Gene name - oskar
Cytological map position - 85B
Function - assembly of germ plasm
Keywords - posterior group
Symbol - osk
Genetic map position - 3-48
Classification - novel
Cellular location - oocyte protein
oskar serves two functions during Drosophila development (Lehmann, 1986). osk is responsible for assembling the germ plasm, a specialized cytoplasm required for germ cell formation. The germ plasm contains polar granules made up of proteins ( including Fat facets, Oskar, Vasa, Staufen and Tudor), mRNAs, including Oskar, (but not Vasa, Staufen or Tudor), and mitochondrial coded ribosomal RNA and polysomes.
During the syncytial phase of the early embryo the germ plasm induces germ cell fate on a number of zygotic nuclei adjacent to the posterior pole. Ectopic pole cells are induced in flies with genetically engineered excess amounts of oskar, or when Oskar mRNA is ectopically localized. The formation of ectopic pole cells depends not only on oskar but on two other genes as well: vasa and tudor. Oskar functions upstream of these latter two genes, and Staufen acts as the tether for Oskar mRNA at the posterior pole.
Exactly how Oskar assembles the germ plasm is unknown. Oskar does not possess RNA recognition motifs, however Vasa, a protein closely associated with Oskar, is an ATP dependent RNA helicase with RNA binding motifs. However it works, it is clear that Oskar has a major role in determining germ cell fate, since this determination is made in the early hours of development. Over the course of development, these germ cells will find their way to the mature ovaries and testis of the adults where in their turn they will produce eggs and sperm to ensure the existence of the next generation. This early sex cell determination is one of the wonders of biology; the developmental distance between the first cells of the embryo and the mature germ cells is marvelously brief. This is an extraordinary developmental protocol, one designed to guarantee that germ cells are not subject to an complex lineage prior maturation.
oskar's second developmental function is also concerned with polar plasm. Nanos mRNA becomes localized to the germ plasm. Through the capacity of Nanos to inhibit translation of Hunchback mRNA, the abdominal fate of the fly is determined. Nanos does not bind Hunchback mRNA. This role is reserved for Pumilio, which appears to bring Nanos into the complex (Murata, 1995). Again, the role of Oskar in this function is not fully understood, but Oskar's capacity to assemble the germ plasm is involved.
Thus Oskar has a dual function: determination of germ cell fate and determination of posterior polarity (Lehmann, 1994). Oskar resembles the CEO of a large firm. The work of such an officer is important, but it isn't easy to see exactly what that work entails or how it is accomplished. Oskar has been termed an anchor, something that directs the assembly of the pole plasm. But the immediate targets of Oskar are not yet understood, nor is it clear how, and with what other components in the developmental hierarchy, these as yet unknown targets interact.
The precise restriction of proteins to specific domains within a cell plays an important role in early development and differentiation. An efficient way to localize and concentrate proteins is by localization of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination. A central issue is how localized mRNAs are derepressed. Regulatory elements for both RNA localization and translational repression are situated in the 3' UTR of OSK mRNA, as they are in NOS. In the case of OSK, premature translation is prevented by Bruno, a 68-kD protein encoded by the arrest (aret) locus. Bruno recognizes a repeated conserved sequence (BRE, for Bruno response element) in the osk 3' UTR, and colocalizes with OSK mRNA to the posterior pole. In contrast to NOS, however, 3' UTR-mediated localization at the posterior pole is not sufficient for translation, as heterologous transcripts localized under the control of the full-length OSK 3' UTR are not translated. This indicates that the OSK 3' UTR, although it may participate, is not sufficient for translational activation, and that sequences elsewhere in the transcript are required for translation of OSK mRNA (Gunkel, 1998).
When OSK mRNA reaches the posterior pole of the Drosophila oocyte, its translation is derepressed by an active process that requires a specific element in the 5' region of the mRNA. This novel type of element is a translational derepressor element, whose functional interaction with the previously identified repressor region in the OSK 3' UTR is required for activation of Oskar mRNA translation at the posterior pole. The derepressor element only functions at the posterior pole, suggesting that a locally restricted interaction between trans-acting factors and the derepressor element may be the link between mRNA localization and translational activation. Specific interaction of two proteins with the OSK mRNA 5' region is shown; one of these also recognizes the 3' repressor element. p50 is a BRE binding protein that recognizes 3' repressor motifs similar to those recognized by Bruno. p50 functions as a second translational repressor independent of Bruno. The involvement of a second repressor protein in OSK translational control is not unexpected. Indeed, aubergine (aub), a gene required for efficient OSK mRNA translation, is required even when Bruno-mediated repression is alleviated by mutations in the BRE, leading to the suggestion that the aub gene product enhances translation by counteracting the action of a second repressor. It is interesting to note that the requirement for aub function in OSK translation is conferred not only by the OSK 3' UTR but also involves the 5' end of OSK mRNA. Consistent with this possible involvement of the OSK 5' end in translational repression, it is found that in transgenic flies containing an inefficient BRE, premature translation increases when the 5' end is truncated. Understanding the extent to which the 5' end of the OSKtranscript might contribute to overall translational repression will require mutations that selectively disrupt 5' repressor function without simultaneously affecting derepressor function (Gunkel, 1998).
The second protein interacting with the 5' end, p68, could act as a transcriptional activator. p68 is shown to be independent of Bruno. So far it has not been possible to define a p50-binding specificity distinct from that of p68 and to abolish selectively the binding of one or the other protein. Hence, the data do not allow the affirmation that p50 functions as a repressor, not only by binding to the BRE, but also through its interaction with the OSK 5', or that p68 is the derepressor protein. There are several mechanisms by which OSK could be activated at the posterior pole. The translation repressor proteins Bruno and p50 could be degraded by an activity localized at the posterior pole or else be displaced competitively by a derepressor protein. Alternatively, Oskar protein expression could be activated by concentration of the mRNA, resulting in the accumulation of small amounts of Oskar protein by leaky translation, thus initiating a positive feedback loop in which Oskar protein stimulates its own translation. None of these mechanisms is involved in the initial event of translational derepression. In the absence of the 5' derepressor element, OSK transcripts remain repressed, arguing against a passive, local repressor inactivation model. Therefore, the mode of action of the derepressor element is distinct from that of previously described cases, in which repression is released passively by inactivation of a repressor protein and no additional RNA elements are required. The derepressor element does not coincide with the BRE, suggesting that a competitive displacement of the repressor protein from the BRE is unlikely to be the mechanism leading to derepression. Finally, a combination of leaky translation and positive feedback of Oskar protein on its own translation as a mechanism for derepression is unlikely, as reporter transcripts can be derepressed in the absence of endogenous Oskar. Thus mechanisms by which 3' UTR-binding proteins repress translation are still not understood and it is unclear how the 5' derepressor element overcomes translational repression. The fact that transcripts lacking the derepressor element are localized but not translated demonstrates that the element plays little or no role in RNA localization and that localization does not suffice for translational derepression (Gunkel, 1998).
Translational recruitment of OSK mRNA is always accompanied by posterior localization of the mRNA, indicating that localization may trigger the release from translational repression. It is suggested that RNA localization directs osk transcripts into a cytoplasmic subcompartment containing trans-acting factors that interact specifically with the 5' element to mediate derepression. The spatial restriction of the derepression machinery could be achieved by prelocalization of at least some of the components to the posterior pole, or by the localized activation of uniformly distributed factors. During the early stages of oogenesis, OSK mRNA initially fills the entire cytoplasm of the growing oocyte and yet no Oskar protein is detected, even in the posterior region. This suggests that the derepressor proteins are expressed or activated only at certain stages of oocyte development, possibly through signals from the posterior pole. The existence of localized derepressors is supported by the observation that reporter transcripts bearing the BCD 3' UTR into which the OSK repressor element is inserted are localized to the anterior ofoocytes of embryos and not derepressed, even when they contain the derepressor element. The DEAD-box RNA helicase Vasa (whose SDS-PAGE mobility is similar to that of p68), the 120-kD double-stranded RNA-binding protein Staufen, and Aubergine, whose gene has not yet been cloned, play a role in the translation of OSK mRNA. On the basis of the data presented in this report, Staufen and Aubergine could be required to overcome p50-mediated repression, as both are necessary for osk translation, even in the absence of BRE-mediated repression (Gunkel, 1998).
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 two Oskar isoforms, Long Osk and Short Osk. Evidence is presented that this coupling is not sufficient to restrict Oskar to the posterior pole of the oocyte. 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. 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 (Vanzo, 2002).
At mid-oogenesis, the transport of OSK mRNA to the posterior pole of the oocyte requires the plus-end motor Kinesin I, a polarized microtubule network and an intact actin cytoskeleton. The polarized and sustained transport of OSK mRNA can also account for its maintenance at the posterior pole of the oocyte from stage 7 to 10. In contrast, at stage 10, an active process of mRNA maintenance must exist, as the polarized microtubule network is disassembled and a subcortical array of microtubules forms and promotes vigorous cytoplasmic streaming. Indeed, several lines of evidence indicate that, as of stage 10, Osk protein maintains OSK mRNA localization. (1) OSK mRNA delocalizes during stage 10 in the three osk nonsense mutants osk54, osk84 and osk346, predicted to produce truncated Osk peptides of 179, 253 and 323 amino acids, respectively. However, the failure to detect these peptides by Western analysis suggests that they are unstable and the nonsense osk alleles are protein null. (2) The maintenance of a transgenic NOS-OSK3'UTR mRNA at the posterior pole of stage 10B oocytes requires Osk protein. (3) Posterior accumulation of fluorescent OSK mRNA, injected into living oocytes at stage 10-11, occurs by a trapping mechanism dependent on endogenous Osk protein. Although it supports expression of Short Osk, oskM1R transgenic mRNA detaches from the posterior pole during late stage 10. In contrast, Long Osk, which is dispensable for pole plasm formation, is competent and required to persistently confine OSK mRNA at the posterior pole of the oocyte during late oogenesis until early embryogenesis. Thus, OSK mRNA maintenance is an active process mediated by Long Osk. Maintenance of the three nonsense OSK mRNAs is rescued in heterozygous females. This rescue in trans can only be attributed to the Long Osk isoform encoded by the wild-type osk gene. Consistent with this, Long Osk can also maintain localization of the transgenic oskM1R mRNA, which encodes only Short Osk (Vanzo, 2002).
In the absence of Long Osk, Short Osk also detaches from the posterior cortex of stage 10 oocytes, in concert with oskM1R mRNA. Both Short Osk and OSK mRNA delocalize in dense aggregates, suggesting that they might be associated. Consistent with this, they co-localize in the same released aggregates. It is noteworthy that the delocalizing pattern of oskM1R mRNA is significantly different from that of osk84 mRNA, which diffuses without forming aggregates. Because osk84 mRNA encodes an unstable Osk peptide, it is concluded that aggregate formation is dependent on Short Osk. The pole plasm protein Vasa, which is a component of the polar granules, the germline granules of Drosophila, is also detected in these aggregates. This suggests that the aggregates contain nascent but untethered polar granules, whose assembly might be initiated by Short Osk-mediated clustering of OSK mRNA. The ability of Short Osk to package macro-molecular complexes is supported by the observation that it can oligomerize, in a yeast two-hybrid assay. Given the underexpression of Long Osk relative to Short Osk in wild-type ovaries, multimerization of Short Osk could also explain the apparent non-stoichiometric competence of Long Osk to anchor Short Osk at the oocyte cortex (Vanzo, 2002).
Because both Long and Short Osk can sequester OSK mRNA, it is likely that the same region in the two isoforms mediates RNA association. However, no RNA-binding activity has been reported for Osk, which does not exhibit any predicted RNA-binding motif in its coding sequence. Thus, the association of the two Osk isoforms with OSK mRNA most probably involves adaptor(s). One such candidate could be Staufen, a double-strand RNA-binding protein suspected to bind OSK mRNA directly. Staufen is required for posterior maintenance of OSK mRNA, as revealed by its delocalization in the temperature-sensitive mutant stauC8. In ovaries expressing each Osk isoform individually, Staufen either co-localizes with the Long Osk/OSK mRNA complex at the posterior pole of the oocyte or co-segregates with the Short Osk/OSK mRNA complex in the released aggregates, as expected of an adaptor factor. Staufen binds to Short Osk in a yeast two-hybrid assay, but binds quite poorly to Long Osk, which does not reflect the robust ability of Long Osk to maintain OSK mRNA localization that this study reveals. Thus, whether the association of the two Osk isoforms with OSK mRNA relies on a direct interaction with Staufen or with another adaptor factor remains to be elucidated (Vanzo, 2002).
The observation that Long Osk but not Short Osk can anchor at the cortex suggests that the N-terminal extension of Long Osk mediates anchoring. Surprisingly, the extension is not sufficient for this function, as revealed by its failure to maintain an Osk-ß-galactosidase fusion in Osk protein-null oocytes at the onset of oocyte streaming. This suggests that at least two separate docking modules, one in the N-terminal extension of Long Osk and a second in the region shared by the two Osk isoforms, cooperate to form a robust anchoring domain. Two observations support this hypothesis: (1) the Osk-ß-galactosidase fusion remains localized during stages 8 to 10 of oogenesis in Osk protein-null oocytes, whereas native ß-galactosidase translated from a posterior localized RNA fails to accumulate at this location; (2) even in the absence of Long Osk, a residual amount of Short Osk remains localized at the posterior pole of the oocyte during late oogenesis and supports substantial posterior patterning and partial fertility of the progeny. Thus, two docking modules appear to be involved in Long Osk anchoring, but neither alone is sufficient for this process (Vanzo, 2002).
An important result of this work is that Long Osk anchors Short Osk, the pole plasm-inducing isoform, at the posterior pole of oocyte. Strikingly, although it contains the entire Short Osk sequence, Long Osk can not recruit pole plasm components. It has been proposed that the N-terminal extension of Long Osk exerts an inhibitory effect on downstream protein-interaction domains. It is proposed that this inhibition is caused by folding of the robust anchoring domain of Long Osk, masking the pole plasm-recruiting activity of this isoform. By contrast, the absence of the N-terminal anchoring module would allow Short Osk to nucleate pole plasm assembly. Hence, anchoring and pole plasm nucleation might be structurally mutually exclusive activities (Vanzo, 2002).
The results show that, in the absence of Long Osk-anchoring activity, which causes a massive dispersion of Short Osk from the posterior pole of the oocyte, effective abdominal patterning in the embryo can nonetheless be achieved. Consistent with this, strong impairment of OSK mRNA localization and translation in mutants in Tropomyosin II, an actin-binding protein, and Barentsz, a putative component of the transport machinery, has little consequence on abdominal development. This demonstrates that, with regard to abdomen formation, a substantial excess of Short Osk is present at the posterior pole of the wild-type oocyte. However, these results indicate that dispersion of untethered Short Osk from the posterior of oskM1R oocytes can hinder anterior development. A contribution of the weak premature translation of oskM1R to these anterior patterning defects cannot be excluded. However, the complete suppression of these defects by co-expression of oskM139L (which makes the long isoform), demonstrates that the anchoring activity of Long Osk can restrict the pole plasm-inducing activity of Short Osk to the posterior. Anchoring of the bulk of Short Osk to the posterior pole would lead to titration of limiting pole plasm components from any residual ectopically localized Osk (Vanzo, 2002).
In contrast, Short Osk delocalization causes a significant reduction of germ cell precursors and fertility of the progeny. These results provide yet another demonstration of the correlation between Osk protein dose and the number of pole cells formed. Indeed, whereas overexpression of Osk at the posterior pole increases pole cell number, its underexpression, caused by defects in RNA localization or translation, impedes pole cell formation. In this analysis, restoration of Short Osk anchoring by co-expression of Long Osk enhances Short Osk accumulation and restores germ-plasm integrity. This demonstrates that Long Osk guarantees accumulation of high levels of Short Osk in the subcortical region of the egg that is subsequently incorporated into pole cells. It has been shown that when the D. virilis Osk homolog is expressed in Drosophila melanogaster oocytes, it efficiently rescues the posterior patterning defects of osk mutants, but does not support pole cell formation in the embryos. Transgenic D. virilis Osk fails to maintain OSK mRNA localization in D. melanogaster, which led to a hypothesis that virilis Osk is not competent to anchor at the posterior pole of the D. melanogaster oocyte. This conclusion is entirely consistent with the demonstration that Osk-mediated anchoring of the pole plasm is a critical step during Drosophila germline formation (Vanzo, 2002).
Given that both efficient germ cell formation and, to a lesser extent, proper patterning rely on Long Osk-mediated anchoring of the pole plasm, an important issue in the future will be the characterization of the mechanism by which Long Osk is tethered to the cortex (Vanzo, 2002).
Bases in 5' UTR - 14
Exons - four
Bases in 3' UTR - 1045
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. 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 (Markussen, F. H. (1995).
There are no known oskar homologs.
Finding which elements of a protein are conserved in evolution is one of the first approaches to determining important protein functional domains. There are two broad domains of Oskar proteins conserved between D. melanogaster and D. virilus: one 82 amino acid stretch in the central portion of the molecule and the carboxy-terminal 220 amino acids. The two proteins share over 70% identity.
The Drosophila virilis oskar homolog, virosk, was examined as a transgene in Drosophila melanogaster flies. Cis-acting signals for the localization of Oskar mRNA are conserved, although the Virosk transcript also transiently accumulates at novel intermediate sites. The Virosk protein, however, shows substantial differences from Oskar: while virosk is able to rescue body patterning in a D. melanogaster oskar mutant, it is impaired in both mRNA maintenance and pole cell formation. Furthermore, virosk induces a dominant maternal-effect lethality when introduced into a wild-type background, and interferes with the posterior maintenance of the endogenous Oskar transcript in early embryogenesis. Virosk protein is unable to anchor at the posterior pole of the early embryo; this defect could account for all of the characteristics of virosk mentioned above (Webster, 1994).
The colocalization of morphogenetic signals involved in germ cell formation and in the specification of the body axis is not unique to Drosophila but is also found in Caenorhabditis elegans and Xenopus (Lehmann, 1994).
The establishment of the germline is a critical, yet surprisingly evolutionarily labile, event in the development of sexually reproducing animals. In the fly Drosophila, germ cells acquire their fate early during development through the inheritance of the germ plasm, a specialized maternal cytoplasm localized at the posterior pole of the oocyte. The gene oskar (osk) is both necessary and sufficient for assembling this substance. Both maternal germ plasm and oskar are evolutionary novelties within the insects, as the germline is specified by zygotic induction in basally branching insects, and osk has until now only been detected in dipterans. In order to understand the origin of these evolutionary novelties, comparative genomics, parental RNAi, and gene expression analyses was used in multiple insect species. It was found that the origin of osk and its role in specifying the germline coincided with the innovation of maternal germ plasm and pole cells at the base of the holometabolous insects and that losses of osk are correlated with changes in germline determination strategies within the Holometabola. These results indicate that the invention of the novel gene osk was a key innovation that allowed the transition from the ancestral late zygotic mode of germline induction to a maternally controlled establishment of the germline found in many holometabolous insect species. It is proposed that the ancestral role of osk was to connect an upstream network ancestrally involved in mRNA localization and translational control to a downstream regulatory network ancestrally involved in executing the germ cell program (Lynch, 2011).
date revised: 6 December 99
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