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

Gene name - oskar

Synonyms -

Cytological map position - 85B

Function - assembly of germ plasm

Keywords - posterior group

Symbol - osk

FlyBase ID:FBgn0003015

Genetic map position - 3-48

Classification - novel

Cellular location - oocyte protein



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Kanke, M., Jambor, H., Reich, J., Marches, B., Gstir, R., Ryu, Y.H., Ephrussi, A. and Macdonald, P.M. (2015). oskarRNA plays multiple noncoding roles to support oogenesis and maintain integrity of the germline/soma distinction. RNA [Epub ahead of print]. PubMed ID: 25862242
Summary:
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. This study shows 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, was to sequester the translational regulator Bruno (Bru), which itself controlled translation of osk mRNA. This defined a novel regulatory circuit, with Bru restricting the activity of osk, and osk in turn restricting the activity of Bru. Other functional elements, which did not bind Bru and were positioned close to the 3' end of the RNA, acted in the oocyte and were essential. Despite the different roles played by the different types of elements contributing to RNA function, mutation of any led to accumulation of the germline regulatory factors in the follicle cells.

Kanke, M. and Macdonald, P. M. (2015). Translational activation of Oskar mRNA: Reevaluation of the role and importance of a 5' regulatory element. PLoS One 10: e0125849. PubMed ID: 25938537
Summary:
Local translation of oskar (osk) mRNA at the posterior pole of the Drosophila oocyte is essential for axial patterning of the embryo, and is achieved by a program of translational repression, mRNA localization, and translational activation. Multiple forms of repression are used to prevent Oskar protein from accumulating at sites other than the oocyte posterior. Activation is mediated by several types of cis-acting elements, which presumably control different forms of activation. This study characterized a 5' element, positioned in the coding region for the Long Osk isoform and in the extended 5' UTR for translation of the Short Osk isoform. This element was previously thought to be essential for osk mRNA translation, with a role in posterior-specific release from repression. From this work, which includes assays which separate the effects of mutations on RNA regulatory elements and protein coding capacity, it was found that the element is not essential, and the study concludes that there is no evidence supporting a role for the element only at the posterior of the oocyte. The 5' element has a redundant role, and is only required when Long Osk is not translated from the same mRNA. Mutations in the element do disrupt the anchoring function of Long Osk protein through their effects on the amino acid sequence, a confounding influence on interpretation of previous experiments.

Simon, B., Masiewicz, P., Ephrussi, A. and Carlomagno, T. (2015). The structure of the SOLE element of oskar mRNA. RNA [Epub ahead of print]. PubMed ID: 26089324.
Summary:
mRNA localization by active transport is a regulated process that requires association of mRNPs with protein motors for transport along either the microtubule or the actin cytoskeleton. oskar mRNA localization at the posterior pole of the Drosophila oocyte requires a specific mRNA sequence, termed the SOLE, which comprises nucleotides of both exon 1 and exon 2 and is assembled upon splicing. The SOLE folds into a stem-loop structure. Both SOLE RNA and the exon junction complex (EJC) are required for oskar mRNA transport along the microtubules by kinesin. The SOLE RNA likely constitutes a recognition element for a yet unknown protein, which either belongs to the EJC or functions as a bridge between the EJC and the mRNA. This study determined the solution structure of the SOLE RNA by Nuclear Magnetic Resonance spectroscopy. The SOLE forms a continuous helical structure, including a few noncanonical base pairs, capped by a pentanucleotide loop. The helix displays a widened major groove, which could accommodate a protein partner. In addition, the apical helical segment undergoes complex dynamics, with potential functional significance.

Jeske, M., Bordi, M., Glatt, S., Muller, S., Rybin, V., Muller, C. W. and Ephrussi, A. (2015). The crystal structure of the Drosophila germline inducer Oskar identifies two domains with distinct Vasa helicase- and RNA-binding activities. Cell Rep 12: 587-598. PubMed ID: 26190108
Summary:
In many animals, the germ plasm segregates germline from soma during early development. Oskar protein is known for its ability to induce germ plasm formation and germ cells in Drosophila. However, the molecular basis of germ plasm formation remains unclear. This study shows that Oskar is an RNA-binding protein in vivo, crosslinking to nanos, polar granule component, and germ cell-less mRNAs, each of which has a role in germline formation. Furthermore, high-resolution crystal structures are presented of the two Oskar domains. RNA-binding maps in vitro to the C-terminal domain, which shows structural similarity to SGNH hydrolases. The highly conserved N-terminal LOTUS domain forms dimers and mediates Oskar interaction with the germline-specific RNA helicase Vasa in vitro. These findings suggest a dual function of Oskar in RNA and Vasa binding, providing molecular clues to its germ plasm function.

Yang, N., Yu, Z., Hu, M., Wang, M., Lehmann, R. and Xu, R.M. (2015). Structure of Drosophila Oskar reveals a novel RNA binding protein. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 26324911
Summary:
Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. This study reports the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix-fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, it was found that Osk-C binds the 3'UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3'UTRs, and provide structural insights into a novel protein-RNA interaction motif involving a hydrolase-related domain.

Macdonald, P. M., Kanke, M. and Kenny, A. (2016). Community effects in regulation of translation. Elife 5 [Epub ahead of print]. PubMed ID: 27104756
Summary:
Certain forms of translational regulation, and translation itself, rely on long-range interactions between proteins bound to the different ends of mRNAs. A widespread assumption is that such interactions occur only in cis, between the two ends of a single transcript. However, certain translational regulatory defects of the Drosophila oskar (osk) mRNA can be rescued in trans. It is proposed that inter-transcript interactions, promoted by assembly of the mRNAs in particles, allow regulatory elements to act in trans. This study confirms predictions of that model and shows that disruption of Polypyrimidine tract binding protein (PTB) dependent particle assembly inhibits rescue in trans. Communication between transcripts is not limited to different osk mRNAs, as regulation imposed by cis-acting elements embedded in the osk mRNA spreads to gurken mRNA. It is concluded that community effects exist in translational regulation.
Broyer, R., Monfort, E. and Wilhelm, J. E. (2016). Cup regulates oskar mRNA stability during oogenesis. Dev Biol [Epub ahead of print]. PubMed ID: 27554167
Summary:
The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. This study found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form.
Veeranan-Karmegam, R., Boggupalli, D. P., Liu, G. and Gonsalvez, G. B. (2016). A new isoform of Drosophila non-muscle Tropomyosin 1 interacts with Kinesin-1 and functions in oskar mRNA localization. J Cell Sci 129: 4252-4264. PubMed ID: 27802167
Summary:
Recent studies have revealed that diverse cell types use mRNA localization as a means to establish polarity. Despite the prevalence of this phenomenon, much less is known regarding the mechanism by which mRNAs are localized. The Drosophila melanogaster oocyte provides a useful model for examining the process of mRNA localization. oskar (osk) mRNA is localized at the posterior of the oocyte, thus restricting the expression of Oskar protein to this site. The localization of osk mRNA is microtubule dependent and requires the plus-end-directed motor Kinesin-1. Unlike most Kinesin-1 cargoes, localization of osk mRNA requires the Kinesin heavy chain (Khc) motor subunit, but not the Kinesin light chain (Klc) adaptor. This report, demonstrates that a newly discovered isoform of Tropomyosin 1, referred to as Tm1C, directly interacts with Khc and functions in concert with this microtubule motor to localize osk mRNA. Apart from osk mRNA localization, several additional Khc-dependent processes in the oocyte are unaffected upon loss of Tm1C. These results therefore suggest that the Tm1C-Khc interaction is specific for the osk localization pathway.
Ryu, Y. H., Kenny, A., Gim, Y., Snee, M. and Macdonald, P. M. (2017). Multiple cis-acting signals, some weak by necessity, collectively direct robust transport of Oskar mRNA to the oocyte. J Cell Sci. PubMed ID: 28760927
Summary:
Localization of mRNAs can involve multiple steps, each with its own cis-acting localization signals and transport factors. How is the transition between different steps orchestrated? This study shows that the initial step in localization of Drosophila oskar mRNA - transport from nurse cells to the oocyte - relies on multiple cis-acting signals. Some of these are binding sites for the translational control factor Bruno, suggesting that Bruno plays an additional role in mRNA transport. Although transport of oskar mRNA is essential and robust, the localization activity of individual transport signals is weak. Notably, increasing the strength of individual transport signals, or adding a strong transport signal, disrupts the later stages of oskar mRNA localization. It is proposed that the oskar transport signals are weak by necessity; their weakness facilitates transfer of the oskar mRNA from the oocyte transport machinery to the machinery for posterior localization.
BIOLOGICAL OVERVIEW

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).

Oskar anchoring restricts pole plasm formation to the posterior of the Drosophila oocyte

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).

RNA sequences required for the noncoding function of oskar RNA also mediate regulation of Oskar protein expression by Bicoid Stability Factor

The Drosophila oskar (osk) mRNA is unusual in having both coding and noncoding functions. As an mRNA, osk encodes a protein which is deployed specifically at the posterior of the oocyte. This spatially-restricted deployment relies on a program of mRNA localization and both repression and activation of translation, all dependent on regulatory elements located primarily in the 3' untranslated region (UTR) of the mRNA. The 3' UTR also mediates the noncoding function of osk, which is essential for progression through oogenesis. Mutations which most strongly disrupt the noncoding function are positioned in a short region (the C region) near the 3' end of the mRNA, in close proximity to elements required for activation of translation. Bicoid Stability Factor (BSF) binds specifically to the C region of the mRNA. Both knockdown of bsf and mutation of BSF binding sites in osk mRNA have the same consequences: Osk expression is largely eliminated late in oogenesis, with both mRNA localization and translation disrupted. Although the C region of the osk 3′ UTR is required for the noncoding function, BSF binding does not appear to be essential for that function (Ryu, 2015).

One way to categorize RNAs is by their coding potential, or lack thereof. Members of one group, the mRNAs, have long open reading frames and are translated, thereby performing a coding function. The other group, consisting of RNAs without long open reading frames, has many members with no consistent size or organization. Such noncoding RNAs perform a wide variety of structural, regulatory and enzymatic functions. Often, these coding and noncoding roles are mutually exclusive. Most of the exceptions involve small ORFs, which can encode short peptides, in long noncoding RNAs (lncRNAs). Rarely, more dramatic overlap in function has been observed for conventional mRNAs with long open reading frames. The Xenopus VegT mRNA encodes a transcription factor required for endoderm formation in the embryo. The same mRNA also has a structural role in organization of the cytokeratin cytoskeleton. Depletion of VegT mRNA leads to fragmentation of the cytokeratin network in the vegetal cortex of the oocyte. Sequences within much of the mRNA appear to act redundantly in controlling the organization of the cytokeratin network, with a functional element contained within a 300 nt portion of the 3′ UTR sufficient to induce depolymerization of cytokeratin filaments (Ryu, 2015).

A second mRNA with essential coding and noncoding functions is oskar (osk), from Drosophila. Osk protein is expressed specifically at the posterior pole of the oocyte and early embryo, where it is responsible for embryonic body patterning and germ cell formation. In the absence of Osk protein, oogenesis progresses normally except for the failure to assemble posterior pole plasm in the oocyte. Although eggs are produced, the embryos fail to form abdominal segments and die. This coding role for osk places substantial constraints on the mRNA sequence. The open reading frame is constrained by the need to encode Osk protein. In addition, noncoding regions are constrained by the elaborate regulation required to restrict Osk protein expression to a discrete subcellular domain: misexpression of Osk is just as lethal as loss of Osk. The osk mRNA is also needed, independent of its coding role, for progression through oogenesis. In the absence of osk mRNA a variety of defects emerge in the organization of the egg chamber, with oogenesis arrested and no eggs produced ( Jenny, 2006; Kanke, 2015). These defects are present well before the developmental stage when Osk protein first appears, and the osk RNA function does not require the osk coding region. Instead, the osk mRNA 3' UTR mediates the noncoding function, placing constraints on the sequence of that region of the mRNA (Ryu, 2015).

Deployment of Osk protein specifically at the posterior pole of the oocyte involves a complex and coordinated program of mRNA localization and translational control. osk mRNA is transcribed in the nurse cells and transported into the oocyte through cytoplasmic bridges. Within the oocyte, osk mRNA is transiently enriched at different positions, culminating in persistent posterior localization starting at stage 9; this is when Osk protein first accumulates. Translational repression serves to prevent expression from osk mRNA that has not yet been localized, or has failed to become localized. Once osk mRNA is localized, translational activation must then override repression and allow Osk protein to be made. Many factors and regulatory elements are required for this regulation, with most of the elements positioned in the 3'UTR. Among the elements are a number of binding sites for Bru (BREs and others), clustered in two regions of the 3'UTR: the AB region (close to the coding region), and the C region (close to the 3' end). Mutation of all the BREs disrupts translational repression, revealing the role of Bru as a repressor. By contrast, mutation of only the C region cluster of BREs disrupts translational activation, implicating Bru in activation, as well as repressio in the C region also disrupts translational activation (Ryu, 2015).

The noncoding role of osk mRNA is mediated by the 3' UTR. Of greater importance to the noncoding requirement for osk mRNA are sequences positioned close to the mRNA 3' end in the C region, including the Bru binding sites that activate translation. These C region Bru binding sites contribute to sequestration of Bru, but also play a separate and essential role in osk noncoding function. Additional sequences essential for the noncoding function, which do not bind Bru, are positioned nearby. Some of the sequences in this region appear to act by binding poly(A) binding protein (PABP). However, the mutations which most strongly disrupt osk RNA function are not PABP binding sites, and the factor expected to bind them has not been identified (Kanke, 2015; Ryu, 2015 and references therein).

To better understand the roles of the C region of the osk mRNA this study sought proteins which bind specifically to the essential sequences. Bicoid Stability Factor (BSF), a protein previously found to act in stabilizing the bicoid mRNA, binds to the osk C region, with binding dependent on sequences most critical for osk RNA function early in oogenesis. Surprisingly, it was found that the same sequences are also required again, late in oogenesis, for regulation of osk expression. BSF mediates this later function, as shown in two complementary approaches. However, binding of BSF to the C region does not appear to be responsible for the early function, as certain mutations which substantially reduce BSF binding have no effect on the noncoding role of osk mRNA. Why regulatory and functional elements should be superimposed in the RNA sequence is an intriguing question, as the osk 3′ UTR is quite large and thus does not seem to be constrained in size (Ryu, 2015).

Long Oskar controls mitochondrial inheritance in Drosophila melanogaster

Inherited mtDNA mutations cause severe human disease. In most species, mitochondria are inherited maternally through mechanisms that are poorly understood. Genes that specifically control the inheritance of mitochondria in the germline are unknown. This study shows that the long isoform of the protein Oskar regulates the maternal inheritance of mitochondria in Drosophila melanogaster. During oogenesis mitochondria accumulate at the oocyte posterior, concurrent with the bulk streaming and churning of the oocyte cytoplasm. Long Oskar traps and maintains mitochondria at the posterior at the site of primordial germ cell (PGC) formation through an actin-dependent mechanism. Mutating long oskar strongly reduces the number of mtDNA molecules inherited by PGCs. Therefore, Long Oskar ensures germline transmission of mitochondria to the next generation. These results provide molecular insight into how mitochondria are passed from mother to offspring, as well as how they are positioned and asymmetrically partitioned within polarized cells (Hurd, 2016).

Germ cells are the means by which sexually reproducing organisms transmit genetic material to subsequent generations to ensure the continuance of the species. Consequently, the formation and specification of germ cells is one of the most important events in development. PGC formation can occur either through the cytoplasmic inheritance of maternally deposited determinants, called germ plasm, or through inductive cell-signaling events. In D. melanogaster, PGCs are formed by the deposition of germ plasm at the posterior of the embryo. The germ plasm has long been known to be rich in mitochondria. In fact, in mammals one of the names for germline granules is the intermitochondrial cement. The reason for this curious association, however, has been unclear until now (Hurd, 2016).

This study shows that mitochondria accumulate in the germ plasm to ensure the transmission of their genomes to the next generation. In D. melanogaster, most mitochondria are transported to the germ plasm during cytoplasmic streaming in developing oocytes and maintained there by an actin-dependent mechanism. Long Oskar controls mitochondrial anchoring at the posterior and is not only necessary but also sufficient to tether mitochondria wherever it is expressed. Mutating long oskar decreases the number of mitochondrial genomes transmitted to the next generation, demonstrating that Long Oskar is important for mtDNA inheritance. Long Oskar mutants also have reduced numbers of PGCs and frequently impaired oogenesis. The current data suggest that a potential cause of this is a failure to enrich mitochondria at the posterior. However, it remains to be determined whether the reduction in the number of mitochondria at the posterior and in PGCs affects PGC survival, formation, or division. Long Oskar-mediated mitochondrial enrichment could also play a role in the formation, biogenesis, and/or anchoring of germ plasm to the posterior prior to PGC formation. Alternatively, the defects in long oskar mutants could be due to some other function of Long Oskar independent of its role in trapping mitochondria at the posterior (Hurd, 2016).

Previous studies have analyzed mitochondrial distribution during earlier stages of Drosophila oogenesis. They show that mitochondria initially enter the oocyte traveling on microtubules and once there coalesce into a single mass resembling a structure called the Balbiani body. Recent data suggest that selective replication of mtDNA may restrict the transmission of deleterious mtDNA mutations at this time. Further experiments showed that Balbiani body mitochondria associate with the posterior until stage 7, when the oocyte repolarizes its microtubule network. This study analyzed mitochondrial distribution at later stages of oogenesis. The vast majority of mitochondria passed into PGCs accumulate during and after stage 10b, and thus may be predominantly nurse cell derived and Balbiani body independent. In the absence of Long Oskar a small amount of mitochondria do enter the PGCs, however, and it is possible that these could constitute a different pool that entered the oocyte at an earlier stage. Direct visualization of mitochondrial populations are needed to determine whether specific sources of mitochondria reach the posterior pole or whether they are randomly selected from the oocyte pool (Hurd, 2016).

Mitochondrial transport is often an active process in which motor proteins and their adapters move mitochondria along the cytoskeleton. Interestingly, this is not likely the case in D. melanogaster stage 10 oocytes. Instead, it was found that mitochondria move apparently passively, caught in the bulk flow of the oocyte cytoplasm, to localize to the oocyte posterior. This mode of localization is not unique to mitochondria; germ plasm RNAs, such as nanos, also use it to localize to the embryo posterior. Cytoplasmic streaming occurs in a wide variety of other contexts, across a range of organisms and developmental stages. Given the current findings it will be interesting to investigate whether cytoplasmic streaming is used in other contexts as a means of mitochondrial transport or asymmetric localization (Hurd, 2016).

How Long Oskar uses the actin cytoskeleton to anchor mitochondria remains unclear. Oskar is present in two forms, Short and Long. Short Oskar is an integral member of germ plasm and is both necessary and sufficient to form functional PGCs. In stark contrast, Long Oskar is distinctly localized to endocytic membranes and is not required for PGC formation per se (Tanaka, 2011b; Vanzo, 2007). Long Oskar may instead function to help anchor the germ plasm by promoting yolk endocytosis and remodeling of the actin cytoskeleton. Unexpectedly, this study did not identify any endocytic proteins in Long Oskar co-immunoprecipitation experiments. Instead, the most abundant Long Oskar interacting proteins identified were actin and actin-binding proteins including surprisingly a number of muscle-specific actinomyosin proteins. This leaves open the possibility that Long Oskar, and more specifically its N-terminal domain, nucleates actin directly or regulates proteins that modify actin. If so, Long Oskar would likely represent a new type of actin-modifying protein, as its N-terminal domain bears no sequence homology to any actin-modifying protein in Drosophila or elsewhere. Overexpression of Long Oskar in S2R+ cells caused gross alteration to the F-actin cytoskeleton, which is also consistent with Long Oskar binding the actin cytoskeleton and possibly competing with other actin cytoskeletal binding proteins. Further experiments will be required to determine exactly how Long Oskar alters the actin cytoskeleton and whether cytoskeletal-mediated mitochondrial localization requires endosomal components (Hurd, 2016).

The actin cytoskeleton is necessary for mitochondrial retention at the posterior pole of the embryo. Defects in mitochondrial interactions with the cytoskeleton are associated with many neurodegenerative diseases. Interestingly, disruption of F-actin with actin-depolymerizing drugs affects mitochondrial retention, but not transport, in Drosophila neurons. Furthermore, in vertebrate axonal neurons mitochondria have been shown to interact with actin microfilaments. As both Oskar and TmII are reported to be expressed in Drosophila neurons, it would be interesting to determine whether these two proteins similarly anchor mitochondria in this cell type. Further high-resolution imaging is also required to determine the regulation and dynamics of this potentially general mechanism of mitochondrial retention (Hurd, 2016).

Long Oskar acts as the main mechanism of mitochondrial inheritance in PGCs. Whether mitochondria that localize to the posterior and represent the majority of those inherited, are chosen at random, or are selected based on fitness, health, or some other attribute remains to be determined. In yeast, such a 'fitness'-based mechanism of inheritance has been observed. There, bundles of F-actin extend from the bud tip to the mother cell and serve as tracks for mitochondrial movement. Far from static, these actin cables are continuously moving away from the bud. Therefore, for mitochondria to be inherited into daughter cells they must 'crawl upstream' against the opposing movement of the actin cables, creating a fitness test such that only the healthiest mitochondria make it and are inherited. It is possible that a similar situation also occurs at the posterior of Drosophila oocytes. Although mitochondria appear to be statically anchored at the posterior in the embryo, the current analysis does not exclude the possibility that they are undergoing short-range movements on actin filaments. Indeed, purifying selection against deleterious mtDNA mutations has been observed in the Drosophila germline. It will be interesting to explore whether the accumulation and inheritance of mitochondria serves as a mechanism to test fitness and/or select against those that carry harmful mutations (Hurd, 2016).

Most organisms inherit mitochondria uniparentally. The reason for this remains unclear. Recent evidence suggests that inheritance of paternal mtDNA can be harmful. Consistent with this, multiple pathways have been described in Drosophila preventing the transmission of paternal mtDNA. Clearly, understanding mechanistically how mitochondria are transmitted and the genes that regulate this process is a key step in ultimately determining why this unusual mode of inheritance is so prevalent in nature (Hurd, 2016).


GENE STRUCTURE

oskar and pumilio are separated by 140 kb which includes an intervening gene PRD7 (Kim-Ha, 1991).
Genomic length - 3.3 kb

Bases in 5' UTR - 14

Exons - four

Bases in 3' UTR - 1045


PROTEIN STRUCTURE

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).

Amino Acids - 606

Structural Domains

There are no known oskar homologs.


EVOLUTIONARY 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).

Patterns of molecular evolution of the germ line specification gene oskar suggest that a novel domain may contribute to functional divergence in Drosophila

In several metazoans including flies of the genus Drosophila, germ line specification occurs through the inheritance of maternally deposited cytoplasmic determinants, collectively called germ plasm. The novel insect gene oskar (see Oskar precomputed BLAST) is at the top of the Drosophila germ line specification pathway, and also plays an important role in posterior patterning. A novel N-terminal domain of Oskar (the Long Oskar domain) evolved in Drosophilids, but the role of this domain in Oskar functional evolution is unknown. Trans-species transgenesis experiments have shown that Oskar orthologs from different Drosophila species have functionally diverged, but the underlying selective pressures and molecular changes have not been investigated. As a first step toward understanding how Oskar function could have evolved, molecular evolution analysis was applied to Oskar sequences from the completely sequenced genomes of 16 Drosophila species from the Sophophora subgenus, Drosophila virilis and Drosophila immigrans. Overall, this gene is subject to purifying selection, but individual predicted structural and functional domains are subject to heterogeneous selection pressures. Specifically, two domains, the Drosophila-specific Long Osk domain and the region that interacts with the germ plasm protein Lasp, are evolving at a faster rate than other regions of Oskar. Further, evidence is provided that positive selection may have acted on specific sites within these two domains on the D. virilis branch. This domain-based analysis suggests that changes in the Long Osk and Lasp-binding domains are strong candidates for the molecular basis of functional divergence between the Oskar proteins of D. melanogaster and D. virilis. This molecular evolutionary analysis thus represents an important step towards understanding the role of an evolutionarily and developmentally critical gene in germ plasm evolution and assembly (Ahuja, 2014).


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

date revised: 10 February 2014

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