BIOLOGICAL OVERVIEW

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 stau^C8. 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).

Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire

KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. This study demonstrates that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. These results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification (Huang, 2014).

This study reveals a previously unknown transcriptional role for Enok in regulating the polarized localization of Osk during oogenesis through promoting the expression of spir and mael. Spir and Mael are required for the properly polarized MT network in oocytes from stages 8 to 10A. However, protein levels of both decreased at later stages of oogenesis, allowing reorganization of the MT network and fast ooplasmic streaming. The persistent presence of Spir extending into stage 11 led to loss of ooplasmic streaming and resulted in female infertility. These findings suggest that the temporal regulation of spir expression is crucial for oogenesis, and, interestingly, Enok protein levels were also reduced in egg chambers during stages 10-13 compared with stages 1-9. While the stability of Spir or the translation of spir mRNA may also be a target for regulation, the results suggest that Enok is involved in the dynamic modulation of spir transcript. Furthermore, the results demonstrate the importance of Enok for expression of spir and mael in both ovaries and S2 cells, suggesting that Enok may play a similar role in other Spir- or Mael-dependent processes such as heart development (Huang, 2014).

Notably, Mael is also important for the piRNA-mediated silencing of transposons in germline cells. Mutations in genes involved in the piRNA pathway, including aub and armitage (armi), result in axis specification defects in oocytes as well as persistent DNA damage and checkpoint activation in germline cells. The activation of DNA damage signaling is suggested to cause axis specification defects in oocytes, as the disruption of Osk localization in piRNA pathway mutants can be suppressed by mutations in mei-41 or mnk, which encode ATR or checkpoint kinase 2, respectively. However, mutation in mnk cannot suppress the loss of posteriorly localized Osk in the mael mutant oocyte, indicating that the oocyte polarization defect in the mael mutant is independent of DNA damage signaling. Therefore, although the possibility that the piRNA pathway is affected in enok mutants due to down-regulation of mael cannot be excluded, the Osk localization defect in the enok mutant oocyte is likely independent of mei-41 and mnk (Huang, 2014).

In addition to the osk mRNA localization defect, both spir and mael mutants affect dorsal-ventral (D/V) axis formation in oocytes. However, no defects in the D/V patterning were observed in the eggshells of enok mutant germline clone embryos. Interestingly, among the spir mutant alleles that disrupt formation of germ plasm, only strong alleles result in dorsalized eggshells and embryos, while females with weak alleles produce eggs with normal D/V patterning. Since the enok¹ and enok² ovaries still express ~25% of the wild-type levels of spir mRNA, enok mutants may behave like weak spir mutants. Similarly, the ~40% reduction in mael mRNA levels in enok mutants as compared with the wild-type control may not have significant effects on the D/V axis specification (Huang, 2014).

Redundancy in HAT functions has been reported for both Moz and Sas3, the mammalian and yeast homologs of Enok, respectively. In yeast, deletion of either GCN5 (encoding the catalytic subunit of ADA and SAGA HAT complexes) or SAS3 is viable. However, simultaneously deleting GCN5 and SAS3 is lethal due to loss of the HAT activity of the two proteins, suggesting that Gcn5 and Sas3 can compensate for each other in acetylating histone residues. Indeed, while deleting SAS3 alone had no effect on the global levels of H3K9Ac and H3K14Ac, disrupting the HAT activity of Sas3 in the gcn5Δ background greatly reduced the bulk levels of H3K9Ac and H3K14Ac in yeast. Also, mammalian Moz targets H3K9 in vivo and regulates the expression of Hox genes, but the global H3K9Ac levels are not significantly affected in the homozygous Moz mutant, indicating that other HATs have overlapping substrate specificity with Moz. In flies, a previous study had reported that the H3K23Ac levels were reduced 35% in nejire (nej) mutant embryos, which lack functional CBP/p300. However, knocking down nej by dsRNA in S2 cells severely reduced levels of H3K27Ac but had no obvious effect on global levels of H3K23Ac. This study showed that the global H3K23Ac levels decreased 85% upon enok dsRNA treatment in S2 cells. This study also showed that the H3K23Ac levels are highly dependent on Enok in early and late embryos, larvae, adult follicle cells and nurse cells, and mature oocytes. Therefore, although Nej may also contribute to the acetylation of H3K23, the results indicate that, in contrast to its mammalian and yeast homologs, Enok uniquely functions as the major HAT for establishing the H3K23Ac mark in vivo (Huang, 2014).

The H3K23 residue has been shown to stabilize the interaction between H3K27me3 and the chromodomain of Polycomb. Therefore, acetylation of H3K23 may affect the recognition of H3K27me3 by the Polycomb complex. Another study showed that the plant homeodomain (PHD)-bromodomain of TRIM24, a coactivator for estrogen receptor α in humans, binds to unmodified H3K4 and acetylated H3K23 within the same H3 tail. Also, the levels of H3K23Ac at two ecdysone-inducible genes, Eip74EF and Eip75B, have been shown to correlate with the transcriptional activity of these two genes at the pupal stage, suggesting the involvement of H3K23Ac in ecdysone-induced transcriptional activation. This study further provided evidence for the activating role of the Enok-mediated H3K23Ac mark in transcriptional regulation (Huang, 2014).

In mammals, MOZ functions as a key regulator of hematopoiesis. Interestingly, one of the genes encoding mammalian homologs of Spir, spir-1, is expressed in the fetal liver and adult spleen, indicating the expression of spir-1 in hematopoietic cells. Thus, it will be intriguing to investigate whether the Drosophila Enok-Spir pathway is conserved in mammals and whether Spir-1 functions in hematopoiesis. Taken together, the results demonstrate that Enok functions as an H3K23 acetyltransferase and regulates Osk localization, linking polarization of the oocyte to histone modification (Huang, 2014).

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 LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage

DEAD-box RNA helicases play important roles in a wide range of metabolic processes. Regulatory proteins can stimulate or block the activity of DEAD-box helicases. This study shows that LOTUS (Limkain, Oskar, and Tudor containing proteins 5 and 7) domains present in the germline proteins Oskar, TDRD5 (Tudor domain-containing 5; Tejas), and TDRD7 (Tapas) bind and stimulate the germline-specific DEAD-box RNA helicase Vasa. Crystal structure of the LOTUS domain of Oskar in complex with the C-terminal RecA-like domain of Vasa reveals that the LOTUS domain occupies a surface on a DEAD-box helicase not implicated previously in the regulation of the enzyme's activity. It was shown that, in vivo, the localization of Drosophila Vasa to the nuage and germ plasm depends on its interaction with LOTUS domain proteins. The binding and stimulation of Vasa DEAD-box helicases by LOTUS domains are widely conserved (Jeske, 2017).

This study provides molecular insight into the function of animal LOTUS domain proteins, factors involved in diverse germline functions. The DEAD-box helicase Vasa interacts with the LOTUS domains of Oskar, TDRD5/Tejas, and TDRD7/Tapas but not with MARF1. In Drosophila, interaction with LOTUS domain proteins is required for Vasa localization to the nuage and germ plasm. Structural and functional analyses of the LOTUS-Vasa interaction uncovered a key role of a C-terminal extension present in only a subset of LOTUS domains, pointing to two LOTUS domain subclasses with distinct functions in animals. The eLOTUS domain of Oskar, TDRD5, and TDRD7 not only interacts with Vasa but also stimulates its helicase activity. The mLOTUS domains present in MARF1 lack this extension and very likely have a distinct role within the germline that will need to be addressed in the future. While Drosophila TDRD5 (Tejas) and TDRD7 (Tapas) contain a single eLOTUS domain, some TDRD5 and TDRD7 proteins from other animals harbor mLOTUS domains in addition to their N-terminal eLOTUS domain. Whether the mLOTUS domains from MARF1, TDRD5, and TDRD7 have related activities or are functionally distinct remains to be determined (Jeske, 2017).

The Drosophila eLOTUS domain proteins Oskar, Tejas, and Tapas have been considered to be scaffolding proteins whose function is to recruit Vasa and other germline factors to germ plasm or the nuage. While LOTUS domains were originally predicted to be RNA-binding domains, attempts to detect any RNA-binding activity of the eLOTUS domain of Oskar have failed. The present study uncovered a conserved function of eLOTUS domains in binding and stimulating a DEAD-box RNA helicase, thus attributing an active regulatory role to Oskar, Tejas, and Tapas in the germline. The stimulation of the ATPase activity of Vasa by the eLOTUS domain seems universal, but its consequence and function within the germline are unknown. In Drosophila, Vasa stimulation by Tejas and/or Tapas in the nuage might be involved in the piRNA pathway, whereas Vasa stimulation by Oskar in the pole plasm likely has a distinct role. Vasa was suggested to activate translation of mRNAs in the egg chamber through recruitment of eIF5B, which catalyzes ribosomal subunit joining to form elongation-competent ribosomes. Vasa has been shown to physically interact with eIF5B in yeast two-hybrid assays and pull-down experiments from lysates. A Vasa region that extends C-terminally from the helicase core was shown to be required for the eIF5B interaction, which raised the question of whether eLOTUS and eIF5B jointly or mutually exclusively bind to Vasa. Attempts were made to test this in GST pull-down assays with recombinant proteins. However, surprisingly, no interaction of Vasa with GST-eIF5B or any change in Vasa's ATPase activity in the presence of eIF5B was detected. It is concluded that Vasa and eIF5B do not physically interact and that the recruitment of eIF5B by Vasa might be mediated through RNA or other proteins. It is equally plausible that Vasa's role in translation might be that of a DEAD-box RNA helicase involved in remodeling RNA-protein complexes. Given its importance in germline biology, the mechanism by which Vasa promotes translation of mRNAs merits thorough re-examination (Jeske, 2017).

In the nuage, Vasa is essential for the secondary piRNA biogenesis pathway, also known as the Ping-Pong cycle. Bombyx Vasa associates with the Piwi proteins Siwi and Ago3, two major players in the Ping-Pong cycle in the germ plasm. Within the Ping-Pong cycle, Siwi is loaded with piRNAs, and the complex binds and cleaves transposon mRNAs in an orientation antisense to piRNAs. The cleavage products are then loaded into Ago3, and the complex recognizes and cleaves piRNA cluster transcripts, leading to specific amplification of piRNAs that target transposon mRNAs present in the cell. Vasa is required for the safe handover of transposon mRNA fragments from Siwi to Ago3. Furthermore, the ATPase activity of Vasa is necessary for the release of transposon RNAs from Siwi-piRNA complexes after cleavage. It is therefore possible that stimulation of Vasa by the Tejas and/or Tapas eLOTUS domains is required for high efficiency of the Ping-Pong cycle. The higher activity of Tejas compared with Tapas that was detected might be reflected in vivo by its dominant role in transposon silencing within the nuage (Jeske, 2017).

LOTUS domains are not restricted to animals but are also present in bacteria, fungi, and plants-organisms without a Vasa ortholog. From sequence alignments, it appears that bacterial, fungal, and plant LOTUS domains lack the particular C-terminal extension, and it will be interesting to investigate and compare their function with that of mLOTUS domains of animal proteins, such as MARF1 (Jeske, 2017).

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).

Localization of oskar mRNA by agglomeration in ribonucleoprotein granules

Localization of oskar mRNA to the posterior of the Drosophila oocyte is essential for abdominal patterning and germline development. oskar localization is a multi-step process involving temporally and mechanistically distinct transport modes. Numerous cis-acting elements and trans-acting factors have been identified that mediate earlier motor-dependent transport steps leading to an initial accumulation of oskar at the posterior. Little is known, however, about the requirements for the later localization phase, which depends on cytoplasmic flows and results in the accumulation of large oskar ribonucleoprotein granules, called founder granules, by the end of oogenesis. Using super-resolution microscopy, this study showed that founder granules are agglomerates of smaller oskar transport particles. In contrast to the earlier kinesin-dependent oskar transport, late-phase localization depends on the sequence as well as on the structure of the spliced oskar localization element (SOLE), but not on the adjacent exon junction complex deposition. Late-phase localization also requires the oskar 3' untranslated region (3' UTR), which targets oskar to founder granules. Together, these results show that 3' UTR-mediated targeting together with SOLE-dependent agglomeration leads to accumulation of oskar in large founder granules at the posterior of the oocyte during late stages of oogenesis. In light of previous work showing that oskar transport particles are solid-like condensates, these findings indicate that founder granules form by a process distinct from that of well-characterized ribonucleoprotein granules like germ granules, P bodies, and stress granules. Additionally, they illustrate how an individual mRNA can be adapted to exploit different localization mechanisms depending on the cellular context (Eichler, 2023).

The finding that founder granules appear to be agglomerates of osk RNPs provides new insight into the process by which osk accumulates at the posterior of the oocyte during late stages of oogenesis. osk is transported in RNPs containing 2–4 transcripts. Recent work has shown that these RNPs are initially liquid-like condensates but they rapidly mature to a non-dynamic, solid state that prevents incorporation of additional mRNA molecules. Inducing a more liquid-like state results in formation of large, dynamic condensates at the posterior of late-stage oocytes that subsequently detach, indicating that the solid state is necessary for proper founder granule assembly and anchoring. Th4 observation that founder granules contain multiple physically distinct osk RNPs packed together is consistent with the solid-like properties of these RNPs and indicates that they do not form through the collapse of transport RNPs into larger condensates but rather through an aggregative process. This mechanism contrasts with Drosophila germ granules, whereby pre-formed protein condensates are populated by RNPs containing single transcripts, which then self-assemble within the granules to form homotypic clusters. What limits the agglomeration of osk RNPs into founder granules to the posterior of the oocyte remains unclear. RNP-RNP associations may be fostered by the high posterior concentration of osk RNPs achieved previously by kinesin-dependent transport. Since proteins can partition into osk RNPs after their transition to a solid-like state, proteins recruited to the germ plasm, perhaps by Osk protein itself, could mediate this behavior. Intriguingly, zebrafish germ plasm mRNAs form homotypic RNPs that aggregate into compact structures while retaining their distinct spherical appearance. This similarity with founder granules suggests that agglomeration may be a more generalized mechanism for mRNA compartmentalization (Eichler, 2023).

The function of the earlier acting EJC/SOLE complex in late-phase osk localization was interrogated, and the SOLE, but not the adjacent EJC, was found to be required. Whereas the function of the SOLE in the earlier localization phase relies only on the structure of the proximal stem, both the sequence of the proximal stem and its structure are important for late-phase localization. How the SOLE collaborates with the EJC to promote kinesin-dependent osk motility and what, if any regulatory factor interacts with it are not yet known. The sequence-dependence of the SOLE and lack of requirement for the EJC in late-phase localization suggests a different mode of action, possibly through the binding of a different protein to the proximal stem and recruitment of new RNP components or through RNA-RNA interactions. The change in ovarian physiology with the onset of nurse cell dumping could lead to an exchange of proteins associated with osk, to inhibit kinesin-dependent motility and promote posterior agglomeration (Eichler, 2023).

The failure of osk-K10_3'UTR mRNA to localize at late stages of oogenesis despite the presence of the SOLE indicates that similarly to the earlier phase, late-phase localization depends on both the SOLE and the 3' UTR. This dependence on the 3' UTR for the late accumulation of osk is not for the purpose of hitchhiking, and by swapping the osk and nos 3' UTRs it was shown that the osk 3' UTR specifies association of osk RNPs in founder granules independently of the SOLE. This function of the osk 3' UTR may be conferred by the same 3' UTR-binding proteins that control the formation and/or initial localization of osk transport RNPs and remain associated with osk at the posterior pole, such as Bru1, Stau, or Hrp48. For example, a prion-like domain in Bru1 required for formation of osk transport RNPs could also mediate self-association of these RNPs when they come in contact at the posterior pole. Likewise, mammalian Stau has the propensity to form cytoplasmic aggregates. The requirements for Bru1 and Stau in the earlier phase of osk localization make it difficult to test this idea, however. Additionally, the osk 3' UTR may function to prevent co-condensation of osk RNPs with germ granules through the recruitment of proteins like Hrp48, which maintains the solid-like properties of osk RNPs (Eichler, 2023).

Results from swapping the osk and nos 3' UTRs also suggest that either osk 5' UTR and/or coding sequences other than the SOLE contribute to maintaining osk RNPs in founder granules. Since multivalent interactions are typically required for inclusion of components in phase separated condensates, it is not surprising that binding of founder granule components to multiple sites within osk would be required for the integrity of these granules. Further dissection of the sequence requirements and identification of interacting factors will be necessary to define the mechanisms by which the various osk elements accomplish the different tasks (Eichler, 2023).

The process by which osk mRNA achieves its posterior localization is remarkably complex and labor intensive, involving distinct machineries for transport into the oocyte, movement to the posterior pole during stages 8 to 10, and further accumulation during late stages of oogenesis. Given the dependence of embryonic abdominal patterning and germ cell formation on the amount of osk mRNA localized during oogenesis, the reliance on numerous distinct contributions to osk localization likely provides robustness to processes governing the targeting of osk RNPs to the right location and the accumulation of sufficient osk there. Moreover, the distinct process of assembling founder granules ensures that osk mRNA remains separated from germ granules to promote its degradation in the embryonic germ plasm and minimize its inheritance by pole cells (Eichler, 2023).

GENE STRUCTURE

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

Bases in 5' UTR - 14

Exons - four

Bases in 3' UTR - 1045

PROTEIN STRUCTURE

Two isoforms of Oskar protein are produced by alternative start codon usage. The short isoform, which is translated from the second in-frame AUG of the mRNA, has full Oskar activity. When Oskar RNA is localized, accumulation of Oskar protein requires the functions of Vasa and Tudor, as well as Oskar itself, suggesting a positive feedback mechanism in the induction of pole plasm by Oskar (Markussen, F. H. (1995).

Amino Acids - 606

Structural Domains

There are no known oskar homologs.

EVOLUTIONARY HOMOLOGS

Finding which elements of a protein are conserved in evolution is one of the first approaches to determining important protein functional domains. There are two broad domains of Oskar proteins conserved between D. melanogaster and D. virilus: one 82 amino acid stretch in the central portion of the molecule and the carboxy-terminal 220 amino acids. The two proteins share over 70% identity.

The Drosophila virilis oskar homolog, virosk, was examined as a transgene in Drosophila melanogaster flies. Cis-acting signals for the localization of Oskar mRNA are conserved, although the Virosk transcript also transiently accumulates at novel intermediate sites. The Virosk protein, however, shows substantial differences from Oskar: while virosk is able to rescue body patterning in a D. melanogaster oskar mutant, it is impaired in both mRNA maintenance and pole cell formation. Furthermore, virosk induces a dominant maternal-effect lethality when introduced into a wild-type background, and interferes with the posterior maintenance of the endogenous Oskar transcript in early embryogenesis. Virosk protein is unable to anchor at the posterior pole of the early embryo; this defect could account for all of the characteristics of virosk mentioned above (Webster, 1994).

The colocalization of morphogenetic signals involved in germ cell formation and in the specification of the body axis is not unique to Drosophila but is also found in Caenorhabditis elegans and Xenopus (Lehmann, 1994).

The establishment of the germline is a critical, yet surprisingly evolutionarily labile, event in the development of sexually reproducing animals. In the fly Drosophila, germ cells acquire their fate early during development through the inheritance of the germ plasm, a specialized maternal cytoplasm localized at the posterior pole of the oocyte. The gene oskar (osk) is both necessary and sufficient for assembling this substance. Both maternal germ plasm and oskar are evolutionary novelties within the insects, as the germline is specified by zygotic induction in basally branching insects, and osk has until now only been detected in dipterans. In order to understand the origin of these evolutionary novelties, comparative genomics, parental RNAi, and gene expression analyses was used in multiple insect species. It was found that the origin of osk and its role in specifying the germline coincided with the innovation of maternal germ plasm and pole cells at the base of the holometabolous insects and that losses of osk are correlated with changes in germline determination strategies within the Holometabola. These results indicate that the invention of the novel gene osk was a key innovation that allowed the transition from the ancestral late zygotic mode of germline induction to a maternally controlled establishment of the germline found in many holometabolous insect species. It is proposed that the ancestral role of osk was to connect an upstream network ancestrally involved in mRNA localization and translational control to a downstream regulatory network ancestrally involved in executing the germ cell program (Lynch, 2011).

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

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

Tropomyosin 1-I/C coordinates kinesin-1 and dynein motors during oskar mRNA transport

Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo bidirectional transport by binding to both motors simultaneously. However, it is not known how motor activities are coordinated in such circumstances. In the Drosophila female germline, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar messenger RNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. This study shows through in vitro reconstitution that Tm1-I/C, a tropomyosin-1 isoform, links kinesin-1 in a strongly inhibited state to DDBE-associated oskar mRNA. Nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and structural modeling indicate that Tm1-I/C suppresses kinesin-1 activity by stabilizing its autoinhibited conformation, thus preventing competition with dynein until kinesin-1 is activated in the oocyte. Thus work reveals a new strategy for ensuring sequential activity of microtubule motors (Heber, 2024).

oskar (osk) mRNA localization in the Drosophila egg chamber is an attractive system for studying dual motor transport. Delivery of osk to the posterior pole of the developing oocyte, which drives abdominal patterning and germline formation in the embryo, is driven by the successive activities of dynein and kinesin-1. In early oogenesis, osk mRNA that is synthesized in the nurse cells is transported into the interconnected oocyte by dynein in complex with dynactin and the activating adaptor Bicaudal D (BicD), which is linked to double-stranded mRNA localization signals by the RNA-binding protein Egalitarian (Egl). Association of Egl with BicD and consequent dynein activation are enhanced by binding of Egl to RNA, indicating a role for the cargo in promoting dynein activity. In early oogenesis, microtubule minus ends are nucleated in the oocyte, consistent with the dynein-based delivery of mRNAs into this cell. During mid-oogenesis, the polarity of the microtubule network shifts dramatically, with plus ends pointing towards the oocyte posterior. At this stage, Khc translocates osk to the posterior pole. This process is independent of Klc, raising the question of how Khc is linked to osk and how its motor activity is regulated (Heber, 2024).

Transport of osk RNA by Khc requires the unique I/C isoform of tropomyosin-1, Tm1-I/C (hereafter Tm1). Tm1 binds to a noncanonical but conserved cargo-binding region in the Khc tail and stabilizes interaction of the motor with RNA, suggesting a function as an adaptor. Both Khc and Tm1 are loaded onto osk ribonucleoprotein particles (RNPs) shortly after their export from the nurse cell nuclei, although the motor only appears to become active in the mid-oogenesis oocyte. Similarly, dynein remains associated with osk RNPs during Khc-mediated transport within the oocyte, but is inactivated by displacement of Egl by Staufen. How the two motors are linked simultaneously to osk RNPs, and how Khc is inhibited during dynein-mediated transport into the oocyte, is not known (Heber, 2024).

This study shows that Tm1 inhibits Khc by stabilizing its autoinhibited conformation through a new mechanism involving the motor’s regulatory tail domain and stalk. Tm1 also links Khc to the dynein-transported osk RNP, thereby allowing cotransport of inactive Khc on osk RNA by dynein. In vivo, such a mechanism would avoid competition between the two motors during delivery of osk RNPs to the oocyte by dynein, while ensuring that Khc is available on these structures to mediate their delivery to the oocyte posterior in mid-oogenesis. With its cargo-binding and motor regulatory functions, it is proposed that Tm1 is a noncanonical light chain for kinesin-1 (Heber, 2024).

Tm1 was recently implicated as an RNA adaptor for kinesin-1. The current study reveals a previously unknown role of Tm1 in osk transport. Tm1 is shown to negatively regulates Khc activity, which is proposed to occurs a conformational change in the Khc stalk that stabilizes the Khc motor-tail interaction and thereby enhances autoinhibition (Heber, 2024).

With its functions in cargo binding and motor regulation, it is speculated that Tm1 is an alternative Klc in the Drosophila female germline. Because precise osk RNA localization during oogenesis is critical for development, positive regulators of Khc function such as PAT1 and negative regulators such as Tm1 may have replaced Klc to provide nuanced control of Khc activity. If this hypothesis is correct, Klc should also be dispensable for Tm1-dependent RNA localization in somatic tissues (Heber, 2024).

Tm1 also stimulates association of Khc in a strongly inhibited state with dynein-associated osk RNPs. This mechanism would allow dynein-mediated transport of osk RNPs from the nurse cells to the oocyte to proceed without competition with Khc, while positioning the plus-end directed motor on the RNPs for their posteriorward transport within the ooplasm in mid-oogenesis (Heber, 2024).

Kinesin-1 autoinhibition is not fully understood, in part because of a paucity of structural information for the full-length molecule. It has been proposed that interaction of the tail’s IAK motif with the motor domain plays a key role in kinesin-1 autoinhibition. Consistent with this notion, strongly enhanced motility was obserced of Drosophila Khc when the IAK motif was deleted. However, it was recently shown that Klc inhibits Khc activity independently of the IAK-motor interaction. It was also found that the IAK motif is not needed for inhibition of Khc by Tm1. Instead, it was found that Tm1-mediated inhibition occurs via the Khc AMB domain, a region adjacent to the IAK motif that is essential for osk localization (Heber, 2024).

Structural analyses suggest that Tm1 stabilizes the autoinhibited folded conformation of Khc by inducing rearrangement of the Khc coiled-coil stalk. Therefore, a model is proposed in which both the motor-IAK interaction and interactions within the Tm1-bound stalk that include the AMB domain act synergistically to achieve the stable inhibited conformation of Khc (Heber, 2024).

Two recent studies have proposed compact structural arrangements for mammalian kinesin-1. Those studies used chemical crosslinking and cryo-EM, which are likely to enrich for a homogeneous population of compact conformations of Khc and Khc–Klc tetramers, to provide detailed static structural information. By contrast, in the current study of Drosophila Khc and Khc–Tm1 complexes, NMR and SAXS were employed providing insight into the different conformational states, and thus flexibility, of kinesin-1 by enabling analysis of structures in solution. A previous study observed the compact, inhibited conformation in the isolated Khc, showing that Klc-binding does not induce a new fold of Khc but rather stabilizes the inhibited conformation. This is in agreement with the current model, in which Tm1, a putative alternative Klc, shifts the structural equilibrium of Khc towards the autoinhibited state by stabilizing its compact conformation (Heber, 2024).

Although it is known that many cargo types are transported by the concerted action of dynein and kinesins, the underlying regulatory mechanisms have been elusive. This study has identified one of very few examples of a factor that not only links dynein and kinesin-mediated transport, but also modulates transport through differential motor regulation. Linkage of dynein and kinesin-3 by the dynein-activating adaptor Hook3 has been demonstrated, but the cellular events for which this is relevant are still emerging. Recently, reconstituted coupling of dynein and kinesin-1 by TRAK1 and TRAK2 has provided insight into how the motors are recruited and regulated for mitochondrial transport. However, unlike this study's integration of DDBE and Khc in reconstituted osk RNPs, these systems lacked intact native cargoes and thereby excluded potential cargo-directed positioning and modulation of motor complexes (Heber, 2024).

Several studies have reported that active dynein and kinesin motors engage in a tug-of-war when artificially coupled. This study also observe motor opposition in reconstituted RNPs containing DDBE and Khc, presumably because of the stochastic engagement of autoinhibited Khc with microtubules. However, the observation that Tm1 supports efficient dynein-mediated RNA transport through robust inhibition of Khc highlights the importance of regulatory factors in addition to mechanical coupling in native transport complexes. Supporting the in vivo relevance of negative regulation of Khc during bidirectional transport, kinesin-1-activating IAK mutations were recently shown to impair dynein-mediated transport processes in Aspergillus nidulans Collectively, these observations point to complex interplay between opposite-polarity motors that are bound simultaneously to cargoes. Further reconstitutions of dual motor systems on native cargoes should reveal generalities of dynein-kinesin crosstalk, as well as any cargo-specific regulatory mechanisms (Heber, 2024).

This study provides mechanistic insight into two critical aspects of osk mRNA transport—assembly of the dual motor complex and how Khc activity is suppressed during dynein-mediated delivery of the transcript from the nurse cells to the oocyte. However, it is not understood how Khc takes over from dynein after osk RNPs arrive in the oocyte. Although recent work has shown that inactivation of dynein by the RNA-binding protein Stau is part of this process, how Tm1- and IAK-mediated inhibition of Khc is alleviated to allow delivery of the mRNA to the oocyte posterior is an open question. One candidate to fulfill this role is Ensconsin, which is required for posterior osk localization and is enriched in the oocyte relative to the nurse cells. Strikingly, the human counterpart of Ensconsin (MAP7) was recently shown to stimulate activity of mammalian kinesin-1 in vitro. Other candidate Khc activators include the exon junction complex, which, together with the SOLE RNA structure, is essential for transport of osk to the oocyte posterior. Because Tm1 needs to remain bound to the osk RNP throughout its posterior translocation, it is likely that the activating factor(s) induces a conformational change in the Khc–Tm1 complex rather than dissociation of Tm1. Future investigations of these regulatory mechanisms are likely to elucidate how kinesin-1 activity is orchestrated in other systems (Heber, 2024).

Earlier treatment of Oskar in The Interactive Fly

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.

date revised: 1 March 2024

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.