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: | 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
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
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.
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
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
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
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
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
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
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.
Tiwari, B., Kurtz, P., Jones, A. E., Wylie, A., Amatruda, J. F., Boggupalli, D. P., Gonsalvez, G. B. and Abrams, J. M. (2017). Retrotransposons mimic germ plasm determinants to promote transgenerational inheritance. Curr Biol 27(19): 3010-3016.e3013. PubMed ID: 28966088
Retrotransposons are a pervasive class of mobile elements present in the genomes of virtually all forms of life. In metazoans, these are preferentially active in the germline, which, in turn, mounts defenses that restrain their activity. This study report that certain classes of retrotransposons ensure transgenerational inheritance by invading presumptive germ cells before they are formed. Using sensitized Drosophila and zebrafish models, this study found that diverse classes of retrotransposons migrate to the germ plasm, a specialized region of the oocyte that prefigures germ cells and specifies the germline of descendants in the fertilized egg. In Drosophila, evidence was found for a "stowaway" model, whereby Tahre retroelements traffic to the germ plasm by mimicking oskar RNAs and engaging the Staufen-dependent active transport machinery. Consistent with this, germ plasm determinants attracted retroelement RNAs even when these components were ectopically positioned in bipolar oocytes. Likewise, vertebrate retrotransposons similarly migrated to the germ plasm in zebrafish oocytes. Together, these results suggest that germ plasm targeting represents a fitness strategy adopted by some retrotransposons to ensure transgenerational propagation.
Nieuwburg, R., Nashchekin, D., Jakobs, M., Carter, A. P., Khuc Trong, P., Goldstein, R. E. and St Johnston, D. (2017). Localised dynactin protects growing microtubules to deliver oskar mRNA to the posterior cortex of the Drosophila oocyte. Elife 6. PubMed ID: 29035202
The localisation of oskar mRNA to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Kinesin 1 transports oskar mRNA to the oocyte posterior along a polarised microtubule cytoskeleton that grows from non-centrosomal microtubule organising centres (ncMTOCs) along the anterior/lateral cortex. This study shows that the formation of this polarised microtubule network also requires the posterior regulation of microtubule growth. A missense mutation in the dynactin Arp1 subunit causes most oskar mRNA to localise in the posterior cytoplasm rather than cortically. oskar mRNA transport and anchoring are normal in this mutant, but the microtubules fail to reach the posterior pole. Thus, dynactin acts as an anti-catastrophe factor that extends microtubule growth posteriorly. Kinesin 1 transports dynactin to the oocyte posterior, creating a positive feedback loop that increases the length and persistence of the posterior microtubules that deliver oskar mRNA to the cortex.
Kistler, K. E., Trcek, T., Hurd, T. R., Chen, R., Liang, F. X., Sall, J., Kato, M. and Lehmann, R. (2018). Phase transitioned nuclear Oskar promotes cell division of Drosophila primordial germ cells. Elife 7. PubMed ID: 30260314
Germ granules are non-membranous ribonucleoprotein granules deemed the hubs for post-transcriptional gene regulation and functionally linked to germ cell fate across species. Little is known about the physical properties of germ granules and how these relate to germ cell function. This study examined two types of germ granules in the Drosophila embryo: cytoplasmic germ granules that instruct primordial germ cells (PGCs) formation and nuclear germ granules within early PGCs with unknown function. Cytoplasmic and nuclear germ granules are phase transitioned condensates nucleated by Oskar protein that display liquid as well as hydrogel-like properties. Focusing on nuclear granules, Oskar was found to drive their formation in heterologous cell systems. Multiple, independent Oskar protein domains synergize to promote granule phase separation. Deletion of Oskar's nuclear localization sequence specifically ablates nuclear granules in cell systems. In the embryo, nuclear germ granules promote germ cell divisions thereby increasing PGC number for the next generation.
Eichler, C. E., Hakes, A. C., Hull, B. and Gavis, E. R. (2020). Compartmentalized oskar degradation in the germ plasm safeguards germline development. Elife 9. PubMed ID: 31909715
Partitioning of mRNAs into ribonucleoprotein (RNP) granules supports diverse regulatory programs within the crowded cytoplasm. At least two types of RNP granules populate the germ plasm, a cytoplasmic domain at the posterior of the Drosophila oocyte and embryo. Germ granules deliver mRNAs required for germline development to pole cells, the germ cell progenitors. A second type of RNP granule, here named founder granules, contains oskar mRNA, which encodes the germ plasm organizer. Whereas oskar mRNA is essential for germ plasm assembly during oogenesis, this study shows that it is toxic to pole cells. Founder granules mediate compartmentalized degradation of oskar during embryogenesis to minimize its inheritance by pole cells. Degradation of oskar in founder granules is temporally and mechanistically distinct from degradation of oskar and other mRNAs during the maternal-to-zygotic transition. These results show how compartmentalization in RNP granules differentially controls fates of mRNAs localized within the same cytoplasmic domain.

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).

The transcriptome-wide landscape and modalities of EJC binding in adult Drosophila

Exon junction complex (EJC) assembles after splicing at specific positions upstream of exon-exon junctions in mRNAs of all higher eukaryotes, affecting major regulatory events. In mammalian cell cytoplasm, EJC is essential for efficient RNA surveillance, while in Drosophila, EJC is essential for localization of oskar mRNA. This study has developed a method for isolation of protein complexes and associated RNA targets (ipaRt) to explore the EJC RNA-binding landscape in a transcriptome-wide manner in adult Drosophila. The EJC was found at canonical positions, preferably on mRNAs from genes comprising multiple splice sites and long introns. Moreover, EJC occupancy is highest at junctions adjacent to strong splice sites, CG-rich hexamers, and RNA structures. Highly occupied mRNAs tend to be maternally localized and derive from genes involved in differentiation or development. These modalities, which have not been reported in mammals, specify EJC assembly on a biologically coherent set of transcripts in Drosophila (Obrdlik, 2019).

The exon junction complex (EJC) consists of a heterotetramer core composed of eIF4AIII, Mago, Y14 (Tsunagi), and Barentsz (Btz) and auxiliary factors that form the EJC periphery. The complex assembles on mRNAs during splicing, -20 to -24 nt upstream of exon-exon junctions. EJC assembly is a multi-step process that begins with CWC22-mediated deposition of the DEAD-box helicase eIF4AIII on nascent pre-mRNAs (Alexandrov, 2012; Barbosa, 2012; Steckelberg, 2015) and is followed by recruitment of Mago and Y14, forming a pre-EJC intermediate. The pre-EJC is stably bound to RNA because of the ATPase-inhibiting activity of the (non-RNA-binding) Mago-Y14 heterodimer, which 'locks' eIF4AIII helicase in its RNA-bound state. Once formed, the pre-EJC is completed by recruitment of Barentsz (Btz), forming mature EJCs (Obrdlik, 2019).

The roles of the EJC in post-transcriptional control of gene expression are manifold. In the nucleus, EJC subunits have a role in splicing, mRNA export, and nuclear retention of intron-containing RNAs. In the cytoplasm, the EJC is reported to play a role in translation, nonsense-mediated decay (NMD) , and RNA localization. Although most EJC functions appear conserved, in Drosophila the EJC is not crucial for NMD, but it is essential for oskar mRNA localization within the developing oocyte. To better understand the engagement of the EJC in the fly, a strategy to stabilize mRNA binding proteins (mRBPs) associated with their RNA templates has been developed within multi-protein messenger ribonucleoprotein (mRNP) assemblies, and the EJC mRNA interactome was defined in adult Drosophila melanogaster. Through the use of the crosslinking agent dithio(bis-) succinimidylpropionate (DSP), the method captures stable and transient protein interactions in close proximity and allows definition of the binding sites of specific protein (holo-)complexes associated with their RNA templates (isolation of protein complexes and associated RNA targets [ipaRt]). This analysis of EJC-protected sites defined by ipaRt reveals that in Drosophila, EJC binding occurs at canonical deposition sites, with a median coordinate ~22 nt upstream of exon-exon junctions. Although in mammals EJC-mediated protection outside canonical sides was reported, this study finds that in Drosophila the degree of non-canonical EJC-mediated RNA protection is minimal. In Drosophila RNA polymerase II transcripts protected primarily by the EJC derive from genes involved in differentiation or development, while mRNAs protected primarily by mRBPs derive from genes with homeostatic functions. This analysis suggests that the EJC's bias for transcripts in Drosophila is a consequence of several modalities in the genes' architecture, particularly splice site number and intron length. Moreover, EJC binding is enhanced by adjacent RNA secondary structures and CUG-rich hexamers located 3' to the EJC binding site. These modalities were not identified in previous studies of mammalian EJC binding, reflecting either greater specificity of this method for fully assembled EJCs or differences in EJC binding between flies and human. This study provides a comprehensive transcriptome-wide view of EJC-RNA interactions in a whole organism and unravels RNA modalities that contribute to the unforeseen biological coherence of the bound transcripts (Obrdlik, 2019).

This study has profiled the landscape of EJC binding across the transcriptome of a whole animal, Drosophila melanogaster, and determined the parameters that influence the distribution of the complex on RNAs in the organism. Previous knowledge of EJC-RNA interactions was based on UV-crosslinking experiments in specific cell types grown as homogeneous cultures for the individual studies. Although UV crosslinking remains a method of choice for identification of protein binding sites on nucleic acids, because of the inefficient penetration of UV light into tissues and organisms, the method is most useful when applied to cells in culture. In contrast, this analysis of EJC distribution in the tissues of whole Drosophila flies was made possible by ipaRt, which uses the crosslinking agent DSP to freeze protein-protein interactions within otherwise dynamic RNP complexes, such as the EJC (Obrdlik, 2019).

DSP-mediated covalent bond formation between the RNA helicase eIF4AIII and the Mago-Y14 heterodimer is shown to preserve EJCs in their 'locked' state on mRNAs and that efficient recovery of the bound RNAs does not require their crosslinking to eIF4AIII using UV light. The 'ipaRt' approach, like CLIP and iCLIP, enables highly stringent washing of the samples. In support of the robustness and reliability of the DSP-based assays, this study demonstrated high reproducibility not only among technical but also biological replicates of EJC ipaRt, as well as mRBP footprinting sequencing results (Obrdlik, 2019).

Furthermore, ipaRt allows enables the use of non-RNA-binding subunits of the EJC, such as Mago, as immunoprecipitation baits. This is highly relevant in the context of the EJC, as it has been shown that its RNA-binding subunit, the RNA helicase eIF4AIII, may have other, EJC-independent functions in the cell. ipaRt afford the option of using Mago as a EJC bait, and indeed this is a main reason for the high-quality definition of the EJC binding landscape in the fly cytoplasm that was achieved. The protection site reads obtained from EJC ipaRt map almost exclusively to canonical EJC deposition sites with a median protection ~22 nt of the upstream exon's 3' end. In contrast to mammalian EJC CLIP and RIP studies, in which eIF4AIII served as an immunoprecipitation bait, EJC ipaRt reads mapping to regions distant from canonical deposition sites are of low abundance and sequencing coverage. Although this discrepancy could reflect differences in EJC engagement in humans and Drosophila, it more likely reflects the choice of bait or the cell compartment in which the analysis was executed. Indeed, a recent study in human cells revealed that when the cytoplasmic EJC component Btz was chosen as the bait rather than eIF4AIII, the proportion of non-canonical EJC deposition sites was negligible (Obrdlik, 2019).

Finally, in ipaRt the DSP crosslinker is applied ex vivo during tissue disruption and does not require inhibition of translation in vivo. Therefore ipaRt is considered a method of choice for functional investigations of protein-RNA complexes in fully developed organisms and tissues (Obrdlik, 2019).

Through this analysis, factors were defined that contribute to or inhibit EJC assembly on mRNAs and at individual exon-exon junctions in Drosophila. From this it is deduced that the landscape of EJC binding to RNAs is sculpted through regulation of EJC assembly at two levels in the fly (Obrdlik, 2019).

At the upstream regulatory level, the degree to which EJCs are assembled on an mRNA is dictated by the complexity of the gene's architecture: mRNAs produced from genes of simple architecture are marked by fewer EJCs, while mRNAs from genes of complex architecture, comprising multiple splice sites and long introns, are EJC bound to a higher degree. Given that EJCs assemble on mRNAs concomitantly with splicing, it is not surprising that mRNAs of genes containing a greater number of introns are more likely to be EJC bound. However, the finding that the enhancing effect on EJC binding provoked by large introns is not restricted to flanking junctions but occurs at junctions mRNA-wide is unexpected. Loss-of-function experiments indicate that the EJC participates in exon definition during splicing of long intron-containing genes in Drosophila, particularly in definition of exons proximal to the long introns. The data exclude any significant bias toward EJC assembly in proximity to long-intron splice junctions. Instead they reveal a general enhancement of EJC binding at exon-exon junctions throughout transcripts of long-intron genes. Therefore, it is concluded that stable binding of EJCs within mRNAs of long-intron genes is not the result of EJC engagement in exon definition. Instead, it is proposed that the high degree of EJC binding to long-intron transcripts derives from the increased number and resting time of co-transcriptionally assembled spliceosomes on the nascent transcripts, which would increase the probability of CWC22-dependent eIF4AIII recruitment to pre-mRNAs during splicing (Obrdlik, 2019).

At the downstream regulatory level, after EJC assembly rates at transcripts are defined, deposition of EJCs along mRNA exon-exon junctions is modulated by the structural and sequence context of the splice sites. dsRNA stem structures in exon-exon junctions of Drosophila mRNAs either antagonize EJC assembly when present within canonical EJC deposition sites or enhance EJC assembly when located in the vicinity of the EJC deposition site. Absence of dsRNA within the EJC binding moiety is in agreement with reported preference of EJCs for ssRNA. It remains to be elucidated how and why EJC binding is positively affected when RNA stem structures are found in its direct proximity on the bound template (Obrdlik, 2019).

Although it is likely that the structural context of exon-exon junctions in Drosophila directly influences the degree of EJC assembly, sequence composition-derived effects on EJC binding to mRNA are a consequence of the assigned roles of these sequences during pre-mRNA splicing. This study has demonstrated that exon-exon junctions with strong 5' and strong 3' splice sites (SSs) are biased toward junctions with enhanced EJC binding. For the regulation of weak 5' and 3' SSs, which commonly occur at alternatively spliced junctions, cis-acting splicing regulatory elements (SREs) were shown to be of importance. In light of the negative impact of alternative splicing at the level of EJC mRNA binding, it is not surprising that conventional exonic splice enhancers (ESEs) and exonic splicing silencers (ESSs) hardly affect EJC binding at the level of individual exon-exon junctions. Whether the position-dependent bias mediated by the UUU-triplet- and CUG-triplet-containing hexamers toward inhibited or enhanced EJC binding that this study has discovered in the Drosophila dataset is due to a direct or indirect influence of these hexamers on splicing remains to be addressed. UUU-triplet-containing hexamers, which are strongly biased against EJC binding, could potentially function as yet undefined 5'ESS in Drosophila. Interestingly, CUG-triplet-containing hexamers, which are strongly biased toward enhanced EJC binding, share sequence similarity with a previously predicted CUG containing 5'ESE of short intron splice sites. It appears likely that the CUG-triplet and UUU-triplet hexamers exert their effect on EJC binding as a yet undefined class of SREs (Obrdlik, 2019).

In agreement with reports in mammals, the extent of EJC occupancy varies between mRNAs and exon-exon junctions also in Drosophila. The splice site score next to a junction correlates with increased EJC deposition in the fly, and this relationship between splicing efficiency and EJC deposition has also been proposed in mammalian studies. Analysis of published mammalian Btz iCLIP data revealed several modalities that correlate with the increased binding landscape of the EJC on mRNAs in both mammals and Drosophila, including the large number of introns, high transcript abundance, and sequence context of individual exon-exon junctions. Interestingly, the presence of long introns has a slightly negative effect and the amount of alternative splicing a slightly positive effect on EJC occupancy in mammals; the latter agrees with previous observations. Studies in cultured mammalian cells have reported that EJC-enriched junctions contain a relatively high proportion of 'non-canonical' protection sites, which were enriched for RBP consensus sequences of the SR protein family. Analysis of mammalian Btz iCLIP data confirms that presence of ESEs in upstream exons and 5' intronic splicing enhancers (ISE)s in introns correlates with enhanced EJC binding. Moreover, a group of junctions have been identified in mammals containing AGAA hexamers that are biased for enhanced EJC binding, but their effects are not especially strong near the canonical EJC deposition site. These hexamers match the AGAA-encompassing consensus sequence of the mammalian SR protein SRSF10, known to function as splicing enhancers, and have been found previously in EJC bound exon-exon junctions. Not only do the in silico results agree with these reports and support the proposed cooperative binding of EJC with SR proteins, they also partially explain the EJC's preference in mammals for alternatively spliced mRNAs (Obrdlik, 2019).

One observation deriving from this analysis of published mammalian Btz iCLIP datasets is surprising. Although junctions in Drosophila are observed to be enhanced or inhibited in EJC binding by specific base-pairing probability (bpp) profiles, thus by specific RNA folding categories, it was not possible to detect any striking difference between overall bpp profiles of exon-exon junctions with enhanced or inhibited EJC binding in mammals. Indeed, the only aspect of RNA structure shared by mammals and Drosophila is the negative effect of dsRNA when directly overlapping with the canonical EJC deposition site. In Drosophila, however, the presence of dsRNA close to canonical deposition sites enhances EJC binding, an effect that is not observed in mammalian cells (Obrdlik, 2019).

The findings regarding the differences in the RNA modalities enriched at highly occupied mammalian and Drosophila EJC sites provide insight into the expansion of functions of the EJC during eukaryotic evolution. Spliceosome catalyzed splicing reactions are bidirectional, and efficient formation of exon-exon junctions during RNA maturation is achieved by Prp22-induced release of spliceosomes from mRNAs. The EJC is absent in organisms with low rates of RNA splicing, such as Saccharomyces cerevisiae, but present in organisms with high splicing rates, such as Schizosaccharomyces pombe. This suggests that with the increased demand for splicing accuracy in higher eukaryotes, the EJC evolved to function as an exon-exon junction 'lock' hindering spliceosome reassembly at spliced exon-exon junctions. Because EJC binding in the fly is enhanced at strong splices sites, but is not affected by splicing enhancer elements, and is not biased toward alternatively spliced mRNAs, it is proposed that the EJC preserved its primary function as such a lock in Drosophila. Two recent studies provide evidence that also in mammals bound EJCs hinder spliceosome assembly, suppressing recursive splicing (RS) of RS exons. The previously reported importance of EJC for splicing fidelity, and the current observations on the mode of EJC binding to transcripts in the fly revealing its independence from splicing regulatory elements indeed supports that the EJC's most conserved function is to ensure splicing irreversibility (Obrdlik, 2019).

The EJC further evolved to become a central component of the NMD pathway in mammals, in which more than 95% of all genes are alternatively spliced. This may explain why EJCs in mammals are enriched on alternatively spliced transcripts. In Drosophila, in which only 30% of all genes appear to be alternatively spliced, the EJC is not a component of the main NMD pathway. It is proposed that although the EJC-NMD pathway evolved before segregation of the proto- and deuterostome clades, it gained importance by complementing the faux 3'UTR-NMD pathway during the evolution of vertebrates, for which RNA surveillance and spatiotemporal control of gene expression are essential (Obrdlik, 2019).

Similarly, recruitment of the EJC and interacting proteins upon splicing to facilitate mRNA localization so far seems exclusive to Drosophila. Two Drosophila-specific features that modulate EJC binding, namely, the presence of a large intron within a gene and secondary structure near the junction, are also predictive of mRNA localization. Although the precise strength of association between these features and mRNA localization remains to be verified with larger and more quantitative datasets, previous studies with the SOLE in oskar RNA have shown that RNA structure and EJC binding are indeed crucial for oskar mRNA localization (Obrdlik, 2019).

Competition between kinesin-1 and myosin-V defines Drosophila posterior determination

Local accumulation of oskar (osk) mRNA in the Drosophila oocyte determines the posterior pole of the future embryo. Two major cytoskeletal components, microtubules and actin filaments, together with a microtubule motor, kinesin-1, and an actin motor, myosin-V, are essential for osk mRNA posterior localization. This study used Staufen, an RNA-binding protein that colocalizes with osk mRNA, as a proxy for osk mRNA. Posterior localization of osk/Staufen was shown to be determined by competition between kinesin-1 and myosin-V. While kinesin-1 removes osk/Staufen from the cortex along microtubules, myosin-V anchors osk/Staufen at the cortex. Myosin-V wins over kinesin-1 at the posterior pole due to low microtubule density at this site, while kinesin-1 wins at anterior and lateral positions because they have high density of cortically-anchored microtubules. As a result, posterior determinants are removed from the anterior and lateral cortex but retained at the posterior pole. Thus, posterior determination of Drosophila oocytes is defined by kinesin-myosin competition, whose outcome is primarily determined by cortical microtubule density (Lu, 2020).

It is well established that kinesin-1 is essential for localization of osk/Staufen particles at the posterior pole of the Drosophila oocyte. However, it remained unclear how the compact posterior cap is anchored and retained over time. Cortical F-actin remodeling and Myosin-V, as well as the Arp1 subunit of the dynactin complex, have been all implicated in the osk/Staufen cortical localization. This study combined genetic and optogenetic tools to demonstrate that direct competition between two motors, kinesin-1 and myosin-V, ensures the posterior anchorage of osk/Staufen. Notably, it was demonstrated that the outcome of the competition is primarily determined by the density of cortical microtubules. High microtubule density at the anterior and lateral cortex favors kinesin-driven osk/Staufen cortical exclusion, while low microtubule density at the posterior pole favors myosin-driven cortical retention. Therefore, the kinesin-myosin competition and cortical microtubule density together determine the initial accumulation of osk mRNA at the posterior pole (Lu, 2020).

The cortical exclusion model was first proposed after uniform cortical localization of osk mRNA was observed in the kinesin-null oocytes. In agreement with this model, this study shows that constitutively active kinesin-1 causes osk/Staufen mislocalization in the cytoplasm of the oocyte, whereas reducing microtubule density at the lateral cortex leads to ectopic accumulation of Staufen at the cortex. These data support the model that kinesin-driven cortical exclusion along cortically-attached microtubules plays an essential role in restricting osk/Staufen to the posterior pole (Lu, 2020).

Previously, several groups have proposed that kinesin-1 transports osk/Staufen particles along slightly biased cortical microtubules, resulting in net movement of osk/Staufen from the anterior side to the posterior pole in stage 8-9 oocytes. In fact, kinesin-driven cortical exclusion and kinesin-driven transport towards the posterior pole are not mutually exclusive; they describe the same event of osk/Staufen movement. Within the oocyte, cortical microtubules are anchored to the cortex by their minus-ends while their plus-ends face towards the cytoplasm. Due to the anterior-posterior gradient of cortical microtubule density, more microtubule plus ends are oriented towards the posterior pole. Thus, kinesin-1-driven transport along microtubules is a prerequisite for kinesin-1-driven cortical exclusion. Cortical exclusion of osk/Staufen by kinesin-1 results in biased transport of osk/Staufen towards the posterior pole (Lu, 2020).

This kinesin-myosin competition model is suggested by genetic interaction data from a previous study. Specifically, increasing KHC dosage enhances osk/Staufen mislocalization phenotypes in myosin-V loss-of-function mutants, while reducing KHC dosage by half partially suppresses myosin loss-of-function phenotypes. Furthermore, double MyoV and Khc mutant clones have diffuse cytoplasmic localization of osk mRNA, compared to uniform cortical localization of osk mRNA in Khc single mutant clones. These data strongly imply that in the absence of kinesin-1, myosin-V promiscuously anchors osk/Staufen everywhere in the cortex. This study manipulated the activity of either kinesin-1 or myosin-V and found that proper balance between the activities of these two motors is critical for correct osk/Staufen localization, supporting the model in which kinesin-myosin competition is key to the correct posterior determination in the Drosophila oocyte (Lu, 2020).

The competition between kinesin-1 and myosin-V described in this study is not the first example of such a mechanism for cargo transport and localization. For instance, myosin-V opposes microtubule-dependent transport and provides a dynamic anchor for melanosomes, peroxisomes , recycling endosomes, mitochondria, and synaptic vesicles at sites of local accumulation of F-actin. At these sites, the abundance of F-actin tracks provides an upper hand for myosin-V to win the tug-of-wars over microtubule motors. Kinesin-myosin competition appears to be an evolutionarily conserved mechanism to allow flexible refinement and/or error correction, as motors constantly undergo reversible binding and releasing activity on cytoskeletal filaments (Lu, 2020).

In the oocytes, the machinery responsible for osk/Staufen localization contains the same basic building blocks; however, unlike the other systems, the outcome of the competition is not determined by actin filament density, as F-actin density is uniform along the oocyte cortex. Instead, the outcome of this competition is decided by abundance of microtubule tracks. Higher microtubule density at the anterior and lateral cortex favors kinesin-mediated cortical removal of osk/Staufen, while scarcity of microtubule tracks at the posterior pole favors myosin-V-dependent anchoring. To confirm this model, optogenetic tools were used to recruit a microtubule-depolymerizing kinesin, kinesin-13/Klp10A, to actin cortex, and thus locally modulate cortical microtubule density. Locally decreasing cortical microtubule density causes ectopic accumulation of Staufen at the cortex. The loss of microtubules prevents kinesin-driven cortical exclusion, which allows myosin-V to win the competition and form a patch of cortically localized Staufen. This recruitment of Staufen is reversible and repeatable, indicating this kinesin-myosin competition is continuous, and the outcome of this never-ending battle is decided by the local microtubule concentration (Lu, 2020).

Competition between kinesin-1 and myosin-V is sufficient for initial anchoring at the posterior pole Previously, synthetic motor domains of a plus-end motor, kinesin-1, and a minus-end motor, kinesin-14/Nod, were used to label overall microtubule polarity in Drosophila oocytes and neurons (Kin:βGal and Nod:βGal). As myosin-V is essential for osk/Staufen localization, in this study two synthetic motor constructs (KHC576 and MyoVHMM) were expressed in the oocyte, and their dimerization was induced using a rapalog-dependent dimerization system. Dimerized motors accumulate at the posterior pole, highly resembling the osk/Staufen localization. This posterior accumulation is dependent on the anterior-posterior microtubule gradient; dimerized motors fail to localize at the posterior pole after microtubule depolymerization. Together, using dimerized synthetic motors, this study demonstrated that direct competition (without any cargo binding) between a microtubule motor, kinesin-1, and an actin motor, myosin-V, is sufficient for initial posterior localization in a Drosophila oocyte (Lu, 2020).

In summary, this study has elucidated the anchorage mechanism for initial posterior localization of osk/Staufen during mid-oogenesis. Kinesin-1 competes with myosin-V to control osk/Staufen localization. The outcome of this kinesin-myosin competition is primarily determined by cortical microtubule density. Higher microtubule density at anterior-lateral cortex allows kinesin-1 to win and cortically exclude osk/Staufen, while lower microtubule density at posterior pole favors myosin-V-mediated anchorage at the cortex. Together, two cytoskeletal components (microtubules and F-actin) and two molecular motors (kinesin-1 and myosin-V) govern the posterior determination for future Drosophila embryos (Lu, 2020).

Makorin 1 controls embryonic patterning by alleviating Bruno1-mediated repression of oskar translation

Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila, maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. This study shows that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation of oskar (osk). Mkrn1 interacts with poly(A) binding protein (pAbp) and binds specifically to osk 3' UTR in a region adjacent to A-rich sequences. Using Drosophila S2R+ cultured cells this study shows that this binding site overlaps with a Bruno1 (Bru1) responsive element (BREs) that regulates osk translation. Increased association of the translational repressor Bru1 with osk mRNA was observed upon depletion of Mkrn1, indicating that both proteins compete for osk binding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. It is concluded that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation, most likely by competing with Bru1 to bind to osk 3' UTR (Dold, 2020).

The data indicates that Mkrn1 is essential for oogenesis, embryonic patterning, and germ cell specification. An essential role for Mkrn1 in oogenesis has also been recently reported (Jeong, 2019). By taking advantage of a new allele that specifically disrupts Mkrn1 binding to RNA, this study demonstrates that Mkrn1 exerts its function in embryogenesis and germ cell specification, primarily via regulating osk translation by antagonizing Bru1 binding (Dold, 2020).

Control of osk translation has been studied in depth, revealing a complex spatio-temporal interplay between repressing and activating factors. Relief of translational repression and activation of osk translation is likely to involve multiple redundant mechanisms. For example, Bru1 can be phosphorylated on several residues, and phosphomimetic mutations in these residues inhibit Cup binding in pulldown assays. However, these do not seem to affect translational repression activity in vivo. Stau, Aub, Orb and pAbp have also been implicated in activating osk translation. However, it is unlikely that Mkrn1 controls osk translation by recruiting Stau, as Stau still colocalizes with osk mRNA in Mkrn1W oocytes. Instead, it is proposed that Mkrn1 exerts its positive activity by competing with Bru1 binding to osk 3' UTR (see Makorin 1 controls embryonic patterning by alleviating Bruno1-mediated repression of oskar translation). This is evidenced by the overlap of their binding sites, the increased association of Bru1 to osk mRNA upon Mkrn1 knockdown and by the observation that reducing bru1 dosage is sufficient to partially alleviate osk translational repression (Dold, 2020).

Two distinct Bru1 binding regions (AB and C) are present in the osk 3' UTR and are required for translational repression. However, the C region has an additional function in translational activation. Indeed, it was hypothesized that an activator binds the C region to relieve translational repression. This activator was proposed to either be Bru1 itself, or a different protein that can bind the BRE-C, which is what this study observed for Mkrn1. The results suggest that the interaction of pAbp with the nearby AR region, and the consequent stabilization of Mkrn1 binding, contributes to the role of BRE-C in osk translational activation. Other factors may also be involved. For instance, Bicoid Stability Factor (BSF) binds the C region in vitro at the 3' type II Bru1-binding site, at a similar site to where Mkrn1 binds osk. Deletion of this site impacts embryonic patterning, yet depletion of BSF has no effect on Osk protein expression up to stage 10, indicating that initial activation of osk translation is effective even in the absence of BSF. In this case, only late stage oocytes display reduced Osk accumulation. Therefore, it is possible that a concerted action of Mkrn1 and BSF exists at the osk 3' UTR site to activate translation and sustain it at later stages (Dold, 2020).

The binding of Mkrn1 to mRNA seems to be extremely specific. The binding to osk is dependent on a downstream A-rich sequence and on interaction with pAbp. A few other targets identified in this study also display enrichment for downstream AA nucleotides. and human MKRN1 has recently been shown to associate preferentially to such sequences. Relevant to this, Bru1 binds to grk 3' UTR in addition to osk, and several proteins that associate with Mkrn1 also associate with grk mRNA. However, this study found no evidence that Mkrn1 binds specifically to grk, which lacks poly(A) stretches in the proximity of its Bru1 binding sites, and consistently, no regulatory role of Mkrn1 on Grk translation was observed (Dold, 2020).

In addition to pAbp, it is noteworthy that Mkrn1 associates with other proteins previously implicated in osk localization and translational activation. Its interaction with eIF4G would be consistent with a role in alleviating Cup-mediated repression, as it could recruit eIF4G to the cap-binding complex at the expense of Cup. However, interaction between Mkrn1 and eIF4E was not observed. The association between Mkrn1 and Imp is also intriguing as the osk 3' UTR contains 13 copies of a five-nucleotide motif that interacts with Imp. This region is essential for osk translation but Osk accumulation is unaffected in Imp mutants, suggesting the involvement of another factor that binds these motifs. In contrast to pAbp, no alteration was observed of Mkrn1 binding when Imp was depleted, indicating that Imp is not required to stabilize Mkrn1 on osk mRNA (Dold, 2020).

The molecular links uncovered in this study between Mkrn1 and RNA-dependent processes in Drosophila are consistent with recent high-throughput analysis of mammalian MKRN1 interacting proteins. RNA binding proteins, including PABPC1, PABPC4, and eIF4G1, were highly enriched among the interactors. Moreover, human MKRN1 was also recently shown to bind to RNA, dependent on the PAM2 motif and the interaction with PABPC1. In addition, the short isoform of rat MKRN1 was shown to activate translation but the underlying mechanism remained unknown. Since in vertebrates MKRN genes are highly expressed in gonads and early embryos as well, it is possible that similar molecular mechanisms are employed to regulate gene expression at these stages. Consistent with this, MKRN2 was recently found to be essential for male fertility in mice. Thus, this study provides a mechanism that explains the role of Mkrn1 in translation and constitutes a solid framework for future investigations deciphering the roles of vertebrate MKRNs in post-transcriptional control of gene expression during gametogenesis and early development (Dold, 2020).


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


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.


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: 2 December 2018

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