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Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including messenger ribonucleoprotein complexes (RNPs). Here we show that orchestration ofoskarRNP transport in theDrosophilagermline relies on the interplay of two double-stranded RNA binding proteins, Staufen and the dynein adaptor Egalitarian (Egl). We find that Staufen antagonizes Egl-mediated transport ofoskarmRNA by dynein bothin vitroandin vivo. Following delivery of nurse cell-synthesizedoskarmRNA into the oocyte by dynein, recruitment of Staufen to the RNPs results in dissociation of Egl and a switch to kinesin-1-mediated translocation of the mRNA to its final destination at the posterior pole of the oocyte. We additionally show that Egl associates withstaufen(stau)mRNA in the nurse cells, mediating its enrichment and translation in the ooplasm. Our observations identify a novel feed-forward mechanism, whereby dynein-dependent accumulation ofstaumRNA, and thus protein, in the oocyte enables motor switching onoskarRNPs by downregulating dynein activity.

SummaryAsymmetric localization ofoskarRNP granules to the oocyte posterior is crucial for abdominal patterning and germline formation of theDrosophilaembryo. We show thatoskarRNP granules in the oocyte are condensates with solid-like physical properties. Using purifiedoskarRNA and scaffold proteins Bruno and Hrp48, we confirmin vitrothatoskargranules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of specific proteins. Genetic modification of scaffold granule proteins, or tethering the intrinsically disordered region of human Fused in Sarcoma tooskarmRNA, allowed modulation of granule material propertiesin vivo. The resulting liquid-like properties impairedoskarlocalization and translation with severe consequences on embryonic development. Our study reflects how physiological phase transitions shape RNA-protein condensates to regulate localization and expression of a maternal RNA that instructs germline formation.

DEAD-box RNA helicases play important roles in a wide range of metabolic processes. Regulatory proteins can stimulate or block the activity of DEAD-box helicases. Here, we show that LOTUS (Limkain, Oskar, and Tudor containing proteins 5 and 7) domains present in the germline proteins Oskar, TDRD5 (Tudor domain-containing 5), and TDRD7 bind and stimulate the germline-specific DEAD-box RNA helicase Vasa. Our crystal structure of the LOTUS domain of Oskar in complex with the C-terminal RecA-like domain of Vasa reveals that the LOTUS domain occupies a surface on a DEAD-box helicase not implicated previously in the regulation of the enzyme's activity. We show that, in vivo, the localization of Drosophila Vasa to the nuage and germ plasm depends on its interaction with LOTUS domain proteins. The binding and stimulation of Vasa DEAD-box helicases by LOTUS domains are widely conserved.

When temperature fluctuation threatens the fidelity of Drosophila oogenesis, Klar restrains posterior-ward translocation of oskar mRNA, thereby adapting the rate of oskar delivery to the capacity of the anchoring machinery., 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. We find that the motor regulator Klar β mediates a gain-control process that prevents saturation-based distortions in this positive feedback loop. We demonstrate that, 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, we show 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. We conclude that by preventing defects in oskar anchoring, this mechanism contributes to the developmental robustness of a poikilothermic organism living in a variable temperature environment.

mRNA transport coupled with translational control underlies the intracellular localization of many proteins in eukaryotic cells. This is exemplified in Drosophila, where oskar mRNA transport and translation at the posterior pole of the oocyte direct posterior patterning of the embryo. oskar localization is a multistep process. Within the oocyte, a spliced oskar localization element (SOLE) targets oskar mRNA for plus end-directed transport by kinesin-1 to the posterior pole. However, the signals mediating the initial minus end-directed, dynein-dependent transport of the mRNA from nurse cells into the oocyte have remained unknown. Here, we show that a 67-nt stem–loop in the oskar 3′ UTR promotes oskar mRNA delivery to the developing oocyte and that it shares functional features with the fs(1)K10 oocyte localization signal. Thus, two independent cis-acting signals, the oocyte entry signal (OES) and the SOLE, mediate sequential dynein- and kinesin-dependent phases of oskar mRNA transport during oogenesis. The OES also promotes apical localization of injected RNAs in blastoderm stage embryos, another dynein-mediated process. Similarly, when ectopically expressed in polarized cells of the follicular epithelium or salivary glands, reporter RNAs bearing the oskar OES are apically enriched, demonstrating that this element promotes mRNA localization independently of cell type. Our work sheds new light on how oskar mRNA is trafficked during oogenesis and the RNA features that mediate minus end-directed transport.

Localization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport‐competent mRNPs. We show that the posterior‐targeting kinesin‐1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein‐dependent transport from the nurse cells into the oocyte. We demonstrate that kinesin‐1 recruitment requires the DmTropomyosin1‐I/C isoform, an atypical RNA‐binding tropomyosin that binds directly to dimerizing oskar 3′UTRs. Finally, we show that a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin‐1 and that the motor is activated during mid‐oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo‐dependent motor activation constitutes an optimized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo‐transport processes and between the cargo‐associated dynein and kinesin‐1.

Localization and local translation ofoskarmRNA at the posterior pole of theDrosophilaoocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport-competent mRNPs. We show that the posterior-targeting kinesin-1 is loaded upon nuclear export ofoskarmRNPs, prior to their dynein-dependent transport from the nurse cells into the oocyte. We demonstrate that kinesin-1 recruitment requires theDmTropomyosin1-I/C isoform, an atypical RNA-binding tropomyosin that binds directly to dimerizingoskar3’UTRs. Finally, we show that a small but dynamically changing subset ofoskarmRNPs gets loaded with inactive kinesin-1 and that the motor is activated during mid-oogenesis by the functionalized splicedoskarRNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo-dependent motor activation constitutes an optmized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo-transport processes and between the cargo associated dynein and kinesin-1.

oskar mRNA localization at the oocyte posterior pole is essential for correct patterning of the Drosophila embryo. Here we show at the ultrastructural level that endogenous oskar ribonucleoprotein complexes (RNPs) assemble sequentially with initial recruitment of Hrp48 and the exon junction complex (EJC) to oskar transcripts in the nurse cell nuclei, and subsequent recruitment of Staufen and microtubule motors, following transport to the cytoplasm. oskar particles are non-membrane-bound structures that coalesce as they move from the oocyte anterior to the posterior pole. Our analysis uncovers a role for the EJC component Barentsz in recruiting Tropomyosin II (TmII) to oskar particles in the ooplasm and reveals that TmII is required for kinesin binding to the RNPs. Finally, we show that both kinesin and dynein associate with oskar particles and are the primary microtubule motors responsible for transport of the RNPs within the oocyte.

The Drosophila melanogaster body axes are defined by the precise localization and the restriction of molecular determinants in the oocyte. Polarization of the oocyte during oogenesis is vital for this process. The directed traffic of membranes and proteins is a crucial component of polarity establishment in various cell types and organisms. Here, we investigate the role of the small GTPase Rab6 in the organization of the egg chamber and in asymmetric determinant localization during oogenesis. We show that exocytosis is affected in rab6-null egg chambers, which display a loss of nurse cell plasma membranes. Rab6 is also required for the polarization of the oocyte microtubule cytoskeleton and for the posterior localization of oskar mRNA. We show that, in vivo, Rab6 is found in a complex with Bicaudal-D, and that Rab6 and Bicaudal-D cooperate in oskar mRNA localization. Thus, during Drosophila oogenesis, Rab6-dependent membrane trafficking is doubly required; first, for the general organization and growth of the egg chamber, and second, more specifically, for the polarization of the microtubule cytoskeleton and localization of oskar mRNA. These findings highlight the central role of vesicular trafficking in the establishment of polarity and in determinant localization in Drosophila.

The Drosophila oskar (osk) mRNA is unusual in that it has both coding and noncoding functions. As an mRNA, osk encodes a protein required for embryonic patterning and germ cell formation. Independent of that function, the absence of osk mRNA disrupts formation of the karyosome and blocks progression through oogenesis. Here we show that loss of osk mRNA also affects the distribution of regulatory proteins, relaxing their association with large RNPs within the germline, and allowing them to accumulate in the somatic follicle cells. This and other noncoding functions of the osk mRNA are mediated by multiple sequence elements with distinct roles. One role, provided by numerous binding sites in two distinct regions of the osk 3′ UTR, is to sequester the translational regulator Bruno (Bru), which itself controls translation of osk mRNA. This defines a novel regulatory circuit, with Bru restricting the activity of osk, and osk in turn restricting the activity of Bru. Other functional elements, which do not bind Bru and are positioned close to the 3′ end of the RNA, act in the oocyte and are essential. Despite the different roles played by the different types of elements contributing to RNA function, mutation of any leads to accumulation of the germline regulatory factors in the follicle cells.

Measuring translation in space and time The ribosome translates the information contained within messenger RNAs (mRNAs) into proteins. When and where ribosomes encounter mRNAs can regulate gene expression. Halstead et al. developed an RNA biosensor that allows single molecules of mRNAs that have never been translated to be distinguished from ones that have undergone translation by the ribosome in living cells (see the Perspective by Popp and Maquat). The authors demonstrated the utility of their technique by examining the spatial and temporal regulation of translation in single cells and in Drosophila oocytes during development. Science , this issue p. 1367 ; see also p. 1316

The Drosophila maternal effect gene oskar encodes the posterior determinant responsible for the formation of the posterior pole plasm in the egg, and thus of the abdomen and germline of the future fly. Previously identified oskar mutants give rise to offspring that lack both abdominal segments and a germline, thus defining the `posterior group phenotype'. Common to these classical oskar alleles is that they all produce significant amounts of oskar mRNA. By contrast, two new oskar mutants in which oskar RNA levels are strongly reduced or undetectable are sterile, because of an early arrest of oogenesis. This egg-less phenotype is complemented by oskar nonsense mutant alleles,as well as by oskar transgenes, the protein-coding capacities of which have been annulled. Moreover, we show that expression of the oskar 3′ untranslated region (3′UTR) is sufficient to rescue the egg-less defect of the RNA null mutant. Our analysis thus reveals an unexpected role for oskar RNA during early oogenesis, independent of Oskar protein. These findings indicate that oskar RNA acts as a scaffold or regulatory RNA essential for development of the oocyte.

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. Here we identify the previously reported oskar RNA binding protein p50 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. Our 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.

Targeting proteins to specific domains within the cell is central to the generation of polarity, which underlies many processes including cell fate specification and pattern formation during development. The anteroposterior and dorsoventral axes of the Drosophila melanogaster embryo are determined by the activities of localized maternal gene products. At the posterior pole of the oocyte, Oskar directs the assembly of the pole plasm,and is thus responsible for formation of abdomen and germline in the embryo. Tight restriction of oskar activity is achieved by mRNA localization,localization-dependent translation, anchoring of the RNA and protein, and stabilization of Oskar at the posterior pole. Here we report that the type 1 regulatory subunit of cAMP-dependent protein kinase (Pka-R1)is crucial for the restriction of Oskar protein to the oocyte posterior. Mutations in PKA-R1 cause premature and ectopic accumulation of Oskar protein throughout the oocyte. This phenotype is due to misregulation of PKA catalytic subunit activity and is suppressed by reducing catalytic subunit gene dosage. These data demonstrate that PKA mediates the spatial restriction of Oskar for anteroposterior patterning of the Drosophila embryo and that control of PKA activity by PKA-R1 is crucial in this process.

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

Background: mRNA localization is a powerful and widely employed mechanism for generating cell asymmetry. In Drosophila , localization of mRNAs in the oocyte determines the axes of the future embryo. oskar mRNA localization at the posterior pole is essential and sufficient for the specification of the germline and the abdomen. Its posterior transport along the microtubules is mediated by Kinesin I and several proteins, such as Mago-nashi, which, together with oskar mRNA, form a posterior localization complex. It was recently shown that human Y14, a nuclear protein that associates with mRNAs upon splicing and shuttles to the cytoplasm, interacts with MAGOH, the human homolog of Mago-nashi. Results: Here, we show that Drosophila Y14 interacts with Mago-nashi in vivo. Immunohistochemistry reveals that Y14 is predominantly nuclear and colocalizes with oskar mRNA at the posterior pole. We show that, in y14 mutant oocytes, oskar mRNA localization to the posterior pole is specifically affected, while the cytoskeleton appears to be intact. Conclusions: Our findings indicate that Y14 is part of the oskar mRNA localization complex and that the nuclear shuttling protein Y14 has a specific and direct role in oskar mRNA cytoplasmic localization.

Background: The oskar (osk) gene encodes a determinant of posterior identity in Drosophila , and the localization of osk RNA to the pole plasm at the posterior pole of the oocyte is essential for development of the embryo. The mechanisms by which osk RNA is localized are unknown. Results: To study the mechanisms underlying localization of osk RNA, we have injected fluorescently labelled RNA into oocytes at stages 9, 10 and 11. Injected osk RNA localizes to the pole plasm, reproducing localization of the endogenous RNA. In oocytes at stages 10 and 11, the long-range movement of injected osk RNA is promoted by a vigorous, microtubule-dependent cytoplasmic flow, or ooplasmic streaming. Treatment with colchicine, a microtubule-destabilizing drug, inhibits ooplasmic streaming and prevents localization of the RNA from an injection site distal to the posterior pole. If the RNA is injected close to the posterior pole, however, it localizes even in the presence of colchicine. Similarly, in small oocytes, such as stage 9 oocytes, localization of injected osk RNA is insensitive to colchicine. Conclusions: These results reveal that microtubule-dependent cytoplasmic flows could contribute to the long-range transport of osk RNA, whereas microtubule-independent processes could mediate short-range transport. These results also highlight the role of the osk RNA anchor in the localization process.

The posterior pole plasm of the Drosophila egg contains the determinants of abdominal and germ-cell fates of the embryo. Pole plasm assembly is induced by oskar RNA localized to the posterior pole of the oocyte. Genetics has revealed three additional genes, staufen, vasa, and tudor, that are also essential for pole plasm formation. Staufen protein is required for both oskar RNA localization and translation. Vasa and Tudor are localized dependent on Oskar protein and are required to accumulate Oskar protein stably at the posterior pole. We have explored interactions between these gene products at the molecular level and find that Oskar interacts directly with Vasa and Staufen, in a yeast two-hybrid assay. These interactions also occur in vitro and are affected by mutations in Oskar that abolish pole plasm formation in vivo. Finally, we show that in the pole plasm, Oskar protein, like Vasa and Tudor, is a component of polar granules, the germ-line-specific RNP structures. These results suggest that the Oskar-Vasa interaction constitutes an initial step in polar granule assembly. In addition, we discuss the possible biological role of the Oskar-Staufen interaction.

oskar RNA localization to the posterior pole of the Drosophila melanogaster oocyte requires splicing of the first intron and the exon junction complex (EJC) core proteins. The functional link between splicing, EJC deposition and oskar localization has been unclear. Here we demonstrate that the EJC associates with oskar mRNA upon splicing in vitro and that Drosophila EJC deposition is constitutive and conserved. Our in vivo analysis reveals that splicing creates the spliced oskar localization element (SOLE), whose structural integrity is crucial for ribonucleoprotein motility and localization in the oocyte. Splicing thus has a dual role in oskar mRNA localization: assembling the SOLE and depositing the EJC required for mRNA transport. The SOLE complements the EJC in formation of a functional unit that, together with the oskar 3' UTR, maintains proper kinesin-based motility of oskar mRNPs and posterior mRNA targeting.

mRNA localization coupled with translational control is a highly conserved and widespread mechanism for restricting protein expression to specific sites within eukaryotic cells. In Drosophila, patterning of the embryo requires oskar mRNA transport to the posterior pole of the oocyte and translational repression prior to localization. oskar RNA splicing and the 3′ untranslated region (UTR) are required for posterior enrichment of the mRNA. However, reporter RNAs harboring the oskar 3′ UTR can localize by hitchhiking with endogenous oskar transcripts. Here we show that the oskar 3′ UTR contains a stem–loop structure that promotes RNA dimerization in vitro and hitchhiking in vivo. Mutations in the loop that abolish in vitro dimerization interfere with reporter RNA localization, and restoring loop complementarity restores hitchhiking. Our analysis provides insight into the molecular basis of RNA hitchhiking, whereby localization-incompetent RNA molecules can become locally enriched in the cytoplasm, by virtue of their association with transport-competent RNAs.

mRNA localization coupled with translational control is a widespread and conserved strategy that allows the localized production of proteins within eukaryotic cells. In Drosophila, oskar (osk) mRNA localization and translation at the posterior pole of the oocyte are essential for proper patterning of the embryo. Several P body components are involved in osk mRNA localization and translational repression, suggesting a link between P bodies and osk RNPs. In cultured mammalian cells, Ge-1 protein is required for P body formation. Combining genetic, biochemical and immunohistochemical approaches, we show that, in vivo, Drosophila Ge-1 (dGe-1) is an essential gene encoding a P body component that promotes formation of these structures in the germline. dGe-1 partially colocalizes with osk mRNA and is required for osk RNP integrity. Our analysis reveals that although under normal conditions dGe-1 function is not essential for osk mRNA localization, it becomes critical when other components of the localization machinery, such as staufen, Drosophila decapping protein 1 and barentsz are limiting. Our findings suggest an important role of dGe-1 in optimization of the osk mRNA localization process required for patterning the Drosophila embryo.

(Cell 139, 983–998; November 25, 2009)In this paper, we used cryoimmuno-electron microscopy and live-cell imaging to investigate the sequential assembly of oskar mRNA into an mRNP competent for transport from the Drosophila nurse cells to the oocyte posterior pole. We have recently identified instances in all of the figures where the cryoimmuno-EM data were inappropriately manipulated by the first author. The manipulations do not affect the live-cell imaging data. We are in the process of reanalyzing the raw experimental cryoimmuno-EM data but can already state that the published conclusions are not fully consistent with the raw data. We are therefore retracting the paper. We sincerely apologize for any inconvenience that this might have caused.

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. We identified the nucleo–cytoplasmic shuttling protein PTB (polypyrimidine tract-binding protein)/hnRNP I 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.

During Drosophila oogenesis, oskar mRNA is transported to the posterior pole of the oocyte, where it is locally translated and induces germ-plasm assembly. Oskar protein recruits all of the components necessary for the establishment of posterior embryonic structures and of the germline. Tight localization of Oskar is essential, as its ectopic expression causes severe patterning defects. Here, we show that the Drosophila homolog of mammalian Lasp1 protein, an actin-binding protein previously implicated in cell migration in vertebrate cell culture, contributes to the accumulation of Oskar protein at the posterior pole of the embryo. The reduced number of primordial germ cells in embryos derived from lasp mutant females can be rescued only with a form of Lasp that is capable of interacting with Oskar,revealing the physiological importance of the Lasp-Oskar interaction.

SummaryPrior 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, we directly demonstrate the mechanism proposed for Cup-mediated repression: 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.

oskar messenger RNA localization at the posterior pole of the Drosophila oocyte is essential for germline and abdomen formation in the future embryo. The nuclear shuttling proteins Y14/Tsunagi and Mago nashi are required for oskar mRNA localization, and they co-localize with oskar mRNA at the posterior pole of the oocyte. Their human homologues, Y14/RBM8 and Magoh, are core components of the exon-exon junction complex (EJC). The EJC is deposited on mRNAs in a splicing-dependent manner, 20-24 nucleotides upstream of exon-exon junctions, independently of the RNA sequence. This indicates a possible role of splicing in oskar mRNA localization, challenging the established notion that the oskar 3' untranslated region (3'UTR) is sufficient for this process. Here we show that splicing at the first exon-exon junction of oskar RNA is essential for oskar mRNA localization at the posterior pole. We revisit the issue of sufficiency of the oskar 3'UTR for posterior localization and show that the localization of unrelated transcripts bearing the oskar 3'UTR is mediated by endogenous oskar mRNA. Our results reveal an important new function for splicing: regulation of messenger ribonucleoprotein complex assembly and organization for mRNA cytoplasmic localization.

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. We show that a long poly(A) tail is required for efficient oskar translation, both in vivo and in vitro, but is not sufficient to overcome BRE-mediated repression. Moreover, we show that accumulation of Oskar activity requires the Drosophila homolog of Cytoplasmic Polyadenylation Element Binding protein (CPEB), Orb. As 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. We show that 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. We conclude that Orb-mediated cytoplasmic polyadenylation stimulates oskar translation to achieve the high levels of Oskar protein necessary for posterior patterning and germline differentiation.

Localization of the maternal determinant Oskar at the posterior pole of Drosophila melanogaster oocyte provides the positional information for pole plasm formation. Spatial control of Oskar expression is achieved through the tight coupling of mRNA localization to translational control, such that only posterior-localized oskar mRNA is translated, producing the two Oskar isoforms Long Osk and Short Osk. We present evidence that this coupling is not sufficient to restrict Oskar to the posterior pole of the oocyte. We show that Long Osk anchors both oskar mRNA and Short Osk, the isoform active in pole plasm assembly, at the posterior pole. In the absence of anchoring by Long Osk, Short Osk disperses into the bulk cytoplasm during late oogenesis, impairing pole cell formation in the embryo. In addition, the pool of untethered Short Osk causes anteroposterior patterning defects, owing to the dispersion of pole plasm and its abdomen-inducing activity throughout the oocyte. We show that the N-terminal extension of Long Osk is necessary but not sufficient for posterior anchoring, arguing for multiple docking elements in Oskar. This study reveals cortical anchoring of the posterior determinant Oskar as a crucial step in pole plasm assembly and restriction, required for proper development of Drosophila melanogaster.

Recent advances shed light on the cellular processes that cooperate during oogenesis to produce a fully patterned egg, containing all the maternal information required for embryonic development. Progress has been made in defining the early steps in oocyte specification and it has been shown that progression of oogenesis is controlled by a meiotic checkpoint and requires active maintenance of the oocyte cell fate. The function of Gurken signalling in patterning the dorsal-ventral axis later in oogenesis is better understood. Anterior-posterior patterning of the embryo requires activities of bicoid and oskar mRNAs, localised within the oocyte. A microtubule motor, Kinesin, is directly implicated in localisation of oskar mRNA to the posterior pole of the oocyte.

The precise restriction of proteins to specific domains within a cell plays an important role in early development and differentiation. An efficient way to localize and concentrate proteins is by localization of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination. A central issue is how localized mRNAs are derepressed. In this study we demonstrate that, when oskar mRNA reaches the posterior pole of the Drosophila oocyte, its translation is derepressed by an active process that requires a specific element in the 5′ region of the mRNA. We demonstrate that this novel type of element is a translational derepressor element, whose functional interaction with the previously identified repressor region in the oskar 3′ UTR is required for activation of oskar mRNA translation at the posterior pole. The derepressor element only functions at the posterior pole, suggesting that a locally restricted interaction betweentrans-acting factors and the derepressor element may be the link between mRNA localization and translational activation. We also show specific interaction of two proteins with the oskar mRNA 5′ region; one of these also recognizes the 3′ repressor element. We discuss the possible involvement of these factors as well as known genes in the process of localization-dependent translation.

At the posterior pole of the Drosophila oocyte, oskar induces a tightly localized assembly of pole plasm. This spatial restriction of oskar activity has been thought to be achieved by the localization of oskar mRNA, since mislocalization of the RNA to the anterior induces anterior pole plasm. However, ectopic pole plasm does not form in mutant ovaries where oskar mRNA is not localized, suggesting that the unlocalized mRNA is inactive. As a first step towards understanding how oskar activity is restricted to the posterior pole, we analyzed oskar translation in wild type and mutants. We show that the targeting of oskar activity to the posterior pole involves two steps of spatial restriction, cytoskeleton-dependent localization of the mRNA and localization-dependent translation. Furthermore, our experiments demonstrate that two isoforms of Oskar protein are produced by alternative start codon usage. The short isoform, which is translated from the second in-frame AUG of the mRNA, has full oskar activity. Finally, we show that when oskar RNA is localized, accumulation of Oskar protein requires the functions of vasa and tudor, as well as oskar itself, suggesting a positive feedback mechanism in the induction of pole plasm by oskar.

THE localization of oskar (osk) RNA to the posterior pole of the developing fruit fly (Drosophila) oocyte induces the assembly of pole plasm, causing development of the abdomen and germ line1,2. Failure to localize oskar RNA results in embryos that lack abdomen and germ cells. Conversely, mis-targeting of oskar RNA to the anterior of the oocyte causes formation of ectopic abdomen and germ cells at the anterior pole3. Maternal mutants that have reduced pole plasm activity produce sterile adults with normal abdominal development, suggesting that germ cells are more sensitive than abdomen to defects in pole plasm assembly4. Thus mutations in genes that reduce oskar RNA localization or activity can be recovered as viable sterile adults. In a screen for mutants defective in germ cell formation, we isolated nine alleles of the tropomyosin II gene5. Here we show that mutations in tropomyosin II (TmII) virtually abolish oskar RNA localization to the posterior pole, suggesting an involvement of the actin network in oskar RNA localization.

The oskar gene directs germ plasm assembly and controls the number of germ cell precursors formed at the posterior pole of the Drosophila embryo. Mislocalization of oskar RNA to the anterior pole leads to induction of germ cells at the anterior. Of the eight genes necessary for germ cell formation at the posterior, only three, oskar, vasa and tudor, are essential at an ectopic site.

Oskar is one of seven Drosophila maternal-effect genes that are necessary for germline and abdomen formation. We have cloned oskar and show that oskar RNA is localized to the posterior pole of the oocyte when germ plasm forms. This polar distribution of oskar RNA is established during oogenesis in three phases: accumulation in the oocyte, transport toward the posterior, and finally maintenance at the posterior pole of the oocyte. The colocalization of oskar and nanos in wild-type and bicaudal embryos suggests that oskar directs localization of the posterior determinant nanos. We propose that the pole plasm is assembled stepwise and that continued interaction among its components is required for germ cell determination.

Intracellular mRNA localization is an effective mechanism for protein targeting leading to functional polarization of the cell. The mechanisms controlling mRNA localization and specifically how the actin and microtubule (MT) cytoskeletons cooperate in this process are not well understood. In Drosophila, Oskar protein accumulation at the posterior pole of the oocyte is required for embryonic development 1, 2, 3 and 4 and is achieved by the transport of oskar mRNA and its exclusive translation at the posterior pole 1, 5, 6 and 7. oskar mRNA localization requires the activity of the MT-based motor Kinesin [8], as well as the formation of a transport-competent ribonucleoprotein (RNP) complex [9]. Here, we show that didum, encoding the Drosophila actin-based motor Myosin-V 10 and 11, is a new posterior group gene that promotes posterior accumulation of Oskar. Myosin-V associates with the oskar mRNA transport complex preferentially at the oocyte cortex, revealing a short-range actomyosin-based mechanism that mediates the local entrapment of oskar at the posterior pole. Our results also show that Myosin-V interacts with Kinesin heavy chain and counterbalances Kinesin function, preventing ectopic accumulation of oskar in the cytoplasm. Our findings reveal that a balance of microtubule- and actin-based motor activities regulates oskar mRNA localization in the Drosophila oocyte.

SummaryIn Drosophila, localized activity of oskar at the posterior pole of the oocyte induces germline and abdomen formation in the embryo. Oskar has two isoforms, a short isoform encoding the patterning determinant and a long isoform of unknown function. Here, we show by immuno-electron microscopy that the two Oskar isoforms have different subcellular localizations in the oocyte: Short Oskar mainly localizes to polar granules, and Long Oskar is specifically associated with endocytic membranes along the posterior cortex. Our cell biological and genetic analyses reveal that Oskar stimulates endocytosis, and that its two isoforms are required to regulate this process. Furthermore, we describe long F-actin projections at the oocyte posterior pole that are induced by and intermingled with Oskar protein. We propose that Oskar maintains its localization at the posterior pole through dual functions in regulating endocytosis and F-actin dynamics.

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. We have established a cell-free translation system derived from Drosophila ovaries, which faithfully reproduces critical features of mRNA translation in vivo, namely cap structure and poly(A) tail dependence. We show that 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, we show that 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. The approach that we describe opens new avenues to investigate translational regulation in Drosophila oogenesis at a biochemical level.

Par-1 kinase is critical for polarization of the Drosophila melanogaster oocyte and the one-cell Caenorhabditis elegans embryo. Although Par-1 localizes specifically to the posterior pole in both cells, neither its targets nor its function at the posterior pole have been elucidated. Here we show that Drosophila Par-1 phosphorylates the posterior determinant Oskar (Osk) and demonstrate genetically that Par-1 is required for accumulation of Osk protein. We show in cell-free extracts that Osk protein is intrinsically unstable and that it is stabilized after phosphorylation by Par-1. Our data indicate that posteriorly localized Par-1 regulates posterior patterning by stabilizing Osk.