and oskar mRNA localization in the oocyte, and alteration of gurken localization also alters Imp distribution. Imp binds gurken mRNA with high affinity in vitro; thus, the colocalization with gurken mRNA in vivo is likely to be the result of direct binding. Imp mutants support apparently normal regulation of gurken and oskar mRNAs. However, loss of Imp activity partially suppresses a gurken misexpression phenotype, indicating that Imp does act in control of gurken expression but has a largely redundant role that is only revealed when normal gurken expression is perturbed. Overexpression of Imp disrupts localization of gurken mRNA as well as localization and translational regulation of oskar mRNA. The opposing effects of reduced and elevated Imp activity on gurken mRNA expression indicate a role in gurken mRNA regulation (Geng, 2006).

Imp is the Drosophila homolog of a family of proteins that act in posttranscriptional regulation in a variety of animals. One of the founding members of the family, ZBP-1, binds to a localization element in the chicken beta-actin mRNA and appears to direct localization to the leading edge of embryonic fibroblasts. Another founding member, the Xenopus Vg1RBP/VERA protein, binds to signals directing localization of Vg1 and VegT mRNAs to the vegetal pole of the oocyte. Mammalian homologs, the Imp proteins, have been suggested to act in mRNA localization, mRNA stability, and translational regulation. A recent report (Munro, 2006) examined the RNA binding properties of Drosophila Imp protein, focusing specifically on the osk mRNA and its possible regulation by Imp. Although mutation of candidate Imp binding sites in the osk mRNA did block accumulation of Osk protein, loss of Imp activity did not cause a similar defect. This study shows that Imp interacts with Sqd and Hrp48, two proteins that regulate expression of osk and grk mRNAs. Mutation of the Imp gene does not substantially alter grk or osk expression. Nevertheless, the Imp mutant partially suppresses a grk misexpression phenotype, arguing that it does contribute to grk regulation but may act redundantly and does not have an essential role. Consistent with this interpretation, overexpression of Imp interferes with localization of grk mRNA (Geng, 2006).

Deployment of proteins that control patterning in the oocyte relies on coordinated programs of mRNA localization and translational control. Many RNA binding proteins contribute to these programs, and some interact with one another in regulatory RNPs. This study has shown that Imp is associated in an RNA-dependent manner with Sqd and Hrp48 and is thus part of a complex whose other members have clearly established roles in control of grk and osk expression. Imp does not have an essential role in regulation of either grk or osk mRNAs; both mRNAs are expressed with no obvious defects in Imp mutant ovaries. However, loss of Imp activity does partially suppress the grk misexpression defect in fs(1)K10 mutant oocytes, providing strong evidence that Imp contributes to regulation of grk. This view is reinforced by the colocalization of Imp with grk mRNA in vivo. Imp's role must be largely redundant, only becoming detectable when grk expression is perturbed. Overexpression of Imp has a much more dramatic effect, transiently blocking the dorsal localization of grk mRNA and disrupting localization and translational control of osk mRNA (Geng, 2006).

BREs mediate both repression and activation of oskar mRNA translation and act in trans

Asymmetric positioning of proteins within cells is crucial for cell polarization and function. Deployment of Oskar protein at the posterior pole of the Drosophila oocyte relies on localization of the oskar mRNA, repression of its translation prior to localization, and finally activation of translation. Translational repression is mediated by BREs, regulatory elements positioned in two clusters near both ends of the oskar mRNA 3' UTR. This study shows that some BREs are bifunctional: both clusters of BREs contribute to translational repression, and the 3' cluster has an additional role in release from BRE-dependent repression. Remarkably, both BRE functions can be provided in trans by an oskar mRNA with wild-type BREs that is itself unable to encode Oskar protein. Regulation in trans is likely enabled by assembly of oskar transcripts in cytoplasmic RNPs. Concentration of transcripts in such RNPs is common, and trans regulation of mRNAs may therefore be widespread (Reveal, 2010).

Three types of regulatory elements have been implicated in activation of osk mRNA translation: a 5′ activating element, the IBEs, and now the subset of BREs in the osk 3′ UTR C region. The BREs present an unusual case, being involved in both repression and activation. In principle, a repressive element could be thought to play a passive role in the activation that relieves repression: the element would need to be unoccupied or unproductively bound for activation to occur. For the BREs the role is active, not passive. In the context of the osk C transgene, repression occurs because the AB region BREs are intact. However, despite proper localization of the osk C mRNA to the posterior pole of the oocyte, the normal activation of translation to allow Osk protein expression at that site is defective and Osk protein levels are reduced. Thus, the C region BREs are required to release the mRNA from repression conferred by the AB region BREs. In the context of wild-type osk mRNA, both AB and C region BREs contribute to repression, and so the C region BREs must switch roles, first repressing and later activating. The activating function of C region BREs could be due to position in the mRNA. For example, activation might only occur when BREs are close to the poly(A) tail. Given the absence of a change in poly(A) tail length when activation is defective, any effect on the poly(A) tail itself would have to be more nuanced under this scenario. Activation could involve cooperation between the BRE-binding factor and another activating factor that binds only in the C region. At present, no protein is known to have that property (Reveal, 2010).

The best candidate for the factor that binds to the BREs to mediate activation is Bru, since the BREs were identified and defined by their ability to bind Bru. Moreover, mutation of the type II Bru-binding sites in the C region also disrupts activation. Therefore, if the activator is not Bru, it must be a protein or proteins with the ability to bind to the two different types of sites. Mutants lacking Bru function arrest oogenesis at a very early stage, obscuring any potential role in activation of osk mRNA translation (Reveal, 2010).

Certain defects in translational regulation of osk mRNA can, remarkably, be suppressed by the presence of an osk mRNA with wild-type regulatory elements. This phenomenon is reminiscent of transvection, in which regulatory elements controlling transcription of one allele of a gene can influence transcription of the second allele on the homologous chromosome. A similar model is suggested for translational regulation in trans, in which regulation imposed on one molecule via direct binding of regulatory factors is then conferred on another molecule via association of the mRNAs. Evidence for a physical association between osk transcripts has come from the demonstration that reporter mRNAs containing the osk 3′ UTR (which is necessary but not sufficient for localization) localize to the posterior pole of the oocyte only if endogenous osk mRNA is also present. This 'piggybacking' of the reporter mRNA relies on the PTB protein. PTB binds to multiple sites in the osk mRNA, forming a large aggregate in vitro. Thus, it appears that PTB links multiple osk transcripts to form large RNP particles in vivo. Piggybacking for mRNA localization provides an example of a trans effect in posttranscriptional regulation. For piggybacking, all that is necessary is the physical linkage: directed movement of one osk mRNA molecule to its destination at the posterior pole of the oocyte would confer the same movement on any other molecule in the same RNP particle (Reveal, 2010).

Would physical linkage alone be sufficient to confer all of the different forms of translational regulation on all osk mRNAs in the same RNP particle? At least one type of regulation (activation by the IBEs) is not conferred in trans, providing an example where physical linkage is not sufficient. However, under the current models for Bru/BRE-dependent repression, physical linkage could be sufficient for trans regulation by BREs. One model for repression involves the formation of silencing particles which in some manner limit accessibility to ribosomes. Presumably, any mRNA recruited to the particles would also be protected from ribosomes. A second model for repression involves recruitment of Cup to the osk mRNA by Bru. Cup binds to, and inactivates, eIF4E, thus interfering with initiation of translation. If the inactivated molecule of eIF4E is bound to the osk mRNA cap, then translation initiation is blocked. A weak point of this model has been the necessity that, for repression to be specific, both RNA contacts of the Bru/Cup/eIF4E complex would have to be with the same mRNA molecule: eIF4E would have to bind the cap of a particular osk transcript, and Bru would have to bind the BREs of the same transcript. What would prevent the Cup newly recruited by Bru to osk mRNA from inactivating the eIF4E bound to the cap of a different mRNA? In the context of an RNP containing predominantly osk mRNAs, inactivation of eIF4E by Cup would interfere with translation of any member of the local population of transcripts, even if the Bru/Cup/eIF4E ternary complex bridges two mRNAs. By this scenario, trans regulation would be an inherent feature of the mechanism. The specificity of such trans regulation would be limited by the degree to which the local population of transcripts is homogeneous, and crossregulation between different species of mRNAs would be possible. Recent characterization of sponge bodies has shown that osk mRNA is compartmentalized in the oocyte, with large reticulated sponge bodies having osk distributed in discrete domains. Compartmentalization of osk mRNA could impose selectivity on trans regulation, preventing features of osk regulation from being conferred promiscuously on other mRNAs. Assembly of mRNAs in large RNP particles is common, and elucidation of the rules dictating which types of translational regulation can and cannot be exerted in trans should have broad relevance (Reveal, 2010).

Control of localization-dependent Oskar protein accumulation after the initiation of translation

The appearance of Oskar protein occurs coincident with localization of oskar mRNA to the posterior pole of the Drosophila oocyte, and earlier accumulation of the protein is prevented by translational repression. The nascent polypeptide-associated complex (NAC) is required for correct localization of oskar mRNA. The timing of the defects suggests that, if NAC acts directly via an interaction with nascent Oskar protein, oskar mRNA should be undergoing translation prior to its localization. Polysome analysis confirms that oskar mRNA is associated with polysomes even in the absence of localization of the mRNA or accumulation of Oskar protein. Thus, the mechanisms that prevent accumulation of Oskar protein until it can be secured at the posterior pole of the oocyte include regulated degradation or inhibition of translational elongation (Braat, 2004).

Multiple RNA binding proteins are required for oskar repression, including Bruno (Bru), Bicaudal C (BicC), p50, Apontic (Apt), and Me31B, and at least a subset of these bind to a part of the osk 3' untranslated region (UTR) that contains Bruno Response Elements (BREs). Additional factors that are candidates to act in translational regulation of osk mRNA are identified by mutants with a bicaudal phenotype, since this phenotype is frequently associated with inappropriate expression of Osk. The original bicaudal (bic) locus encodes the ß subunit of the nascent polypeptide-associated complex (NAC; Markesich, 2000). Intact NAC, which contains an additional subunit, alpha, acts to prevent inappropriate interactions of the nascent peptide with signal recognition particle (SRP) and to ensure that nonsecretory proteins are not targeted to the endoplasmic reticulum (ER) (Braat, 2004).

Reduction of either alpha or ß NAC expression has an identical effect on localization of osk mRNA and accumulation of Osk protein during oogenesis. If the NAC action is direct and entails an interaction with nascent Osk peptide, osk mRNA should be associated with ribosomes at a time and place when it is thought to be translationally repressed. Indeed, by polysome analysis it has been found that a significant fraction of osk mRNA is associated with polysomes, even when Osk protein accumulation is dramatically reduced. Thus the apparent repression of osk mRNA translation includes, at least in part, a regulatory event that occurs after the initiation of translation (Braat, 2004).

Data is presented implicating NAC in the proper localization of osk mRNA. Although each NAC subunit has been suggested to have additional but separate roles, the similar phenotypes of the two mutants argues that the reduction of NAC activity is the primary defect. How does NAC contribute to osk mRNA localization? The possibilities are most usefully divided into direct and indirect models (Braat, 2004).

If NAC acts directly, then the complex would interact with the nascent Osk polypeptide as it exits from the ribosome. The one strong prediction of any direct model is that osk mRNA be associated with ribosomes at the stage when the localization defects appear. It is not possible to directly test that prediction, but it was shown that osk mRNA is polysomal in a mutant that accumulates very little Osk protein. Thus the phenomenon of translational repression of osk mRNA need not act exclusively at the level of initiation of translation. Consequently, it is probable that osk mRNA is indeed associated with ribosomes at the developmental stage when reduction of NAC activity causes defects in osk mRNA localization (Braat, 2004).

Given the known activity of NAC in mammalian cells, where its interaction with the nascent peptide prevents inappropriate binding of SRP and introduction into the secretory pathway, a direct role for NAC in osk mRNA localization could be to prevent Osk protein from association with ER. No association of osk mRNA with ER was detected in the NAC mutants, but the binding of NAC to nascent polypeptides may govern or restrict a wider range of interactions than has been demonstrated. If so, the absence of NAC might allow the nascent Osk peptide to make a different type of inappropriate interaction, which could direct the osk mRNA on polysomes to the large discrete bodies observed in the ooplasm of the mutants. The bodies appear to migrate slowly to the posterior pole, suggesting that the osk mRNA contained in the bodies can still interact with the localization machinery (Braat, 2004).

An indirect role for NAC in osk mRNA localization would involve an effect on the expression or processing of a factor required for osk mRNA localization. This indirect role could require either known or novel activities of NAC. It is noteworthy that the mRNA localization defects observed are unlike any that have been described previously, making it unlikely that the phenotype arises from indirect effects via the known localization factors (Braat, 2004).

Translation of osk mRNA has been thought to begin only after it is localized to the posterior pole of the oocyte, but the results now indicate that translation can be initiated prior to localization. Thus there appears to be a system to degrade the nascent protein, and translational elongation may be dramatically inhibited or blocked. Evidence for control after initiation does not challenge the conclusion that Bru-dependent repression of osk translation occurs at the level of initiation, but instead reveals that regulation can occur at multiple stages. Since disruption of the Bru-dependent mechanism leads to excessive Osk accumulation (although the effect can be modest), the postinitiation form of control does not appear to be sufficient for repression. The use of two forms of control may facilitate the highly efficient restriction of Osk protein accumulation prior to the posterior localization of osk mRNA (Braat, 2004).

Any model for regulated Osk protein stability must address the fact that removal of 3′UTR regulatory elements allows the protein to accumulate at any position in the oocyte or the nurse cells. Thus the simplest model, that a stabilizing condition exists exclusively at the posterior pole of the oocyte, is quite unlikely as a sole mechanism (Braat, 2004).

An effect on Osk protein stability mediated by the 3′UTR could best be explained by two models. In the targeting model, a regulatory element in the 3′UTR would recruit a factor that targets the growing polypeptide for proteolysis: recruitment of the targeting factor to the mRNA would be allowed prior to localization, and the binding protein or degradation factor would be displaced from the mRNA or inactivated upon posterior localization. In the pausing model, a protein bound to a 3′UTR regulatory element would inhibit the elongation phase of translation, increasing the time during which incomplete forms of Osk protein are exposed during synthesis. The pausing model of course demands that incomplete Osk proteins be unstable, either because of the inherent instability of an Osk polypeptide that has not assumed the folding of the mature protein or because of the action of factors that specifically recognize the partial protein and target it for degradation. Thus both models rely on regulatory elements in the osk 3′UTR, and the pausing model also requires a specific property -- instability when incomplete -- of the protein encoded by the regulated protein. Similar models have been proposed for regulation of the nanos mRNA, which is also polysome-associated under conditions in which the Nanos protein does not accumulate (Braat, 2004).

In addition to osk mRNA, there are several other examples in which protein accumulation is prevented by a regulatory event that does not disrupt the assembly of polysomes on the transcript. In Drosophila, the nos mRNA displays this behavior, as do the lin-14 and lin-28 mRNAs of C. elegans. In no case has the mechanism responsible for preventing protein accumulation been elucidated. However, the C. elegans mRNAs are notable in that a trans-acting RNA, the lin-4 microRNA, mediates the effect. Recently, it has been shown that mutants defective in RNA interference lead to premature accumulation of Osk protein, as well as a variety of other defects. RNA interference could have an indirect action on translational control of osk mRNA. Alternatively, microRNAs might act directly, as in the translational control of lin-14 and lin-28. The demonstration that regulation of Osk accumulation occurs independent of polysome association strongly suggests a direct role. Proof will require demonstration of direct interactions of microRNAs with osk, and evidence that their selective disruption interferes with translational regulation (Braat, 2004).

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 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).

A repeated IMP-binding motif controls oskar mRNA translation and anchoring independently of Drosophila melanogaster IMP

Zip code-binding protein 1 (ZBP-1) and its Xenopus laevis homologue, Vg1 RNA and endoplasmic reticulum-associated protein (VERA)/Vg1 RNA-binding protein (RBP), bind repeated motifs in the 3' untranslated regions (UTRs) of localized mRNAs. Although these motifs are required for RNA localization, the necessity of ZBP-1/VERA remains unresolved. The role of ZBP-1/VERA was addressed through analysis of the Drosophila melanogaster homologue IGF-II mRNA-binding protein (IMP). Using systematic evolution of ligands by exponential enrichment, the IMP-binding element (IBE) UUUAY, was identified as a motif that occurs 13 times in the oskar 3'UTR. IMP colocalizes with oskar mRNA at the oocyte posterior, and this depends on the IBEs. Furthermore, mutation of all, or subsets of, the IBEs prevents oskar mRNA translation and anchoring at the posterior. However, oocytes lacking IMP localize and translate oskar mRNA normally, illustrating that one cannot necessarily infer the function of an RBP from mutations in its binding sites. Thus, the translational activation of oskar mRNA must depend on the binding of another factor to the IBEs, and IMP may serve a different purpose, such as masking IBEs in RNAs where they occur by chance. These findings establish a parallel requirement for IBEs in the regulation of localized maternal mRNAs in D. melanogaster and X. laevis (Monro, 2006).

IMP contains the four signature KH-type RNA-binding domains and the glutamine-rich COOH terminus that are present in the vertebrate orthologues. Affinity-purified antibodies against IMP reveal the protein in nurse cells and the oocyte early in oogenesis. However, the high concentration of IMP in the follicle cells blocks the penetration of the antibody into the oocyte after stage 4. Therefore, IMP localization was evaluated in a homozygous, viable, and fertile GFP-IMP protein trap line. GFP-IMP is enriched around the nurse cell nuclei and accumulates in the oocyte as soon as it is specified in the germarium, where it shows a uniform distribution until stage 7. IMP accumulates transiently at the anterior of the oocyte during stages 8-9 and then localizes in a crescent at the posterior pole at stage 9, where it remains for the duration of oogenesis. This pattern of localization is very similar to that observed for osk mRNA and Stau protein, which colocalize with IMP throughout oogenesis (Monro, 2006).

To ascertain whether IMP localization depends on osk, whether it is perturbed in various mutants that affect the posterior accumulation of osk mRNA and protein was examined. IMP does not localize to the posterior of the oocyte in staufen, barentsz, and hrp48 mutants, which block the transport of oskar mRNA to the posterior pole. Furthermore, IMP colocalizes with osk RNA to an ectopic dot in the center of the oocyte in a par-1 mutant that disrupts microtubule polarity. Together, these results demonstrate that the localization of IMP to the oocyte posterior pole requires the localization of osk mRNA (Monro, 2006).

IMP could localize to the posterior through a direct interaction with osk mRNA or protein or could be recruited to the posterior by a downstream component of the pole plasm. To distinguish between these possibilities, IMP localization was examined in a strong vasa hypomorph (vasaPD/Df(2L) TW2), which prevents the posterior recruitment of Vasa by Osk and disrupts all subsequent steps in pole plasm assembly. IMP localizes normally to the posterior of these oocytes, suggesting that its posterior accumulation depends on osk directly. Finally, whether IMP localization depends on Osk protein rather than osk mRNA was addressed by examining a nonsense mutation (osk54/Df) that disrupts the anchoring, but not the initial localization, of osk mRNA. IMP still localizes to the posterior of these oocytes at stage 9, but the posterior crescent is weaker than in wild type (WT) and disappears at stage 10. Thus, IMP behaves like osk mRNA in every mutant combination examined, suggesting that it localizes to the posterior in association with the mRNA (Monro, 2006).

The object of this study was to address whether D. melanogaster IMP is required for mRNA localization, because previous studies of its vertebrate homologues, ZBP-1 and VERA/Vg1RBP, had not resolved this question definitively. This study demonstrated that IMP binds directly to osk mRNA at well defined sites that are required for osk translation and anchoring. The best evidence that these sites are bona fide IBEs is that IMP is not recruited to the posterior by osk mRNA in which all 13 IBEs have been mutated with a single base change. Indeed, this is one of the only cases where it has been possible to demonstrate that an RBP interacts in vivo with well defined elements identified biochemically in vitro. In vitro, mutant RNA still competes for binding of IMP, albeit less effectively than the WT osk RNA, suggesting that the 3'UTR may contain other lower affinity sites. However, these sites are not involved in the recruitment of IMP to the posterior in vivo, nor are they sufficient for translational activation. Although the IBEs are thus bona fide in vivo IMP-binding sites, their role in osk RNA translation and anchoring is independent of IMP, which is not required for these activities (Monro, 2006).

Two outcomes of this investigation seem particularly surprising: (1) IBEs are required not for the initial localization of osk mRNA, but instead for its translational activation once it is localized and its subsequent anchoring at the posterior pole; (2) osk mRNA localization-dependent translation and anchoring require the IBEs in its 3'UTR, but not IMP itself (Monro, 2006).

Because Osk protein defines where the pole plasm forms, and hence where the pole cells and abdomen develop, it is essential that osk mRNA is only translated at the oocyte posterior. Indeed, translational control is arguably more important than localization in restricting Osk to the posterior, as normally only 18% of osk mRNA is actually localized, and osk mRNA localization mutants such as barentsz produce a normal abdomen. The translational repression of unlocalized osk mRNA occurs in different ways, depending on the stage of oogenesis. Mutants in RNA interference pathway components cause premature translation of osk mRNA during early oogenesis. Repression at later stages does not depend on these components, but instead requires the binding of Bruno and Hrp48 to three elements in the 3'UTR called Bruno response elements. This repression may occur at the level of translation initiation through the binding of Bruno to Cup protein and of Cup to the Cap-binding protein eIF4E, implying that the 5' and 3' ends of the mRNA are linked (Monro, 2006).

Much less is known about how osk mRNA translation is derepressed at the posterior, apart from the findings that a 297-nt element at the 5' end is required for the localization-dependent activation of a reporter RNA fused to the osk 3'UTR and that the osk 3'UTR, although sufficient to repress the translation of heterologous coding sequences, is insufficient to activate their translation at the posterior. The current data now provide direct evidence that the osk 3'UTR, through its IBEs, is required for translational derepression. Therefore, like activation, repression involves both the 5' and 3' ends. Moreover, three osk transgenes with only 3 out of 13 sites mutated at a single base prevent osk translational derepression. These are much more subtle mutations than the deletions that have previously been used to define osk derepression elements and these will be useful for identifying the corresponding derepressor proteins (Monro, 2006).

Although the CPEB homologue, Orb, and the RISC component, Aubergine, have been proposed to play a role in osk translational activation, mutants in these proteins also affect the initial localization of osk mRNA to the posterior, and this may account for the observed reduction in Osk protein levels. The only mutant combination that produces a similar phenotype to osk13TTgAY, with only 3 out of 13 sites mutated at a single base, is stau-null mutants that have been rescued by a transgene-expressing Stau protein that lacks the fifth double-stranded RNA-binding domain. However, Stau is unlikely to be the putative factor that interacts with the IBEs in the osk 3'UTR to activate translation, both because Stau recognizes double-stranded RNA rather than short-sequence motifs and because the IBE mutations prevent osk mRNA translation without affecting Stau localization to the posterior pole at stage 9 (Monro, 2006).

This brings us to the most significant outcome of this investigation: osk RNA translational activation and anchoring is disrupted by mutants in the IBEs, but not by the loss of IMP itself. The possibility that the IBE mutations prevent osk mRNA derepression and IMP localization indirectly by altering the structure of the RNA seems extremely unlikely, since single-base substitutions within three nonoverlapping sets of three IBEs in widely separated regions of the >1-kb osk 3'UTR produce an identical and very specific defect in translation, without affecting any of the earlier functions of the 3'UTR, such as the maintenance of oocyte fate, the transport of the mRNA from the nurse cells into the oocyte, the translational repression of unlocalized mRNA, or its localization to the posterior pole. Thus, none of these mutations disrupt the binding of any of the factors that mediate these earlier steps, including Staufen, which is thought to recognize the secondary structure of the RNA through the interaction of its double-stranded RNA-binding domains with multiple stem loops. This strongly argues against the possibility that the single base changes to the IBEs inhibit osk RNA translation through a nonspecific effect on RNA folding. This leads to the conclusion that the IBEs play a direct role in the derepression of osk mRNA translation (Monro, 2006).

Because IMP itself is not necessary for derepression, this implies that the IBEs are also recognized by another factor, called factor X. IMP and factor X could function redundantly to derepress osk translation, i.e., the two proteins might share osk's IBEs and compensate for each other's loss. However, factor X cannot be a ZBP-1/VERA family member because, unlike mammals, no such relatives are evident in the D. melanogaster genome (Monro, 2006).

Alternatively, IMP and factor X might function independently, i.e., osk derepression might occur exclusively through factor X binding. Rather than implementing osk's translational derepression, IMP's actual function might be to compete with factor X for IBE binding. In support of this, it was found that overexpression of IMP reduces Osk protein levels at the posterior. Although the purpose of IMP competition is presently unclear, one possibility is that IMP serves to bind, and thereby mask, IBEs that occur by chance in RNAs, for which factor X binding would be unnecessary or even detrimental. According to this view, competition with IMP would restrict factor X binding to those mRNAs, such as osk, that contain many copies of IBEs clustered within a restricted region. In the absence of IMP, factor X could bind to mRNAs with fewer IBEs and inappropriately regulate their translation. This may explain why embryos from imp-null oocytes always die, but from defects that appear unrelated to Osk function (Monro, 2006).

This analysis of the interaction of IMP with osk mRNA closely parallels that of ZBP-1 and VERA/Vg1RBP with ß-actin and Vg1 mRNA, respectively. (1) In each case, the protein has been shown to colocalize with the localized mRNA and can be UV cross-linked to it in extracts; (2) the precise binding sites of each protein have been determined and reveal that each protein recognizes a repeated motif in the target mRNA; (3) the function of these sites has then been analyzed by introducing specific point mutations that abrogate the binding of the protein, and these have been found to have a dramatic effect on translation or localization. This study has gone one step further to compare the phenotype of the IBE mutants with that of mutations in IMP itself. The observation that the former gives a fully penetrant defect in osk mRNA translation, whereas the latter has no phenotype in the germline, conclusively demonstrates that IMP is not responsible for the function of the IBEs in the osk 3'UTR. This is important in light of the observation that many RBPs have been implicated in the posttranscriptional regulation of particular mRNAs by studying the effects of mutations in their binding sites. These results highlight the potential limitations of this approach by demonstrating that one cannot necessarily infer the function of a protein from the phenotype of mutations in the cis-acting sequences that it recognizes (Monro, 2006).

The clear similarities between the localizations and functions of Vg1 and VegT mRNAs in X. laevis oocytes, and of osk mRNA in D. melanogaster oocytes, suggest that binding motifs for ZBP-1 proteins have a fundamental role in embryogenesis. Vg1, VegT, and osk localize as mRNAs to one pole of the oocyte, which is the site where the germ or pole plasm forms, and all three proteins play key roles in the formation of the primary body axis. These findings extend this parallel by showing that the localized expression of all three proteins also depends on a repeated RNA motif, defined by its interaction with IMP or its homologues. Because the results rule out a function for IMP in the regulation of osk mRNA, this calls into question the role of VERA/Vg1RBP1 in the localization of Vg1 and Veg T mRNAs, and it may therefore be worth considering the possibility that there is also a factor X in X. laevis (Monro, 2006).

An essential cytoplasmic function for the nuclear poly(A) binding protein, PABP2, in poly(A) tail length control and early development in Drosophila

Translational control of maternal mRNA through regulation of poly(A) tail length is crucial during early development. The nuclear poly(A) binding protein, PABP2, was identified biochemically from its role in nuclear polyadenylation. This study analyzed the in vivo function of PABP2 in Drosophila. PABP2 is required in vivo for polyadenylation, and Pabp2 function, including poly(A) polymerase stimulation, is essential for viability. An unanticipated cytoplasmic function is demonstrated for PABP2 during early development. In contrast to its role in nuclear polyadenylation, cytoplasmic PABP2 acts to shorten the poly(A) tails of specific mRNAs. PABP2, together with the deadenylase CCR4, regulates the poly(A) tails of oskar and cyclin B mRNAs, both of which are also controlled by cytoplasmic polyadenylation. Both Cyclin B protein levels and embryonic development depend upon this regulation. These results identify a regulator of maternal mRNA poly(A) tail length and highlight the importance of this mode of translational control (Benoit, 2005).

During early development in most species, regulation of gene expression is strictly posttranscriptional. One major posttranscriptional regulatory mechanism involves variations in poly(A) tail length, which regulate mRNA expression by affecting both mRNA stability and translation. Cytoplasmic changes in mRNA poly(A) tail length by deadenylation and polyadenylation play, thus, an essential role in controlling the production of key proteins during early development. While cytoplasmic polyadenylation has been studied extensively, the mechanisms underlying the control of poly(A) tail length in the cytoplasm are unknown (Benoit, 2005).

In Xenopus oocytes, cytoplasmic poly(A) tail elongation requires cis elements, including the cytoplasmic polyadenylation element (CPE) located in the 3' UTR of mRNAs and the nuclear polyadenylation signal, AAUAAA. CPEs are bound by the CPE binding protein (CPEB; see Drosophila CPEB, Orb), a primary factor in cytoplasmic polyadenylation, which also requires a poly(A) polymerase (PAP) and a complex that binds the AAUAAA element, called the Cleavage and Polyadenylation Specificity Factor (CPSF). Cytoplasmic elongation of the poly(A) tail leads to translational activation by remodeling the mRNP: in the repressed state, a translational repressor called Maskin binds to CPEB and eIF4E, the cap binding initiation factor, and precludes the eIF4E-eIF4G interaction that is required for translation initiation. When polyadenylation occurs, the elongated poly(A) tail is bound by the cytoplasmic poly(A) binding protein, PABP, which then interacts with eIF4G. This promotes the association between eIF4E and eIF4G, thereby allowing translation initiation (Benoit, 2005).

The role of the poly(A) tail in translational control in Drosophila is more controversial. In early embryos, a long poly(A) tail is both necessary and sufficient to induce translation of bicoid and Toll mRNAs, which encode the anterior morphogen and a determinant of dorsoventral polarity, respectively). Translation of the posterior determinant oskar (osk) mRNA, is also highly regulated during oogenesis. Translation is repressed until mid-oogenesis, and subsequently in the oocyte, as osk mRNA is being transported to the posterior pole. During this transport, a major translational repressor is Bruno, whose mechanism of action was found to be independent of the 5' cap and the poly(A) tail in vitro. A recent study, however, has identified a new translational repressor of osk mRNA, called Cup, which interacts with both Bruno and eIF4E. This strongly suggests that Cup/Bruno-mediated translational repression is cap-dependent, acting to prevent the eIF4E-eIF4G interaction in a manner similar to Maskin. While the mechanism underlying the release of Bruno repression at the posterior pole is unknown, accumulation of Osk protein at the posterior of the oocyte requires osk mRNA cytoplasmic polyadenylation involving Orb, the Drosophila homolog of CPEB and Drosophila PAP (Benoit, 2005 and references therein).

Before cytoplasmic regulation can occur, poly(A) tails are added to mRNAs in the nucleus in a cotranscriptional reaction involving endonucleolytic cleavage followed by polyadenylation. In mammals, the reaction involves two signals flanking the cleavage site, the upstream polyadenylation signal, AAUAAA, and a downstream GU-rich element. Cleavage requires several cleavage factors, including CPSF, and polyadenylation of cleaved RNAs can be recapitulated in vitro with CPSF, PAP, and the nuclear poly(A) binding protein, PABP2 (PABPN1 in mammals). While PAP has a very low affinity for, and binds aspecifically to, RNA, specificity is achieved through the recognition of the AAUAAA element by CPSF, which then tethers PAP to the RNA by direct protein-protein interaction. While CPSF thus stimulates PAP, complete stimulation occurs only in the additional presence of PABP2, which binds the poly(A) tail when it has reached ten residues in size. At this point, the reaction becomes processive, and a complete poly(A) tail is synthesized very rapidly and without dissociation of PAP from the RNA. PABP2 has a second function in nuclear polyadenylation, namely, to control poly(A) tail length: once the poly(A) tail has reached full length (250 residues in mammalian cells), the reaction becomes slow and distributive. These two functions of PABP2 in nuclear polyadenylation are carried out by different domains of the protein, and they can be uncoupled by point mutations. These data have led to a model in which multiple PABP2 proteins coat the growing poly(A) tail, with only one of them directly interacting with PAP (Benoit, 2005).

Although PABP2 is nuclear at steady-state levels in somatic cells, it shuttles from nuclear to cytoplasmic compartments. While a possible role for PABP2 in mRNA export has not been investigated, PABP2 has been found to be associated with an mRNA during its docking at the nuclear pore, and it was present on the cytoplasmic side of the nuclear envelope. This suggests that the exchange between nuclear PABP2 and cytoplasmic PABP on poly(A) tails occurs in the cytoplasm (Benoit, 2005).

This study used Pabp2 mutants to address the in vivo role of PABP2 in Drosophila.PABP2 has a role in poly(A) tail lengthening in somatic tissues, and that this function is essential for viability. The cytoplasmic presence of PABP2 is described in oocytes and early embryos (Benoit, 1999; full text of article), and it is shown that cytoplasmic PABP2 binds to poly(A) tails at these stages and shortens poly(A) tails of specific mRNAs, in conjunction with the deadenylase CCR4. Cytoplasmic poly(A) tail length control by PABP2 is essential for development, as embryos depleted of PABP2 show early developmental arrest, with elongated poly(A) tails of key maternal mRNAs (Benoit, 2005).

An important set of biochemical data has led to a precise description of PABP2 function in nuclear polyadenylation (Kühn, 2004). In in vitro polyadenylation assays, PABP2 has two distinct roles: it stimulates PAP to make polyadenylation processive, and it controls poly(A) tail length, with polyadenylation becoming distributive once the tail has reached 250 nucleotides. This length control involves a measurement of the poly(A) tail by PABP2. Drosophila PABP2 tested in mammalian polyadenylation-reconstituted assays also shows these two functions. Using a null Pabp2 allele, this study shows that poly(A) tails measured either on total or on individual mRNAs are shorter in Pabp2 mutants than they are in wild-type, consistent with a role for PABP2 in poly(A) tail elongation. The short poly(A) tails in mutant embryos appear to result from deadenylation of existing mRNAs and progressive reduction of new mRNA synthesis as the PABP2 level decreases. Poly(A) tails of newly synthesized Hsp70 mRNA were of similar size in the Pabp255 mutant and in wild-type, although they were present in a small amount in the mutant and were thought to be synthesized with the remaining maternal PABP2. The finding that newly synthesized mRNAs with short poly(A) tails do not accumulate when PABP2 is limiting suggests that PABP2 is absolutely required for polyadenylation and that PAP is unable to produce stable poly(A) tails in the absence of PABP2. In agreement with this, it was found that PABP2 is essential for viability, and specifically for cell viability, since Pabp255 mutant somatic or germline clones were found to not survive. Moreover, lethality in the absence of PAPB2 may be caused by a lack of PAP stimulation, since a Pabp2 transgene bearing a point mutation that prevents PAP stimulation is unable to rescue the lethality of the null allele Pabp255. Taken together, these results strongly suggest that the function of PABP2 in mRNA polyadenylation is essential, and that PAP in the absence of PABP2 is incapable of producing stable polyadenylated mRNAs (Benoit, 2005).

One important conclusion presented in this paper is the identification of an unexpected function for PABP2 in regulating poly(A) tail length of cytoplasmic mRNAs during early development. Using two hypomorphic Pabp2 alleles, it was found that a reduced amount of PABP2 leads to elongated poly(A) tails in two mRNAs regulated by cytoplasmic polyadenylation. Three sets of data indicate that this function of PABP2 is cytoplasmic: (1) the poly(A) tail elongation phenotype on the involved mRNAs is the opposite of the Pabp2 mutant phenotype on total mRNAs, also visible in the same RNA preparations on the control sop mRNA; (2) a reduced level of PABP2 restores longer poly(A) tails on osk and cyc B mRNAs, but not on sop mRNA, in orbmel ovaries in which cytoplasmic polyadenylation is impaired; (3) PABP2 is cytoplasmic in oocytes, and in early embryos prior to the onset of zygotic transcription, it binds poly(A) tails of mRNAs that are also bound by the cytoplasmic proteins Orb and PABP, and it is recruited into cytoplasmic cyc B mRNA particles (Benoit, 2005).

This cytoplasmic function of PABP2 is essential for early development. PABP2 is required to shorten poly(A) tails of, at least, osk and cyc B mRNAs, and in Pabp2 mutant germline clones the lengthening of cyc B poly(A) tails correlates with higher levels of Cyc B protein and with embryonic phenotypes similar to those produced by a high dosage of maternal Cyc B. Misregulation of other maternal mRNAs could also contribute to the lethality of embryos from these germline clones, since cytoplasmic PABP2 probably regulates several of them. The maternal-effect embryonic lethality of Pabp26 is strongly rescued by the Pabp2-I61S transgene, which lacks the nuclear function of PAP stimulation; this suggests that this lethality results from a defect in the cytoplasmic function of PABP2. In addition, the synergistic effect of the simultaneous decrease in PABP2 and CCR4 amounts in the female germline, which leads to important embryonic lethality and elongated poly(A) tails of osk and cyc B mRNAs, also indicates an essential function of cytoplasmic PABP2 in shortening poly(A) tails at these stages. Finally, consistent with PABP2 playing a major role in the cytoplasm during early development is the recent identification of a cytoplasmic PABP2 specific to embryos in Xenopus and mouse (Benoit, 2005).

Several lines of evidence suggest that PABP2 regulates poly(A) tail length in the cytoplasm by using a different mechanism than that used during nuclear polyadenylation. Termination of poly(A) tail elongation during nuclear polyadenylation is thought to result from a PABP2-dependent remodeling of the polyadenylation complex that blocks PAP stimulation. This remodeling depends on the complete coating of the poly(A) tail by PABP2. In sharp contrast, studies of cytoplasmic polyadenylation in Drosophila embryos suggest that the reaction is not processive and does not involve PAP stimulation by PABP2. Cytoplasmic polyadenylation of bicoid mRNA in embryos is slow, with poly(A) tail elongation depending on the level of PAP. Very long poly(A) tails are produced by overexpression of PAP, without poly(A) tail length control. Consistent with this, it was found that cytoplasmic PABP and PABP2 are present on the same mRNA poly(A) tails in ovary and early embryo extracts, thereby precluding complete coating of the poly(A) tail by PABP2 (Benoit, 2005).

In yeast, poly(A) tail length control involves deadenylation by the PAN (Pan2/Pan3) deadenylase, which is activated by poly(A) tail bound PABP. It is proposed that, similarly, during early Drosophila development, cytoplasmic PABP2 controls the poly(A) tail length of key mRNAs whose turnover and translatability are specifically regulated, by modulating the activity of a deadenylase. Poly(A) tail length control of these mRNAs would thus be achieved by the balance between cytoplasmic polyadenylation and deadenylation. A major deadenylation complex in Drosophila is the CCR4/NOT complex, in which CCR4 is the deadenylase (Temme, 2004). ccr4 function is essential in the female germline, where it regulates poly(A) tail lengths of cyc B mRNA and other cell cycle regulators. This study found that Pabp2 and ccr4 act in conjunction in shortening poly(A) tails of specific mRNAs, consistent with a possible role of PABP2 in stimulation of CCR4 activity. In yeast, deadenylation by the CCR4/NOT complex is inhibited in vitro by PABP. If this regulation is conserved in metazoans, the presence of PABP2 on poly(A) tails could modulate this effect of PABP (Benoit, 2005).

zucchini and squash encode two putative nucleases required for rasiRNA production in the Drosophila germline: zucchini and squash are required early during oogenesis for the translational silencing of osk mRNA and at later stages for proper expression of the Grk protein

RNAi is a widespread mechanism by which organisms regulate gene expression and defend their genomes against viruses and transposable elements. This study reports the identification of Drosophila zucchini (zuc) and squash (squ), which function in germline RNAi processes. Zuc and Squ contain domains with homologies to nucleases. Mutant females are sterile and show dorsoventral patterning defects during oogenesis. In addition, Oskar protein is ectopically expressed in early oocytes, where it is normally silenced by RNAi mechanisms. Zuc and Squ localize to the perinuclear nuage and interact with Aubergine, a PIWI class protein. Mutations in zuc and squ induce the upregulation of Het-A and Tart, two telomere-specific transposable elements, and the expression of Stellate protein in the Drosophila germline. These defects are due to the inability of zuc and squ mutants to produce repeat-associated small interfering RNAs (Pane, 2007).

RNAi has been shown to be involved in axial polarization in the Drosophila germline. In this species, establishment of dorsal-ventral (DV) and anterior-posterior (AP) axes is achieved through the localized translation of specific mRNAs. The protein products of gurken (grk) and oskar (osk) genes are essential for this process. Early during oogenesis, grk RNA encoding a TGFα-like molecule is localized to the posterior of the oocyte, where it signals the posterior fate to the adjacent follicle cells. Following the reorganization of the microtubule cytoskeleton at stage 8, the oocyte nucleus and grk RNA are relocalized to the dorsal-anterior corner of the oocyte. Grk protein now induces dorsal cell fates in the surrounding epithelial cells. In contrast to Grk, which is expressed throughout oogenesis, osk mRNA is kept silenced early during oocyte development. At later stages, Osk protein is found at the posterior of the oocytes, where it directs the organization of the germ plasm as well as abdomen formation of the future embryo. The silencing of oskar translation from stage 1 to 6 is controlled by a set of genes, including armitage (armi), maelstrom (mael), spindle-E (spn-E), and aubergine (aub), which have been shown to be required for RNAi phenomena. Mutations in these genes induce ectopic expression of Osk at early stages of oocyte development. This observation revealed a connection between the RNAi machinery and the establishment of the AP axis during Drosophila oogenesis. armi encodes the homolog of Arabidopsis SDE-3 helicase, which plays a role in post-transcriptional gene silencing (PTGS), a mechanism closely related to RNAi. mael encodes an evolutionarily conserved protein that is required for the proper localization of Ago2 and Dicer, two components of the RNAi machinery. aub and spn-E encode a member of the PIWI class of Argonaute proteins and a DExH RNA helicase, respectively. Aub and spn-E are involved in the silencing of some classes of transposable elements and tandem repeats in the germline, in heterochromatin formation, in double-stranded RNA (dsRNA)-mediated RNAi in embryos, and in the defense against viruses. Interestingly, spn-E and aub are also involved in telomere regulation. In most eukaryotes, the telomeres are maintained through the action of telomerase, the enzyme that ensures the addition of six- to eight-nucleotide arrays to the chromosome ends. However, in Drosophila, telomere elongation occurs after the transposition of non-long-terminal repeat (non-LTR) HeT-A, TAHRE, and TART retrotransposons. Mutations in spn-E and aub cause the upregulation of Het-A and TART expression in the germline, which, in turn, increases the frequency of telomeric element attachments to chromosome ends (Pane, 2007).

This study shows that the genes zucchini and squash are required early during oogenesis for the translational silencing of osk mRNA and at later stages for proper expression of the Grk protein. It is proposed that insufficient levels of Grk protein in zuc and squ mutants are at least partially due to activity of a checkpoint that affects Grk translation, similar to the effects of DNA repair mutants in meiotic oocytes. zuc encodes a member of the phospholipase-D/nuclease family, while squ encodes a protein with limited similarity to RNAase HII. Like Aub, Mael, and Armi proteins, Zuc and Squ localize to nuage, an electron-dense structure surrounding the nurse cell nuclei implicated in RNAi and RNA processing and transport. Zuc and Squ physically interact with Aub, thus pointing to a direct role for these proteins in the RNAi mechanisms. In further support of this conclusion, it has been demonstrated that zuc and squ are required for the biogenesis of rasiRNAs in ovaries and testes. Accordingly, mutations in these genes abolish the production of this class of siRNAs and lead to the deregulation of transposable elements and tandem repeats in the Drosophila germline (Pane, 2007).

zucchini and squash cause dorso-ventral patterning defects and egg chamber abnormalities during oogenesis: zuc and squ were identified in a screen for female sterile mutations on chromosome II of Drosophila. zuc and squ mutant females are viable, but produce eggs with a range of DV patterning defects. Flies with the most severe allele of zuc, zucHM27, lay few eggs, all of which are completely ventralized and often collapsed, whereas those with the weaker alleles, zucSG63 and zucRS49, produce some eggs with a more normal eggshell phenotype in addition to the ventralized eggs). In addition, a P element insertion in the coding region of the gene also acts as a strong loss-of-function allele with ventralized eggshell phenotypes. Three independent alleles of squ were recovered from the screen, namely squHE47, squPP32, and squHK3, and these alleles also generate a range of ventralized eggshell phenotypes (Pane, 2007).

Similar eggshell phenotypes have been described for mutations in other spindle class genes, which include both DNA repair enzymes such as spindle-B (spn-B) or okra (okr), as well as the RNAi components spn-E, aub, and mael. Similar to the spindle class mutants, several additional developmental defects can be observed in the zuc and squ mutants during oogenesis. In the wild-type oocyte, the nucleus condenses in a compact sphere, known as the karyosome. In contrast, the DNA in the nuclei of zuc and squ oocytes appears dispersed or in separate structures. Since compaction of chromatin in the karyosome occurs at stage 3, the defects observed in zuc and squ egg chambers indicate a function for the genes in the early development of the oocyte. Similar to spnE mutants, in a small number of zuc and squ egg chambers the oocyte is not positioned at the posterior as in wild-type, but is found in the middle of the egg chamber. Finally, fusion of egg chambers can also be observed in zuc mutants, resulting in egg chambers with 30 nurse cells and two oocytes. Many egg chambers in the zuc mutant undergo degeneration at different stages (Pane, 2007).

Grk expression is affected in zuc and squ mutants: The DV patterning defects suggested that the Gurken protein is not properly expressed in the mutant egg chambers. In earlier stages of oogenesis, Grk protein is detected in the oocyte similar to the wild-type egg chambers. At stage 9 in wild-type oocytes, Grk is localized in a cap above the oocyte nucleus, where it specifies the dorsal fate of the adjacent follicle cells. In zuc mutants, the amount of Grk protein found in the dorsal-anterior corner of the oocyte is strongly reduced or absent, suggesting that zuc controls the expression of Grk during mid-oogenesis. To further address this question, the distribution pattern of the grk transcript was analyzed in wild-type and zuc mutant egg chambers. In wild-type, grk mRNA localization mirrors the distribution of the protein and is found in the dorsal-anterior corner of the oocyte. Similarly, in zuc mutant egg chambers, grk mRNA is properly localized during mid-oogenesis. zuc therefore affects accumulation of the Grk protein in mid-oogenesis, most likely affecting the translation of the transcripts. This phenotype is also characteristic of the spindle class mutants in general (Pane, 2007).

In squ mutants, Grk protein also fails to accumulate properly in the oocyte at stage 9. Similar to zuc, analysis of grk transcripts in these mutants revealed that the grk mRNA is correctly localized in the majority of the squ egg chambers in mid-oogenesis. This result suggests that squ is also required for Grk translation (Pane, 2007).

Drosophila PTB promotes formation of high-order RNP particles and represses oskar translation

Local translation of asymmetrically enriched mRNAs is a powerful mechanism for functional polarization of the cell. In Drosophila, exclusive accumulation of Oskar protein at the posterior pole of the oocyte is essential for development of the future embryo. This is achieved by the formation of a dynamic oskar ribonucleoprotein (RNP) complex regulating the transport of oskar mRNA, its translational repression while unlocalized, and its translational activation upon arrival at the posterior pole. The nucleo-cytoplasmic shuttling protein PTB (hephaestus) (polypyrimidine tract-binding protein)/hnRNP I was identified as a new factor associating with the oskar RNP in vivo. While PTB function is largely dispensable for oskar mRNA transport, it is necessary for translational repression of the localizing mRNA. Unexpectedly, a cytoplasmic form of PTB can associate with oskar mRNA and repress its translation, suggesting that nuclear recruitment of PTB to oskar complexes is not required for its regulatory function. Furthermore, PTB binds directly to multiple sites along the oskar 3' untranslated region and mediates assembly of high-order complexes containing multiple oskar RNA molecules in vivo. Thus, PTB is a key structural component of oskar RNP complexes that dually controls formation of high-order RNP particles and translational silencing (Besse, 2009).

The finding that exogenous RNAs fused to the oskar 3'UTR hitchhike on endogenous oskar molecules for their localization at the posterior pole of the oocyte revealed the capacity of oskar to oligomerize in vivo and assemble into high-order RNP particles containing multiple mRNA molecules (Hachet, 2004). Importantly, the oskar 3'UTR is not only sufficient, but also required for in vivo oligomerization, as exogenous RNAs harboring deletions in this region fail to hitchhike on endogenous oskar (Besse, 2009).

PTB was identified as a trans-acting factor required for formation of high-molecular-weight complexes in vitro, and for efficient copackaging of both 3'UTR-containing reporters and endogenous oskar mRNAs in vivo. This property correlates with the strong binding of PTB to multiple sites dispersed throughout the 3'UTR. Interestingly, a chaperone activity has been proposed for the vertebrate PTB, based on its capacity to bridge two separate regions of the FMDV IRES (Song, 2005), on the conformational changes in RNA induced upon its binding (Mitchell, 2003; Pickering, 2004), as well as on its role in remodeling of the Vg1 RNP complex (Lewis, 2008). This function is further supported by a structural analysis revealing that RRM3 and RRM4 of human PTB adopt a fixed and atypical orientation in which the RNA-binding surfaces of these domains are positioned away from each other (Oberstrass, 2005). As a consequence, RRM3 and RRM4 have the capacity to bring distantly located tracts into close proximity and thus, induce looping of the bound RNA. Given that the amino acid composition of these two RNA-binding domains and their linker region is highly conserved, Drosophila PTB likely folds and functions similarly to its mammalian counterpart. In the context of oskar mRNA, PTB binding may therefore induce specific RNA folding required to establish the RNA-RNA or RNA-protein interactions essential for multimerization of oskar mRNA. Alternatively, PTB may itself bridge different oskar RNA molecules and nucleate the assembly of multimolecular complexes (Besse, 2009).

Repression of oskar mRNA translation is a complex process involving both cap-dependent and cap-independent mechanisms, as well as the presence of 5? and 3' regulatory regions. The phenotype of oocytes with reduced PTB levels indicates that Drosophila PTB, while dispensable for the transport of oskar mRNA, is required for translational repression of the localizing mRNA. Mammalian PTB is already known to regulate translation, mainly by promoting cap-independent translation initiation. PTB binds the IRES located on the 5'UTR of cellular and viral RNAs, thus enhancing the recruitment of trans-acting factors and ribosomes (Stoneley, 2004; Jang, 2006; Semler, 2008). Recent reports indicate that PTB can also promote translation of specific mRNAs when bound to their 3'UTRs (Reyes, 2007; Galban, 2008). However, PTB does not exclusively act as a translational activator, as its interaction with the IRES present in unr and bip mRNAs has been shown to down-regulate their activity (Kim, 2000; Cornelis, 2005). The results of this study suggest that Drosophila PTB also acts as a translational repressor when bound to the 3'UTR of a target mRNA (Besse, 2009).

The mechanism by which PTB enhances or represses translation of target mRNAs remains elusive. The most accepted hypothesis, which may also explain the multiple roles of PTB in RNA regulation, is its capacity to act as a chaperone molecule, promoting the folding of RNAs into specific conformations, thereby modulating the binding of other regulatory proteins. Thus, depending on the specific structure adopted by the RNA, the cellular context, and the binding of other regulatory trans-acting factors, PTB may either promote or inhibit recruitment of the translation machinery. In the case of oskar mRNA, however, the results show that PTB is not required for the binding of the two best-characterized oskar translational repressors, namely, Bruno and Hrp48. Although the possibility that PTB influences the activity of these proteins cannot be excluded, PTB function seems beyond the simple recruitment of oskar main translation repressors (Besse, 2009).

Significantly, the results establish an in vivo link between oskar translation repression and multimerization of oskar 3'UTR-containing RNAs. An attractive possibility is that PTB promotes the formation of densely packed oskar RNP particles, thereby rendering the mRNA inaccessible to the translation machinery. A similar cap-independent function has been proposed for the translational repressor Bruno, based on its ability to promote in vitro oligomerization of a 3'UTR fragment containing duplicated Bruno response elements (BREs), together with its capacity to nucleate the assembly of heavy silencing particles. However, it was observed that BREs are neither strictly required nor sufficient for the hitchhiking of oskar 3'UTR-containing RNAs on endogenous oskar in vivo. Therefore, other trans-acting factors and cis-acting sequences likely contribute to the formation of high-order oskar RNPs in the oocyte. In this context, the chaperone activity of PTB may be essential to promote multimerization of oskar molecules, which would ultimately contribute to their complete translational repression. In contrast, the oskar phenotype of ptb mutant oocytes suggests that the assembly of high-order RNP complexes does not play a significant role in oskar mRNA posterior transport (Besse, 2009).

The assembly of RNP complexes competent for mRNA localization and precise translational control has been suggested to occur in a stepwise manner, some factors associating with the mRNA in the nucleus, and being essential for the subsequent recruitment of other components in the cell cytoplasm. For example, nucleolar association of the RNA-binding protein She2p with its mRNA target ash1 was recently proposed to be an essential step in the assembly of translationally silenced localizing ash1 RNP complexes in yeast. Some oskar translation repressors have been shown to localize both in the nucleus and in the cytoplasm of germ cells. However, whether these regulators are recruited to the oskar complex in the nucleus and whether nuclear association of these factors is required for subsequent translation silencing have not been tested so far (Besse, 2009).

PTB belongs to the hnRNP family of nucleo-cytoplasmic shuttling RNA-binding proteins, which regulate different aspects of RNA metabolism both in the nucleus and in the cytoplasm of eukaryotic cells. Consistent with this, it was observed that Drosophila PTB not only colocalizes with oskar in the oocyte cytoplasm, but also strongly accumulates in the nuclei of germ cells. Given that the nuclear association of PTB with Vg1 mRNA has been proposed to control the subsequent localization of these transcripts in the cytoplasm of Xenopus oocytes (Kress, 2004), this study tested whether the association of Drosophila PTB with oskar mRNA in the nuclei of germ cells is required for its translation repression activity. Notably, it was found that a cytoplasmic version of PTB localizes to the posterior pole of wild-type and ptb mutant oocytes and that this localization is oskar-dependent, strongly suggesting that it is still able to associate with oskar mRNA. More importantly, the mutant GFP-PTB-δNLS is competent in oskar translation repression. Although it is possible that endogenous PTB is loaded onto oskar RNP complexes in the nucleus of germ cells, these data suggest that nuclear recruitment of PTB is not a prerequisite for the formation of translationally silenced complexes. This analysis supports a model in which the complex behavior of RNP particles is controlled by the independent association of specific protein modules in different cell compartments. It also provides further evidence for the reorganization of RNP complexes upon translocation into the cytoplasm (Besse, 2009).

Drosophila PAT1 is required for Kinesin-1 to transport cargo and to maximize its motility

Kinesin heavy chain (KHC), the force-generating component of Kinesin-1, is required for the localization of oskar mRNA and the anchoring of the nucleus in the Drosophila oocyte. These events are crucial for the establishment of the anterior-posterior and dorsal-ventral axes. KHC is also essential for the localization of Dynein and for all ooplasmic flows. Interestingly, oocytes without Kinesin light chain show no major defects in these KHC-dependent processes, suggesting that KHC binds its cargoes and is activated by a novel mechanism. This study sheds new light on the molecular mechanism of Kinesin function in the germline. Using a combination of genetic, biochemical and motor-tracking studies, PAT1 (Zheng, 1998), a KLC-like protein that binds APP, is shown to interact with Kinesin-1, functions in the transport of oskar mRNA and Dynein and is required for the efficient motility of KHC along microtubules. This work suggests that the role of PAT1 in cargo transport in the cell is linked to PAT1 function as a positive regulator of Kinesin motility (Loiseau, 2010).

Several lines of evidence suggest that the aberrant localization of oskar mRNA in Pat1 mutants is due to defects in the transport of the transcript. PAT1 is not required for any other step in oskar mRNA biogenesis, as Pat1 mutants do not affect its accumulation in the oocyte, its colocalization with Staufen or its translational regulation. Furthermore, the penetrance of the oskar mislocalization phenotype in Pat1 mutants is weaker at later stages of oogenesis, suggesting that the transcript is not physically detaching and that its anchoring at the posterior is normal. PAT1 is also not required for the polarization of the oocyte or the microtubule cytoskeleton, nor for the Dynein-dependent localization of RNAs such as bicoid (Loiseau, 2010).

Taking into account the homology of PAT1 to the cargo-binding domain of KLC (the TPRs) and the biochemical interactions between PAT1 and Kinesin-1, one possible model for the function of PAT1 in oskar transport is that PAT1 contributes to the binding of the transcript to KHC. Whether PAT1 associates with oskar mRNA is unknown, but it is interesting to note that mammalian PAT1 interacts with zipcode-binding protein 1 (also known as Igf2bp1), the Drosophila homolog of which, IMP, binds oskar mRNA and localizes to the posterior (Munro, 2006). However, oocytes lacking IMP localize oskar mRNA normally, indicating that the putative binding of PAT1 to IMP is not the only mechanism that would mediate the binding of PAT1 to the oskar-localizing complex (Loiseau, 2010).

If PAT1 contributes to the binding of cargo to KHC, it is possible that, at least for some cargoes, it does so in conjunction with KLC. This hypothesis is supported by several findings. Firstly, PAT1 and KLC have a similar domain organization, with high sequence similarity in the KHC- and cargo-binding domains. Secondly, PAT1 and KLC form a complex with KHC. Lastly, Dynein seems to localize normally to the posterior in Pat1 or Klc single mutants, but it accumulates at the anterior/lateral cortex when both genes are mutated, which is equivalent to the Dynein mislocalization phenotype observed in Khc-null mutant oocytes. These results suggest that PAT1 and KLC might redundantly mediate the interaction of Dynein with KHC that is required for its transport to the posterior (Loiseau, 2010).

This seems not to be the case for the transport of oskar mRNA to the posterior pole of the oocyte. Although the oskar mRNA mislocalization phenotype is more highly penetrant in Pat1, Klc double-mutant oocytes than in the single mutants, in contrast to Khc mutant oocytes, oskar mRNA is not found at the anterior/lateral cortex in these double mutants, but in the center and posterior of the oocyte. This suggests that PAT1 and KLC are not essential for KHC to bind oskar mRNA, and that other proteins must contribute to the interaction of oskar with the motor (Loiseau, 2010).

In contrast to the germline, where the absence of KLC results in no major defects in KHC-dependent processes, both Khc and Klc mutant larvae exhibit a neuronal phenotype in which axonal cargoes (e.g. synaptotagmin vesicles) accumulate in 'clogs' in the peripheral nerves. Furthermore, Khc and Klc mutant larvae present locomotion defects, flip their posterior region upwards and paralyze progressively. Preliminary data show that Pat1 mutant axons also exhibit a high number of clusters of synaptotagmin vesicles, suggesting that PAT1 is also required for KHC function in the nervous system. However, Pat1 mutants are viable and Pat1 larvae show no obvious phenotypes. This indicates that Kinesin-1 has PAT1-independent functions that are important for locomotion and viability. In contrast to Khc and Klc mutants, the axonal clogs are not observed in the proximal region of the Pat1 mutant peripheral nerves. This difference between Pat1 and Khc or Klc larvae might explain the absence of paralysis and lethality in Pat1 mutants. For further analysis of PAT1 function in the nervous system, it would be interesting to study whether the transport of molecules localized in a KLC-independent manner (e.g. FMRP or GRIP1) is disrupted in Pat1 or Pat1, Klc double-mutant neurons (Loiseau, 2010).

In vertebrates there are two partially functionally overlapping KLC isoforms. The present study suggests that PAT1 might be a second KLC isoform in Drosophila. In light of the current results it would be interesting to analyze whether KHC can form various complexes with PAT1 and with KLC. These distinct complexes could have overlapping, but not identical, specificities, providing a possible explanation for how one motor coordinates the transport of its many cargoes (Loiseau, 2010).

In vitro motility assays using purified molecules are powerful systems to obtain mechanistic insights into the function of motors, but they might eliminate physiologically relevant factors that regulate their functions. The use of KHC-GFP ovary extracts on defined microtubule tracks allowed analysis of active KHC with and without PAT1 under more physiological conditions. The results show that the velocity and run length of KHC, but not its association time, are reduced in the absence of PAT1. PAT1 is the first protein shown to positively regulate Kinesin speed and run length, without affecting its association time, revealing a novel mechanism of Kinesin regulation (Loiseau, 2010).

In light of the homology of PAT1 to the HR motif of KLC, and because PAT1 is found in a complex with KHC and KLC in co-immunoprecipitation experiments, a simple hypothesis for how PAT1 stimulates KHC motility is by direct physical interaction. Although interactions have been detected between Drosophila KLC and mammalian PAT1 in a yeast two-hybrid assay, no direct interaction has been detected between Drosophila KHC and PAT1 in this assay. This result suggests that either the PAT1 binding to KHC is transient and/or regulated by additional factors [e.g., KLC), or the hypothesis is incorrect and PAT1 does not directly bind KHC (Loiseau, 2010).

The mechanistic details of how PAT1 facilitates Kinesin-dependent transport would be the focus of future research. A potential mechanism for PAT1 to affect the motility of KHC is through the regulation of the KHC oligomerization state. To test this, the fluorescence intensity and pattern of fluorescence decay for each particle tracked were analyzed. Although GFP fluorescence fluctuates, it is estimated that most particles contained one or two active GFP molecules, indicating that each particle primarily contained a single dimer of KHC in both control and Pat1 mutant extracts. Thus, the formation of higher-order oligomers is unlikely to account for the observed difference in velocity. Another possibility is that PAT1 affects Kinesin activity by binding microtubules, as PAT1 was identified in human cells as a weak microtubule-interacting protein. However, this is thought not to be the case, since Drosophila PAT1 did not show a strong affinity for microtubules in sedimentation assays (Loiseau, 2010).

What other mechanisms could account for the effect of PAT1 on Kinesin motility? One possibility is that PAT1 modulates KHC activity by acting on the motor domain. If it is assumed that PAT1 interacts with KHC via the stalk region, as is the case for KLC, how could this PAT1-KHC association act on the motor domain? Analysis of the KHC conformation changes revealed by FRET on cells confirmed that Kinesin-1 is partially folded while undergoing microtubule-based transport. This conformation would allow the stalk, and consequently PAT1, to come into close molecular contact with the neck and motor domains. Once in close contact, PAT1 could induce a change in the structure of the motor domain, for example by affecting the proximity between the two heads, so that they are in proper proximity for processive motility, or by optimizing the internal tension required for the coordinated activity of the two heads. It is also conceivable that the interaction of PAT1 with Kinesin releases an inhibitory mechanism of motor activity, as it has been shown for JIP1 (APLIP1) and UNC-76. In either case, these effects could be achieved directly by PAT1, or by an additional factor that is recruited to the motor in a PAT1-dependent manner, as has been described for the KHC-interacting protein Milton. Further biochemical and biophysical studies are needed to understand exactly how PAT1 achieves this novel regulation of KHC motility (Loiseau, 2010).

Whether PAT1 functions in both cargo binding and regulation of KHC motility is not clear. Genetic analyses suggest that PAT1 and KLC mediate the interaction of Dynein with KHC; Dynein mislocalizes to the anterior/lateral cortex in a similar manner in Pat1, Klc double-mutant and Khc mutant oocytes. In the case of oskar mRNA it is possible that the slower motility of KHC in the absence of PAT1 explains the mislocalization of this transcript in Pat1 oocytes. This hypothesis is supported by several observations. Firstly, in contrast to components of the oskar-localization complex, PAT1 does not seem to accumulate at the posterior and is diffuse in the cytoplasm of the germline. Secondly, the absence of Pat1 enhances the oskar mRNA mislocalization phenotype in oocytes expressing tailless KHC and it reduces the motility of this truncated motor. Thirdly, the oskar mRNA phenotype in Pat1 mutants is similar to that of Khc17 and Khc23, which are mutant alleles that result in a less motile KHC. In these alleles, oskar mRNA localizes to the posterior pole, but there is a small amount that accumulates in the middle of the oocyte. This is due to a delay in the transport of oskar mRNA. This correlation between slowed motor mechanochemistry and ectopic oskar accumulation as a result of slowed oskar transport supports the hypothesis that the oskar phenotype in Pat1 mutants is due to a slower KHC (Loiseau, 2010).

PAT1 was first identified in human cells, where it weakly interacts with microtubules and with APP. This work in human cells showed that PAT1 appears to regulate APP trafficking and processing, although the molecular mechanism of this regulation is not understood. The results support a model in which the role of PAT1 in APP processing is probably linked to PAT1 function in regulating Kinesin. This hypothesis is further supported by recent work showing that mammalian PAT1 is required for the transport of mRNAs in neurons. Thus, the function of PAT1 as a novel regulator of Kinesin motility and cargo transport seems to be evolutionarily conserved (Loiseau, 2010).

Drosophila javelin-like encodes a novel microtubule-associated protein and is required for mRNA localization during oogenesis

Asymmetrical localization of mRNA transcripts during Drosophila oogenesis determines the anteroposterior and dorsoventral axes of the Drosophila embryo. Correct localization of these mRNAs requires both microtubule (MT) and actin networks. This study identified a novel gene, CG43162, that regulates mRNA localization during oogenesis and also affects bristle development. The Drosophila gene javelin-like, which was identified based on its bristle phenotype, is an allele of the CG43162 gene. Gemale mutants for jvl produce ventralized eggs owing to the defects in the localization and translation of gurken mRNA during mid-oogenesis. Mutations in jvl also affect oskar and bicoid mRNA localization. Analysis of cytoskeleton organization in the mutants reveal defects in both MT and actin networks. Jvl protein colocalizes with MT network in Schneider cells, in mammalian cells and in the Drosophila oocyte. Both in the oocyte and in the bristle cells, the protein localizes to a region where MT minus-ends are enriched. Jvl physically interacts with SpnF and is required for its localization. Overexpression of Jvl in the germline affects MT-dependent processes: oocyte growth and oocyte nucleus anchoring. Thus, these results show that a novel MT-associated protein affects mRNA localization in the oocyte by regulating MT organization (Dubin-Bar, 2011).

In order to investigate further the role of Spn-F in MT organization, new proteins that interact with Spn-F or Ik2 were sought. This study led to identification of the gene CG43162 as a novel MT-associated protein, which is part of this complex. Moreover, the study showed that CG43162 encodes the javelin-like (jvl) gene. Several lines of evidence suggest that that jvl encodes the CG43162 gene: (1) Using fine deficiency mapping of jvl mutants showed that jvl is found in CG43162 region; (2) it was shown that downregulation of CG43162 specifically in the bristles led to defects in bristle morphology, similar to the defects found in jvl mutants; 3) furthermore, a mutation in CG43162 (CG43162D590) failed to complement jvl in both ovarian and bristle phenotypes, suggesting that CG43162D590 and jvl are two different alleles of the same gene; and (4) expression of CG43162 protein in oocytes was found to rescue jvl female sterility. Considering all of these results, it is concluded that the CG43162 gene encodes jvl (Dubin-Bar, 2011).

Moreover, it is suggested that Jvl is part of the Spn-F and Ik2 complex, based on the following evidence: (1) Spn-F physically interacts with Jvl (yeast two hybrid and GST pull-down assays); (2) Spn-F physically interacts with Ik2 (Dubin-Bar, 2008); (3) jvl shares similar mRNA localization and bristle defects to spn-F and ik2; (4) Spn-F and Ik2 colocalize with Jvl to MT, where Jvl determines this localization pattern (Dubin-Bar, 2011).

For further analysis of the jvl gene, Jvl protein localization was characterized. For this purpose, the localization of Jvl protein in S2R+ cells and human cells was analyzed. GFP-Jvl fusion protein was localized to the MT network. Next, the localization pattern of Jvl during oogenesis was analyzed. Using an antibody raised against the Jvl protein, it was found that Jvl is localized to the region where the MT minus-ends reside. At early stages of oogenesis, Jvl protein localizes as a tight crescent in the posterior pole of the oocytes. During mid-oogenesis, Jvl protein is localized all around the cortex, with enrichment at the anterior pole. It was also demonstrated that GFP-Jvl colocalizes with MTs in the nurse cells. Moreover, in the bristles, GFP-Jvl is localized asymmetrically, accumulating at the bristle tip, where other MT minus-end markers are found. Considering these results, indicating that Jvl localizes with the MT network in S2R+ and human cells along with its localization in the egg chamber and developing bristle, it is concluded that Jvl protein is associated with the MT network, specifically with the MT minus-ends (Dubin-Bar, 2011).

jvl1 mutants are female fertile. However, flies hemizygous for jvl1 and flies transheterozygous for jvl (jvl1/jvl2) are female sterile. Beside sterility, it was noticed that the jvl mutant females laid eggs with dorsal-ventral defects. Determination of dorsal-ventral polarity of the eggshell depends on Grk protein signaling. In the hemizygous mutants, grk mRNA localizes in the anterior margins of the oocyte and in ectopic sites inside the oocyte. It has been suggested that grk mRNA moves in two distinct steps, both of which require MT and the motor protein Dynein. Each step depends on a different MT network. First grk mRNA moves towards the anterior of the oocyte, where it localizes transiently, and then to its final localization in the dorsal anterior corner of the oocyte. In jvl mutants, grk mRNA does not reach its final localization in the dorsal anterior corner of the oocyte, suggesting that the MT network upon which this step depends might be impaired in jvl mutants. This MT network is specifically associated with the oocyte nucleus and the minus-end in the dorsal-anterior corner of the oocyte. Next, it was found that Grk protein in jvl mutants is also mislocalized. Grk protein is colocalized with ectopic actin puncta close to the anterior of the oocyte. This localization pattern is also observed in Bicaudal-C and trailer-hitch mutants. It has been suggested that the sequestration of Grk in the actin cages interfered with the signaling to the follicle cells; therefore, it is suggested that sequestration of Grk in the actin cages in jvl mutant females similarly led to the dorsal-ventral polarity defects of the eggshell. In addition to the effect on grk mRNA and protein localization, jvl also affects bcd and osk mRNA localization. In wild-type, bcd mRNA is localized to the anterior pole of the oocyte facing the nurse cells, whereas osk mRNA is localized to the opposite posterior pole. The polar localization of these two mRNAs is maintained throughout the rest of oogenesis and well into early embryogenesis. The anterior localization of bcd requires both intact MTs and dynein motor protein function. osk localization to the posterior pole is achieved by two phases of transport: long-range MT-dependent transport by kinesin to the posterior, followed by actomyosin V-dependent positioning at the oocyte cortex (Dubin-Bar, 2011).

What could be the function of Jvl protein during oogenesis? The effects of jvl on grk and bcd mRNA localization, along with the particular changes affecting cytoskeletal organization close to the oocyte nuclear membrane as evident for Nod:KHC:β-gal localization and Tau mislocalization, suggest that jvl might be involved in either transport to the minus-end of MTs or in the organization of the minus-ends of the microtubule around the oocyte nucleus, as been suggested for its interactor, Spn-F (Abdu, 2006). However, it was also noticed that in jvl mutants, osk mRNA and protein are mislocalized. These phenotypes are probably not due to defects in either transport or organization of the MT plus-end, as the plus-end motor protein Kinesin I was properly localized as in the wild type. Examination of the cytoskeleton components of the oocyte shows that both actin and MTs are misorganized in jvl mutants. The MT levels along the anterior cortex of the oocyte were reduced with specific effects on the MT that surrounds the oocyte nucleus. However, ectopic aggregations of the actin cages were found in the middle of the oocyte. The defects in the organization of both actin and MT network, together with the defects in osk mRNA and protein localization, suggest that jvl could provide a connection between the actin and MT network. In summary, these results suggest that jvl plays a role in organization of the MT in the oocyte or in the stabilization of the connection between MT and actin cytoskeleton in the oocyte (Dubin-Bar, 2011).

This study also examined the effects of overexpression of Jvl in the germline. Overexpression of Jvl with different germline-specific Gal 4 affects oocyte growth, oocyte localization and, in later stages, oocyte nucleus localization. Interestingly enough, all of these phenotypes could arise from effects on MT network function (Dubin-Bar, 2011).

Oocyte growth depends on several processes: early in oogenesis, until stage 7, the oocyte grows at approximately the same rate as a single nurse cell. At these stages, oocyte growth is due to the transport of mRNAs and proteins, including products of early pattern-formation genes from the nurse cells to the oocyte. This transport is a microtubule-dependent process. Later in oogenesis, after stage 7, oocyte growth depends on the transport of components such as lipid droplets, mitochondria and other single particles from the nurse cells into the oocyte. This transport is an actin-dependent process. Beginning in stage 8, the oocyte expands through the uptake of yolk from the surrounding follicle cells and hemolymph. Consequently, oocyte growth is more rapid than nurse cell growth. During stage 11, the remaining nurse cell cytoplasm is rapidly transferred to the oocyte, resulting in doubling the oocyte volume. Overexpression of Jvl affects oocyte growth during stage 6 to stage 8, although the egg chamber size seems to be similar to that of wild-type stage 6 to 8 egg chambers. In these stages, oocyte growth depends on the transport of nutrients from the nurse cells to the oocyte, suggesting that overexpression of Jvl disrupted this transport. The fact that Orb protein is not detected in Jvl-overexpressing small oocytes strengthens this possibility (Dubin-Bar, 2011).

Another phenotype that was obtained in moderate overexpression of Jvl is mislocalization of the oocyte nucleus in 15% of stage 9 egg chambers. During early stages of oogenesis, the oocyte nucleus localizes to the posterior pole of the oocyte. After stage 7, following Grk signal and reorganization of the MT network, the nucleus migrates towards the anterodorsal corner of the oocyte. Positioning of the oocyte nucleus involves two anchoring steps: first anchoring to the lateral membrane, which requires dynein but not kinesin motor protein; and, second, after it localizes to the anterodorsal corner, anchoring to the anterior cortex of the oocyte, which requires both dynein and kinesin motor proteins. Moreover, nucleus anchoring also requires correct organization of the MT scaffold that surrounds the oocyte nucleus. Moderate expression of Jvl did not affect nucleus position in stage 8 egg chambers. At this stage, the nucleus was always at the dorsal anterior corner, as in the wild type. This finding implies that anchoring to the lateral cortex and migration of the oocyte nucleus is not affected in Jvl-overexpressing ovaries. However, the anchoring of the nucleus to the anterior membrane was affected. This could be due to misorganization of the MT scaffold that surrounds the nucleus. Thus, these results demonstrate that overexpression of Jvl protein affects MT-dependent processes such as transport of determinants from the nurse cells to the oocyte, and anchoring of oocyte nucleus to the anterior cortex of the oocyte. Taking into account the phenotypes detected in jvl mutants, the finding that Jvl is an MT-associated protein, together with the effects of Jvl overexpression on MT-dependent processes during oogenesis, it seems likely that jvl has a role in MT organization during oogenesis (Dubin-Bar, 2011).

Most importantly, although jvl encodes for a protein with no homology beside insects, its association with MT network in mammalian cells, along with its effect on MT network in Drosophila, may suggest the existence of mammalian protein(s) with a function analogous to Jvl (Dubin-Bar, 2011).

Dynein associates with oskar mRNPs and is required for their efficient net plus-end localization in Drosophila oocytes

In order for eukaryotic cells to function properly, they must establish polarity. The Drosophila oocyte uses mRNA localization to establish polarity and hence provides a genetically tractable model in which to study this process. The spatial restriction of oskar mRNA and its subsequent protein product is necessary for embryonic patterning. The localization of oskar mRNA requires microtubules and microtubule-based motor proteins. Null mutants in Kinesin heavy chain (Khc), the motor subunit of the plus end-directed Kinesin-1, result in oskar mRNA delocalization. Although the majority of oskar particles are non-motile in khc nulls, a small fraction of particles display active motility. Thus, a motor other than Kinesin-1 could conceivably also participate in oskar mRNA localization. This study shows that Dynein heavy chain (Dhc), the motor subunit of the minus end-directed Dynein complex, extensively co-localizes with Khc and oskar mRNA. In addition, immunoprecipitation of the Dynein complex specifically co-precipitated oskar mRNA and Khc. Lastly, germline-specific depletion of Dhc resulted in oskar mRNA and Khc delocalization. These results therefore suggest that efficient posterior localization of oskar mRNA requires the concerted activities of both Dynein and Kinesin-1 (Sanghavi, 2013).

The auto-inhibitory domain and ATP-independent microtubule-binding region of Kinesin heavy chain are major functional domains for transport in the Drosophila germline

The major motor Kinesin-1 provides a key pathway for cell polarization through intracellular transport. Little is known about how Kinesin works in complex cellular surroundings. Several cargos associate with Kinesin via Kinesin light chain (KLC). However, KLC is not required for all Kinesin transport. A putative cargo-binding domain was identified in the C-terminal tail of fungal Kinesin heavy chain (KHC). The tail is conserved in animal KHCs and might therefore represent an alternative KLC-independent cargo-interacting region. By comprehensive functional analysis of the tail during Drosophila oogenesis, an understanding was gained of how KHC achieves specificity in its transport and how it is regulated. This is the first in vivo structural/functional analysis of the tail in animal Kinesins. The study shows that the tail is essential for all functions of KHC except Dynein transport, which is KLC dependent. These tail-dependent KHC activities can be functionally separated from one another by further characterizing domains within the tail. In particular, the data show the following. First, KHC is temporally regulated during oogenesis. Second, the IAK domain has an essential role distinct from its auto-inhibitory function. Third, lack of auto-inhibition in itself is not necessarily detrimental to KHC function. Finally, the ATP-independent microtubule-binding motif is required for cargo localization. These results stress that two unexpected highly conserved domains, namely the auto-inhibitory IAK and the auxiliary microtubule-binding motifs, are crucial for transport by Kinesin-1 and that, although not all cargos are conserved, their transport involves the most conserved domains of animal KHCs (Williams, 2013).

The oocyte allows the analysis of the C-terminal region of KHC in an in vivo context. The results show that the interaction of Kinesin with its cargos and/or the regulation of the motor is complex and relies on more than one region. The tail (aa 850-975) is essential for all functions of KHC in the st9 oocyte except Dynein transport. These functions include the positioning of the nucleus and Gurken protein (and consequently establishment of the DV axis), the localization of oskar, the induction of streaming, and the distribution of actin-recruiting vesicles. Most of these tail-dependent KHC activities can be functionally separated from one another by further characterizing the conserved domains within the tail. The various functional domains are not necessarily involved in cargo binding, but their presence is required for wild-type cargo transport. In particular, the data show the following: (1) a temporal regulation of the impact of KHC activity on cytoplasmic streaming during oogenesis; (2) a novel essential role for the IAK that is distinct from its auto-inhibitory function; (3) that lack of auto-inhibition in itself is not necessarily detrimental to KHC function; and (4) that the AMB motif is required for oskar RNA localization (Williams, 2013).

The localization of Dynein to the posterior requires Kinesin. This study shows that deletion of the tail has a weak effect on the transport of Dynein, whereas further deletion of the region covering coil3 and half of coil2 renders a motor unable to localize Dynein. This observation correlates with the finding that KLC, which together with Pat1 mediates Dynein localization, interacts with coil3 of KHC in a tail-independent manner. It is then likely that Dynein is a posterior cargo of KHC, and that the Dynein complex interacts with KHC via KLCs. In C. elegans, the KLC-binding protein Jip3 binds Dynein light intermediate chain (Dlic). However, jip3/syd mutant oocytes show no defects in the posterior localization of Dynein. Alternatively, KLC might bind the Dynein intermediate chain (DIC), as in mammals. This observation, together with the fact that amino acids 795-839 (including coil3) are conserved in animal KHCs, makes it plausible that, in the oocyte, KHC localizes Dynein via a coil3-dependent KLC-DIC complex (Williams, 2013).

It is important to keep in mind that even though KLC and the KLC-like protein Pat1 are not essential for the localization of oskar and the nucleus, or for the induction of flows, they still contribute to these KHC-dependent processes, albeit in a minor manner. Pat1 mutants have slightly slower flows, and Pat1,Klc double mutants show mild oskar and nucleus localization defects in 78% and 9%, respectively, of the mutant oocytes. These nucleus anchoring defects might correlate with those seen in KHC1-700 oocytes, since KHC1- 700 does not contain the KLC-binding domain; however, the nucleus phenotypes in KHC1-700 may not be statistically significantly different from those observed in KHC1-849 oocytes. (Williams, 2013).

oskar RNA is found at the anterior/lateral regions of the Khc mutant oocyte. Similarly, Khc27 st9 oocytes show a mispositioned nucleus and an aberrant distribution of Gurken protein. Consequently, embryos resulting from Khc27 oocytes have an aberrant anterior-posterior (AP) and DV body plan. Deletion of the tail produces a motor that is unable to localize oskar RNA and thus is unable to support the establishment of the AP axis. Further characterization of the function of conserved domains within the tail suggests that RNA transport activity relies on the AMB site. In addition, 96.5% of the embryos resulting from tailless KHC oocytes have aberrant dorsal appendage (DA) formation. This DV axis defect might be due to more than the tail function in nucleus positioning, since the nucleus is not positioned in 60% of tailless KHC1-849 oocytes. KHC1-849 oocytes are defective for Gurken protein localization, even when the nucleus seems properly positioned. Given that the oocyte nucleus is associated with one of the MT-organizing centers, it is possible that the defects in Gurken signaling, and thus DV axis, in Kinesin mutants are a result of both nucleus mispositioning and the misorganization of the anterior MTs. This is consistent with MT defects observed at the anterior of KHC1-849 and KHC1-700 oocytes. In wild-type and KHC1-975 oocytes, there is an obvious AP gradient of MTs, with a population of enriched MTs close to the anterior/lateral cortex. This gradient can also be seen in some KHC1-849 and KHC1-700 oocytes. However, most of these mutant oocytes show an extension of this anterior 'bright' MT network towards the posterior around the nucleus, as well as the misorganization of MTs in a pattern that resembles the aberrant vesicles often detected at the anterior of Khc mutant oocytes. The region encompassing the KLC-binding domain might also contribute to the establishment of the DV axis, since the number of oocytes with Gurken in an anterior-dorsal crescent drops from 14% in KHC1-849 oocytes to 0% in KHC1-700 oocytes (Williams, 2013).

At first glance, it is unclear why there are nucleus and Gurken localization defects in the Khc null, when plus ends are biased towards the posterior. As nucleus positioning requires the Dynein complex, it follows that KHC function could be indirect for the anterior cargos, for example via the recycling of Dynein. However, it is thought that KHC could be acting directly on nucleus positioning. First, it cannot be discounted that Dynein and Kinesin act independently: Dynein localization to the posterior is abolished in Pat1,Klc double mutants, whereas nucleus positioning is only weakly affected, suggesting that the coordinated action of the two motors is not necessarily required. Second, the MT network is complex, and there seem to be some plus ends towards the anterior cortex that Kinesin may harness. Third, KHC localizes at the nuclear envelope. Fourth, when KHC is missing, alpha-tubulin and Jupiter-GFP [a MT-associated protein fused to GFP are found in dots at the nuclear envelope in a similar punctate pattern to that displayed by KHC (Williams, 2013).

All these preliminary observations might suggest that KHC is acting on a set of MTs that allows positioning of the nucleus in close proximity to the anterior membrane: when KHC is missing, these MTs seem to 'collapse' to the nuclear envelope and their stable existence is not maintained. Taking work on cultured cells into consideration, Kinesin might well bind to the nuclear envelope and transport the nucleus towards the plus ends. However, it is likely that the relative importance of different molecular links between the nuclear envelope and motors depends on the cell type. For example, Drosophila SUN/KASH proteins (Msp-300, Klarsicht and Klaroid) have no essential functions during oogenesis. Mammalian KHC is also known to bind directly to the nucleoporin Ranbp2 via its tail (Williams, 2013).

There are other mutants that show nucleus positioning defects, including skittles (which encodes phosphatidylinositol 4,5- bisphosphate-synthesizing enzyme), trailer hitch (tral) and Bicaudal C (BicC). Among these, tral and BicC mutants have abnormal actin-covered vesicles that look similar to those present in Khc oocytes. This similarity, together with the data showing that Rabenosyn-5 is present in Khc mutant vesicles, suggest that KHC is required for membrane trafficking in the oocyte. This correlates with the function of KHC in other cells and with the observation that, in Khc oocytes, Rab6 vesicles aggregate abnormally around the mispositioned nucleus. The ectopic vesicles that were observe in Khc oocytes seem to nucleate actin, as seen in time-lapse movies of Utrophin-GFP. As suggested for tral and BicC, the formation of 'actin spheres' (as a readout of vesicle trafficking problems) in Khc oocytes might cause defects in Gurken signaling. In fact, Gurken is detected in close proximity to actin-recruiting vesicles in KHC1-938 oocytes (Williams, 2013).

These data stress that the anterior phenotypes observed in Khc mutant oocytes are likely to be the result of a complex relationship between vesicle trafficking, MTs and nucleus location. Ectopic sites of actin also appear in Rab5, Rab6, IKK-related kinase (IκB kinase-like 2 - FlyBase) and spn-F oocytes. They were interpreted as cytoskeleton defects, but might also be the result of ectopic actin nucleation by aberrantly distributed vesicles (Williams, 2013).

Auto-inhibition to limit the consumption of ATP/GTP by motors not bound to cargos is conserved in Myosins and Kinesins. As both protein families share a common ancestor, it is not unexpected that there is a common mechanism to this auto-inhibition, in which the tail folds back to the motor domain. It is clear from research on affecting auto-inhibition in vivo that these motors cannot function correctly, leading to detrimental transport. What was still unknown is whether the defects in transport are a consequence of a lack of inhibition or are due to alternative functions of the motifs involved. This study has compared these two hypotheses directly for the first time. Recently, the stoichiometry of the interaction between the IAK and motor domains has been determined, with one IAK motif per motor dimer required. This has led to the suggestion that the other motif could be free to bind cargo or other regulators of KHC. A mutant IAK with two individual point mutations (IAKPIRS to IAKSIRS, IAKPIRS to IAKPIRSF) shows weak defects in oskar transport and DA formation that are similar to those of Khc hypomorphic alleles, suggesting that these mutations result in inhibition rather than overactivation of transport. Similarly, the IAK seems to facilitate, rather than downregulate, axonal transport of mitochondria. However, these IAK point mutants did not constitute a full null of IAK activity, since when the entire motif are mutagenized the DA defects are much stronger than those observed in the point mutants. In addition, deletion of the IAK phenocopies the deletion of the tail regarding the formation of dorsal structures, with only a slight increase in the number of normal DAs in IAK mutant oocytes (Williams, 2013).

A hypothesis to explain the cargo transport defects observed in KHC1-975ΔIAK oocytes, which are not observed in KHC1- 975ΔHinge2 oocytes, is that KHC1-975ΔIAK has reduced function for these cargoes. That is to say, the IAK motif has an essential activity that is independent of its auto-inhibition function. Interestingly, the streaming speed of KHC1-975ΔIAK is faster than wild type, suggesting that KHC1-975ΔIAK is not defective for all KHC functions. Instead, the increased streaming speed might be due to the number of motors that are active at any one time being higher. This correlates with many more particles of KHC1-849 than of KHC1-975 moving in in vitro assays (Williams, 2013).

The results with KHCΔHinge2 show that auto-inhibition does not play a major role in transport during oogenesis. However, there seems to be a small contribution of auto-inhibition to DA formation, in accordance with work on fungal kinesin showing that maintenance of the folded conformation partially contributes to growth rates. In conclusion, KHC autoinhibition might not be such an important driving factor as previously thought, especially not in the oocyte. It might be interesting to analyze how a lack of auto-inhibition affects KHC function in other cells, such as neurons (Williams, 2013).

The region 850-910 is conserved between all animal and fungal KHCs and contains the N. crassa putative cargo-binding domain. However, KHC1-910 does not support wild-type localization of oskar or wild-type streaming. It is however possible that KHC1-910 is able to bind cargo but is somehow unable to transport it. This could be the case for oskar RNA, since there is a weak accumulation of the transcript at the posterior in KHC1-910 oocytes. If KHC1-910 binds oskar, but its action is constrained, one would expect an enrichment of KHC1-910 at the anterior/lateral cortex, where oskar accumulates. Higher levels are noy detected of KHC1-910 than of KHC1-975 in that region, and thus it is uncertain how small amounts of oskar reach the posterior in KHC1-910 oocytes. The localization of wild-type amounts of oskar RNA to the posterior is rescued when the AMB site is restored in KHC1-938, demonstrating a key role of this domain in cargo localization. This supports the observation that mutations in this region (four arginines) render a severe Khc allele with reduced motor function in neurons. This AMB region binds to MTs in vitro and in cells, perhaps via electrostatic interactions, and seems responsible for an MT polymerization activity of the tail. Furthermore, KHC slides and bundles MTs in cells and, in the case of fungal Kinesin, this MT bundling activity depends on the tail. How does this MT regulatory function of the domain relate to the capacity of KHC1-938 to localize oskar? The MTs of KHC1-938 and KHC1-910 oocytes still form an AP gradient, seemingly of wild-type topology, supported by the posterior accumulation of KHC1-910GFP. Thus, it could be that the AMB site is affecting oskar RNP binding specifically and not via any MT-related activity. This hypothesis is supported by the fact that there are several proteins that interact with the KHC tail, including Kv3.1 (Shaw - FlyBase), which binds the region containing the AMB domain and requires it for its transport (Williams, 2013).

The localization of oskar RNA in KHC1-938 and KHC1-975ΔIAK oocytes is not completely wild type, since 'dots/clouds' of the transcript are observed in close proximity to the posterior. Dots/clouds of oskar at the posterior is a phenotype observed in oocytes with minor MT defects, oskar translation defects. It is not known why there is an oskar 'dot' phenotype in KHC1-938 and KHC1-975ΔIAK oocytes. There are no obvious MT defects at the posterior of these mutants, although the mutant motors are found in the oskar dots, which might suggest the presence of plus ends. This dots phenotype is also seen in Rab6 and Rab11 mutants and it might thus be related to a vesicle trafficking function of KHC. This idea is supported by the findings, since KHC1-975ΔIAK and KHC1-938 oocytes show aberrant actin spheres/vesicles (84% and 45% of KHC1-975ΔIAK and KHC1-938 oocytes, respectively) and dots/clouds of oskar adjacent to the posterior crescent. The relationship between oskar localization, MTs and endocytosis at the posterior is complex, involving various feedback loops. It is possible that defects in vesicle trafficking result in mild defects in cytoskeleton organization, since Rab11 and Rab6 mutant oocytes show mispolarized MTs. Thus, this inefficient oskar localization to a posterior crescent in mutant oocytes might indirectly result from mild cytoskeleton defects at the posterior. Alternatively, KHC1-975ΔIAK-dependent or KHC1-938- dependent ectopic oskar protein and/or ectopic MT plus ends might result in aberrant endocytosis at the posterior (Williams, 2013).

It is interesting to note that although oskar RNA is not a conserved cargo its transport involves a highly conserved domain, i.e. the AMB domain. This, and the findings concerning the IAK domain, show that although not all cargos are conserved their transport involves the most conserved domains of animal KHCs. Thus, both the IAK and AMB domains might play a crucial role in the transport of cargos in other cell types and organisms (Williams, 2013).

Klar ensures thermal robustness of oskar localization by restraining RNP motility

Communication usually applies feedback loop-based filters and amplifiers to ensure undistorted delivery of messages. Such an amplifier acts during Drosophila melanogaster midoogenesis, when oskar messenger ribonucleic acid (mRNA) anchoring depends on its own locally translated protein product. This study found that the motor regulator Klar β mediates a gain-control process that prevents saturation-based distortions in this positive feedback loop. Like oskar mRNA, Klar β localizes to the posterior pole of oocytes in a kinesin-1-dependent manner. By live imaging and semiquantitative fluorescent in situ hybridization, it was shown that Klar β restrains oskar ribonucleoprotein motility and decreases the posterior-ward translocation of oskar mRNA, thereby adapting the rate of oskar delivery to the output of the anchoring machinery. This negative regulatory effect of Klar is particularly important for overriding temperature-induced changes in motility. It is concluded that by preventing defects in oskar anchoring, this mechanism contributes to the developmental robustness of a poikilothermic organism living in a variable temperature environment (Gaspar, 2014).

Germ plasm anchoring is a dynamic state that requires persistent trafficking

Localized cytoplasmic determinants packaged as ribonucleoprotein (RNP) particles direct embryonic patterning and cell fate specification in a wide range of organisms. Once established, the asymmetric distributions of such RNP particles must be maintained, often over considerable developmental time. A striking example is the Drosophila germ plasm, which contains RNP particles whose localization to the posterior of the egg during oogenesis results in their asymmetric inheritance and segregation of germline from somatic fates in the embryo. Although actin-based anchoring mechanisms have been implicated, high-resolution live imaging revealed persistent trafficking of germ plasm RNP particles at the posterior cortex of the Drosophila oocyte. This motility relies on cortical microtubules, is mediated by kinesin and dynein motors, and requires coordination between the microtubule and actin cytoskeletons. Finally, RNP particle motility was shown to be required for long-term germ plasm retention. It is proposed that anchoring is a dynamic state that renders asymmetries robust to developmental time and environmental perturbations (Sinsimer, 2013).

To determine whether motors mediate microtubule-dependent germ plasm RNP particle motility in late-stage oocytes, advantage was taken of mutations that disrupt motor protein activity. The initial localization of osk mRNA during midoogenesis is mediated by the plus-end motor kinesin, and a null mutation in Kinesin heavy chain (Khc), the force-generating component of kinesin, causes mis-localization of germ plasm around the entire oocyte cortex during midoogenesis. Visualization of GFP-Vas in Khc mutant germline clones showed that this aberrant pattern persisted in late-stage oocytes. Despite the paucity of GFP-Vas particles at any one cortical location, examples were observed of dynamic behavior, and these occurred regardless of where on the cortex the particles were located. Quantification of the small population of GFP-Vas particles at the posterior cortex of Khc mutant oocytes showed a 1.7-fold reduction in the motile fraction as compared to wild-type oocytes and a 36% reduction in the median velocity of the motile particles (Sinsimer, 2013).

The finding that germ plasm RNP particle motility is reduced but not abolished in the complete absence of kinesin function suggested the involvement of a second motor. Therefore whether the minus-end motor dynein might be required was tested. Unlike kinesin, dynein is essential during early oogenesis, and complete abrogation of dynein activity precludes egg development. Consequently, the effect of hypomorphic mutations in Dynein heavy chain (Dhc) was examined; these eggs have reduced dynein activity but still complete oogenesis. Quantification of GFP-Vas in late-stage Dhc mutant oocytes showed a 1.6-fold decrease in the motile fraction (19%) as compared to wild-type. Moreover, the median velocity of motile particles in Dhc mutants was half that of wild-type oocytes. In a second approach, dynein activity was disrupted acutely in late-stage oocytes by heat shock-inducible expression of p50/Dynamitin (Dmn), a component of the dynactin complex that interferes with dynein activity when overexpressed. Although heat shock alone had a minor effect on GFP-Vas particle motility, there was a 2.4-fold decrease in the motile fraction when dynein was inactivated as compared to heat-shocked wild-type controls. Moreover, the median velocity of the motile population in late oocytes overexpressing Dmn was reduced by 30% compared to control oocytes. Thus, it is concluded that both kinesin and dynein contribute to germ plasm RNP motility. The comparable loss of motility in null kinesin and hypomorphic dynein mutants, however, suggests that dynein-mediated transport predominates (Sinsimer, 2013).

Both of these motors are also involved in transport events during midoogenesis: dynein for movement of mRNAs from nurse cells to oocyte and anteriorly directed transport within the oocyte, kinesin for posterior transport of osk. Previous work indicated that the bulk of germ plasm mRNA localization occurs not by motor-dependent transport, however, but by diffusion and entrapment of transcripts that enter the oocyte during nurse cell dumping. It has not been possible to resolve particles containing kinesin or dynein in late oocytes using current GFP fusions. Thus, the important question of when and where the association of motors with germ plasm RNP particles occurs awaits the development of new methods for visualization of motors in Drosophila oocytes (Sinsimer, 2013).

The association of localized germ plasm RNP complexes with dynein in late-stage oocytes may serve a second purpose, providing preassembled transport particles for germ cell inheritance in the early embryo. The process of germ cell formation initiates when centrosomes and/or astral microtubules associated with nuclei that migrate to the posterior of the syncytial embryo induce release of germ plasm from the posterior cortex. Recruitment of germ plasm to the centrosomes by dynein-dependent transport on astral microtubules is required for these nuclei to induce germ cell formation and for the inheritance of the germ plasm by the newly formed germ cells. The prior coupling of germ plasm RNP particles to dynein in the oocyte may allow their rapid accumulation on astral microtubules upon release from the cortex (Sinsimer, 2013).

Under conditions of stress such as nutrient deprivation or in the absence of potential mates, female flies will hold mature eggs until conditions improve to increase the likelihood of survival for their progeny. Notably, females can hold mature eggs for at least 15 days without consequence to the viability or fertility of their progeny). Thus, sustaining germ plasm localization through such a delay of fertilization and the onset of embryogenesis is biologically crucial. It is hypothesized that the persistent trafficking of germ plasm might provide a mechanism for retaining germ plasm at the posterior over long periods of time (Sinsimer, 2013).

Because dynein is a major mediator of germ plasm RNP motility and can be manipulated acutely, whether compromising dynein function in held eggs by inducible Dmn overexpression would lead to a progressive loss of germ plasm from the posterior was tested. Advantage was taken of a single-molecule fluorescent in situ hybridization (smFISH) method to detect endogenous nos and osk mRNAs in mature oocytes. smFISH provides a major advance for mRNA analyses during the vitellogenic stages of Drosophila oocytes, which are largely impervious to standard molecular probes. The amount of localized germ plasm was quantified by measuring fluorescence intensity for each probe (Sinsimer, 2013).

More than 70% of mature oocytes from wild-type control or Dmn-overexpressing females, dissected immediately following heat shock, exhibited robust germ plasm accumulation. A spreading of nos and osk along the cortex was observed in some oocytes overexpressing Dmn, likely due to an immediate effect of dynein inhibition on germ plasm retention following the 2 hr heat shock regimen. Examination of wild-type control oocytes held for 18 hr showed little effect of the holding period alone on localization of the germ plasm RNAs. In contrast, inhibition of dynein function in held oocytes led to a dramatic loss of both nos and osk from the posterior cortex, with robust localization persisting in fewer than 30%. Thus, it is concluded that dynein-mediated motility is required for long-term retention of germ plasm at the posterior cortex of the oocyte (Sinsimer, 2013).

Similarly, the effect of MyoV loss on germ plasm components was tested in held oocytes. In mature didum mutant oocytes, nos mRNA was properly localized to the posterior, and unlike the case for Dmn overexpression, nos persisted during the holding period. Together, these data support a model in which enhanced dynein-mediated motility facilitates nos RNP particle accumulation at the posterior. In contrast, osk mRNA was no longer confined to the posterior cortex in didum mutant oocytes but often had a graded or diffuse distribution. Thus, a requirement for MyoV function in entrapment and/or retention of osk extends into late oogenesis. Strikingly, a tight cortical distribution of osk was largely restored in didum mutant oocytes held for 18 hr. This suggests that given sufficient time, microtubule-based motility of osk RNP particles allows localization to recover in the absence of MyoV (Sinsimer, 2013).


oskar: Biological Overview | Evolutionary Homologs | Regulation | Factors affecting Oskar localization | Developmental Biology | Effects of Mutation | References

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