oskar RNA plays multiple noncoding roles to support oogenesis and maintain integrity of the germline/soma distinction

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.     

A stem–loop structure directs oskar mRNA to microtubule minus ends

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.     

The <Emphasis Type="Italic">Drosophila</Emphasis> RNA-binding protein Lark is required for localization of Dmoesin to the oocyte cortex during oogenesis

The RNA-binding protein Lark has an essential maternal role during Drosophila oogenesis. Elimination of maternal expression results in defects in cytoplasmic dumping and actin cytoskeletal organization in nurse cells. The function of this protein is dependent on the activity of one or more N-terminal RNA-binding domains. Here, we report the identification of Dmoesin (Dmoe) as a candidate RNA target of Lark during oogenesis. In addition to actin defects in the nurse cells of lark mutant ovaries, we observed mislocalization of posteriorly localized mRNAs including oskar and germ cell less in the developing oocyte. Anteriorly and dorsally localized mRNAs were not affected. In addition, we observed displacement of the actin cytoskeleton from the oocyte plasma membrane. These phenotypes are reminiscent of mutations in Dmoe and suggested that this RNA maybe a potential target of Lark. We observed a significant decrease in Dmoe protein associated with the membrane of the developing oocyte with no changes in expression or localization within the nurse cells. Evidence for an association between Lark protein and moe RNA during oogenesis comes from results of a microarray-based Ribonomics approach to identify Lark RNA targets. Thus, our results provide evidence that Dmoe RNA is a target of Lark during oogenesis and that it likely regulates either the splicing or translation of this RNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Aubergine is a component of a nanos mRNA localization complex

Localization of nanos (nos) mRNA to the posterior pole of the Drosophila oocyte is essential for abdominal segmentation and germline development during embryogenesis. Posterior localization is mediated by a complex cis-acting localization signal in the nos 3' untranslated region that comprises multiple partially redundant elements. Genetic analysis suggests that this signal is recognized by RNA-binding proteins and associated factors that package nos mRNA into a localization competent ribonucleoprotein complex. However, functional redundancy among localization elements has made the identification of individual localization factors difficult. Indeed, only a single direct-acting nos localization factor, Rumpelstiltskin (Rump), has been identified thus far. Through a sensitized genetic screen, we have now identified the Argonaute family member Aubergine (Aub) as a nos localization factor. Aub interacts with nos mRNA in vivo and co-purifies with Rump in an RNA-dependent manner. Our results support a role for Aub, independent of its function in RNA silencing, as a component of a nos mRNA localization complex.     

New phenotypes associated with the swallow gene of Drosophila: evidence for a general role in oocyte cytoskeletal organization

During oogenesis in Drosophila, several mRNAs and proteins are localized to discrete regions of the developing oocyte, resulting in a mature oocyte with a well-defined anterior–posterior axis. The product of the swallow (sww) gene is required for the localization of two different mRNAs during oogenesis, bicoid (bcd) and Adducin-like/hu-li tai shao (hts). We initiated a detailed characterization of the phenotypes associated with each of eight sww alleles as a means of investigating the role of sww in oogenic patterning. RNA localization defects in various sww mutants were examined by radioactive in situ hybridization to paraffin sections. Using this technique, several previously unreported RNA localization defects have been observed. Although bcd RNA localization is often lost completely in sww oocytes, in a high proportion of cases, bcd RNA is localized inappropriately along the periphery of the mature oocyte. In several sww mutants, a portion of the bcd mRNA population becomes concentrated at the posterior pole of the oocyte during late oogenesis. Several sww mutations also result inoskar RNA localization defects, consistent with a global role for sww in cytoskeletal regulation or organization. A detailed temporal and spatial analysis of hts RNA localization in sww mutants and in drug-treated ovaries reveals many similarities to bcd RNA localization, and implies the two independent localization events are accomplished by the same mechanism. Received: 10 January 2000 / Accepted: 9 March 2000     

Region-Specific Activation of oskar mRNA Translation by Inhibition of Bruno-Mediated Repression

A complex program of translational repression, mRNA localization, and translational activation ensures that Oskar (Osk) protein accumulates only at the posterior pole of the Drosophila oocyte. Inappropriate expression of Osk disrupts embryonic axial patterning, and is lethal. A key factor in translational repression is Bruno (Bru), which binds to regulatory elements in the osk mRNA 3′ UTR. After posterior localization of osk mRNA, repression by Bru must be alleviated. Here we describe an in vivo assay system to monitor the spatial pattern of Bru-dependent repression, separate from the full complexity of osk regulation. This assay reveals a form of translational activation—region-specific activation—which acts regionally in the oocyte, is not mechanistically coupled to mRNA localization, and functions by inhibiting repression by Bru. We also show that Bru dimerizes and identify mutations that disrupt this interaction to test its role in vivo. Loss of dimerization does not disrupt repression, as might have been expected from an existing model for the mechanism of repression. However, loss of dimerization does impair regional activation of translation, suggesting that dimerization may constrain, not promote, repression. Our work provides new insight into the question of how localized mRNAs become translationally active, showing that repression of osk mRNA is locally inactivated by a mechanism acting independent of mRNA localization.     

Bicaudal-D is essential for egg chamber formation and cytoskeletal organization in drosophila oogenesis

Bicaudal-D (Bic-D) is required for the transport of determinant mRNAs and proteins to the presumptive oocyte, an essential step in the differentiation of the oocyte. Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins. We characterized the ovarian phenotypes of females expressing mutant Bic-D proteins (Bic-D(H)) deleted for each of the heptad repeat domains. The altered migration of follicle cells we observe in mutant ovaries suggests that Bic-D functions in the germline and directs the inward migration of somatic follicle cells. In the germarium Bic-D is required for the organization of the egg chamber and the structural integrity of the oocyte and nurse cells. Examination of the polarized microtubule network in Bic-D(H) ovaries shows that Bic-D function is required for both the establishment of the polarized microtubule network and its maintenance throughout oogenesis. To explain the multiple functions suggested by the pleiotropic Bic-D phenotype, we propose that Bic-D protein could form itself a filamentous structure and represent an integral, essential part of the cytoskeleton.     

Distinct mechanisms for mRNA localization during embryonic axis specification in the wasp Nasonia

mRNA localization is a powerful mechanism for targeting factors to different regions of the cell and is used in Drosophila to pattern the early embryo. During oogenesis of the wasp Nasonia, mRNA localization is used extensively to replace the function of the Drosophila bicoid gene for the initiation of patterning along the antero-posterior axis. Nasonia localizes both caudal and nanos to the posterior pole, whereas giant mRNA is localized to the anterior pole of the oocyte; orthodenticle1 (otd1) is localized to both the anterior and posterior poles. The abundance of differentially localized mRNAs during Nasonia oogenesis provided a unique opportunity to study the different mechanisms involved in mRNA localization. Through pharmacological disruption of the microtubule network, we found that both anterior otd1 and giant, as well as posterior caudal mRNA localization was microtubule-dependent. Conversely, posterior otd1 and nanos mRNA localized correctly to the posterior upon microtubule disruption. However, actin is important in anchoring these two posteriorly localized mRNAs to the oosome, the structure containing the pole plasm. Moreover, we find that knocking down the functions of the genes tudor and Bicaudal-D mimics disruption of microtubules, suggesting that tudor's function in Nasonia is different from flies, where it is involved in formation of the pole plasm.     

The Drosophila hnRNPA/B homolog, Hrp48, is specifically required for a distinct step in osk mRNA localization

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, we isolated three missense alleles in the hnRNPA/B homolog, Hrp48. 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.     

Localization,anchoring and translational control of oskar,gurken, bicoid and nanos mRNA during Drosophila oogenesis

mRNA localization, and translation that is regulated spatially and temporally, are key mechanisms in the execution of polarized developmental programs. For over two decades, the Drosophila oocyte has served as a valuable model to study these mechanisms. Genetic and biochemical studies in flies have greatly contributed to the identification and understanding of factors that govern RNA localization and translational control. Embryonic axis formation is mediated through the subcellular localization and precise translational regulation of four key determinant mRNAs during oogenesis encoded by oskar, bicoid, gurken and nanos. In this review we aim to summarize recent insights into the mechanisms governing the asymmetric distribution and translation of these mRNAs.     

Reversible response of protein localization and microtubule organization to nutrient stress during Drosophila early oogenesis

The maturation of animal oocytes is highly sensitive to nutrient availability. During Drosophila oogenesis, a prominent metabolic checkpoint occurs at the onset of yolk uptake (vitellogenesis): under nutrient stress, egg chambers degenerate by apoptosis. To investigate additional responses to nutrient deprivation, we studied the intercellular transport of cytoplasmic components between nurse cells and the oocyte during previtellogenic stages. Using GFP protein-traps, we showed that Ypsilon Schachtel (Yps), a putative RNA binding protein, moved into the oocyte by both microtubule (MT)-dependent and -independent mechanisms, and was retained in the oocyte in a MT-dependent manner. These data suggest that oocyte enrichment is accomplished by a combination of MT-dependent polarized transport and MT-independent flow coupled with MT-dependent trapping within the oocyte. Under nutrient stress, Yps and other components of the oskar ribonucleoprotein complex accumulated in large processing bodies in nurse cells, accompanied by MT reorganization. This response was detected as early as 2 h after starvation, suggesting that young egg chambers rapidly respond to nutrient stress. Moreover, both Yps aggregation and MT reorganization were reversed with re-feeding of females or the addition of exogenous insulin to cultured egg chambers. Our results suggest that egg chambers rapidly mount a stress response by altering intercellular transport upon starvation. This response implies a mechanism for preserving young egg chambers so that egg production can rapidly resume when nutrient availability improves.     

An RNA‐binding atypical tropomyosin recruits kinesin‐1 dynamically to oskar mRNPs

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.     

Interactions of 40LoVe within the ribonucleoprotein complex that forms on the localization element of Xenopus Vg1 mRNA

Proline rich RNA-binding protein (Prrp), which associates with mRNAs that employ the late pathway for localization in Xenopus oocytes, was used as bait in a yeast two-hybrid screen of an expression library. Several independent clones were recovered that correspond to a paralog of 40LoVe, a factor required for proper localization of Vg1 mRNA to the vegetal cortex. 40LoVe is present in at least three alternatively spliced isoforms; however, only one, corresponding to the variant identified in the two-hybrid screen, can be crosslinked to Vg1 mRNA. In vitro binding assays revealed that 40LoVe has high affinity for RNA, but exhibits little binding specificity on its own. Nonetheless, it was only found associated with localized mRNAs in oocytes. 40LoVe also interacts directly with VgRBP71 and VgRBP60/hnRNP I; it is the latter factor that likely determines the binding specificity of 40LoVe. Initially, 40LoVe binds to Vg1 mRNA in the nucleus and remains with the RNA in the cytoplasm. Immunohistochemical staining of oocytes shows that the protein is distributed between the nucleus and cytoplasm, consistent with nucleocytoplasmic shuttling activity. 40LoVe is excluded from the mitochondrial cloud, which is used by RNAs that localize through the early (METRO) pathway in stage I oocytes; nonetheless, it is associated with at least some early pathway RNAs during later stages of oogenesis. A phylogenetic analysis of 2×RBD hnRNP proteins combined with other experimental evidence suggests that 40LoVe is a distant homolog of Drosophila Squid.     

Ypsilon Schachtel, a Drosophila Y-box protein, acts antagonistically to Orb in the oskar mRNA localization and translation pathway.

Subcellular localization of mRNAs within the Drosophila oocyte is an essential step in body patterning. Yps, a Drosophila Y-box protein, is a component of an ovarian ribonucleoprotein complex that also contains Exu, a protein that plays an essential role in mRNA localization. Y-box proteins are known translational regulators, suggesting that this complex might regulate translation as well as mRNA localization. Here we examine the role of the yps gene in these events. We show that yps interacts genetically with orb, a positive regulator of oskar mRNA localization and translation. The nature of the genetic interaction indicates that yps acts antagonistically to orb. We demonstrate that Orb protein is physically associated with both the Yps and Exu proteins, and that this interaction is mediated by RNA. We propose a model wherein Yps and Orb bind competitively to oskar mRNA with opposite effects on translation and RNA localization.     

Expression and cellular localization of the Toucan protein during Drosophila oogenesis

The toucan (toc) gene is required in the germline for somatic cell patterning during Drosophila oogenesis. To better understand the function of toc, we performed a detailed analysis of the distribution of the Toucan protein during oogenesis. Toc expression is restricted to the germline cells and shows a dynamic distribution pattern throughout follicle development. Mislocalization of the Toc protein in mutant follicles in which the microtubule network is altered indicates that microtubules play a role in Toc localization during oogenesis.     

Comparative analysis of the kinetics and dynamics of K10, bicoid,and oskar mRNA localization in the Drosophila oocyte

The localization of mRNAs to discrete cytoplasmic sites is important for the function of many, and perhaps all, cells. Many mRNAs are thought to be localized in a directed fashion along microtubule tracts. This appears to be the case for several mRNAs that are synthesized in Drosophila nurse cells and then transported into, and localized within, the oocyte. In this report, we compare the transport/localization kinetics and dynamics of three such mRNAs, K10, bicoid, and oskar. We generated flies carrying heat shock—K10, -bicoid, or -oskar fusion genes, which allowed us to carry out the molecular genetics equivalent of a pulse chase experiment. Our analyses indicate that K10, bicoid, and oskar mRNA transport and localization are a continuous process involving multiple movements of the same mRNA molecules. The transport and early localization dynamics of the three mRNAs are indistinguishable from each other and, in order, include accumulation in the apical regions of nurse cells, transport to the posterior pole of the oocyte, and movement to the oocyte's anterior cortex at stage 8. We also show that the rate of transport is the same in each case, ∼︁1.1 μm/min. Only after stage 8 are RNA-specific movements seen The similarities in the transport/early localization kinetics and dynamics of K10, bicoid, and oskar mRNAs suggest that such events are mediated by a common set of factors. We also observe that all three mRNAs localize to the apical regions of somatic follicle cells when expressed in such cells, suggesting that the transport/early localization factors are widespread and involved in the localization of mRNAs in many tissues. © 1996 Wiley-Liss, Inc.     

Glorund interactions in the regulation of gurken and oskar mRNAs

Precise temporal and spatial regulation of gene expression during Drosophila oogenesis is essential for patterning the anterior-posterior and dorsal-ventral body axes. Establishment of the anterior-posterior axis requires posterior localization and translational control of both oskar and nanos mRNAs. Establishment of the dorsal-ventral axis depends on the precise restriction of gurken mRNA and protein to the dorsal-anterior corner of the oocyte. We have previously shown that Glorund, the Drosophila hnRNP F/H homolog, contributes to anterior-posterior axis patterning by regulating translation of nanos mRNA, through a direct interaction with its 3′ untranslated region. To investigate the pleiotropy of the glorund mutant phenotype, which includes dorsal-ventral and nuclear morphology defects, we searched for proteins that interact with Glorund. Here we show that Glorund is part of a complex containing the hnRNP protein Hrp48 and the splicing factor Half-pint and plays a role both in mRNA localization and nurse cell chromosome organization, probably by regulating alternative splicing of ovarian tumor. We propose that Glorund is a component of multiple protein complexes and functions both as a translational repressor and splicing regulator for anterior-posterior and dorsal-ventral patterning.