staufen

The translational repressor Cup is required for germ cell development in Drosophila

In Drosophila, germ cell formation depends on inherited maternal factors localized in the posterior pole region of oocytes and early embryos, known as germ plasm. This study reports that heterozygous cup mutant ovaries and embryos have reduced levels of Staufen (Stau), Oskar (Osk) and Vasa (Vas) proteins at the posterior pole. Moreover, Cup interacts with Osk and Vas to ensure anchoring and/or maintenance of germ plasm particles at the posterior pole of oocytes and early embryos. Homozygous cup mutant embryos have a reduced number of germ cells, compared to heterozygous cup mutants, which, in turn, have fewer germ cells than wild-type embryos. In addition, cup and osk interact genetically, because reducing cup copy number further decreases the total number of germ cells observed in heterozygous osk mutant embryos. Finally, cup mRNA and protein were detected within both early and late embryonic germ cells, suggesting a novel role of Cup during germ cell development in Drosophila (Ottone, 2012).

Germ plasm assembly is a stepwise process occurring during oogenesis. Accumulation of osk mRNA at the posterior of egg chambers is necessary for correct germ plasm assembly, which requires a polarized microtubule network, the plus-end motor kinesin I, and the activity of several genes (cappuccino, spire, par-1. mago nashi, barentz, stau, tsunagi, rab11, and valois). Localization of osk mRNA is strictly linked to the control of its translation, as unlocalized osk mRNA is silent. Upon localization at the posterior pole, the relieve of osk translational repression involves several factors, including Orb, Stau, and Aubergine. Localized Osk protein, in turn, triggers a cascade of events that result in the recruitment of all factors, such as Vas, Tud, and Stau proteins and nanos, germ less mRNAs, necessary for the establishment of functional germline structures (Ottone, 2012).

Posterior anchoring of Osk requires the functions of Vas, as well as Osk itself, to direct proper germ plasm assembly. Misexpression of Osk at the anterior pole of oocytes causes ectopic pole plasm formation, indicating that Osk is the key organizer of pole plasm assembly. Moreover, it has been demonstrated that endocytic pathways acting downstream of Osk regulate F-actin dynamics, which in turn are necessary to attach pole plasm components to the oocyte cortex. As far as Cup is concerned, it has been demonstrated that Cup is engaged in translational repression of unlocalized mRNAs, such as osk, gurken, and cyclinA, during early oogenesis (Ottone, 2012).

The current results establish that Cup is also a novel germ plasm component. First, Cup colocalizes with Osk, Stau, and Vas at the posterior pole of stage 10B oocytes. Second, biochemical evidence indicates that Cup interacts with Stau, Osk, and Vas. Vas localization occurs not through its association with localized RNAs, but rather through the interaction with the Osk protein, which represents an essential step in polar granule assembly (Ottone, 2012).

As a consequence of these interactions, Cup protein is mislocalized in osk and vas mutant stage 10 oocytes, demonstrating that Osk and Vas are essential to achieve a correct localization of Cup at the posterior cortex of stage 10 oocytes. This study suggests that the presence of Cup, Osk, Stau, and Vas are required for a correct germ plasm assembly. Moreover, several immuno-precipitation experiments, using anti- Tud and anti-Vas antibodies, identified numerous P-body related proteins, including Cup, as novel polar granule components (Ottone, 2012).

All the results suggest that Cup plays at least an additional role at stage 10 of oogenesis. Cup, besides repressing translation of unlocalized osk mRNA, is necessary to anchor and/or maintain Stau, Osk, and Vas at the posterior cortex. This novel function of Cup is supported by the findings that, when cup gene dosage is reduced, Stau, Osk, and Vas are partially anchored and/or maintained at the posterior pole, even if these proteins are not degraded. Consequently, pole plasm assembly is disturbed and cup mutant females lay embryos with a reduced number of germ cells. Since the role of Cup, a known multi-functional protein during the different stages of egg chamber development, cannot be easily studied in homozygous cup ovaries, it is not surprising that the involvement of Cup in pole plasm assembly remained undiscovered until now (Ottone, 2012).

During embryogenesis, Cup exerts similar functions. In particular, Osk, Stau, and Vas proteins and osk mRNA are not properly maintained and/or anchored at the posterior pole of embryos laid by heterozygous cup mutant mothers. Surprisingly, osk mRNA is increased in heterozygous cup mutant embryos. Since osk mRNA requires sufficient Osk protein to remain tightly linked at the posterior cortex, the reduced amount of Osk protein observed in heterozygous cup embryos, should be not sufficient to maintain all osk mRNA at the embryonic pole and could stimulate, by positive feedback, de novo osk mRNA synthesis. Also, a direct/indirect involvement of Cup in osk mRNA degradation and/or deadenylation cannot be excluded. The findings that Cup has been found together with Osk, when Osk is ectopically localized to the anterior pole of the embryos, and that reducing cup copy number further decreases the total number of germ cells, observed in heterozygous osk mutant embryos, strengthen the idea that Cup is involved in germ cell formation and/or in maintenance of their identity (Ottone, 2012).

Unlike Osk protein, both cup mRNA and protein were detected within germ cells until the end of embryogenesis. These observations suggest that zygotic cup functions, during germ cell formation and maintenance, are not limited to those carried out in combination with Osk. The finding that homozygous cup mutant embryos display a further decrease of germ cell number, in comparison with heterozygous embryos, supports this hypothesis. Whether or not cup zygotic function is involved in the translational repression of specific mRNAs, different from osk, remains to be explored (Ottone, 2012).

Protein Interactions

Localization of Staufen in the posterior pole

Staufen is required to localize Oskar mRNA. However, oskar function is required to stabilize the posterior localization of Staufen protein. In oskar mutants, Staufen only accumulates transiently at the posterior pole. It thus seems that Oskar protein is required to keep Oskar mRNA and Staufen protein at the posterior pole (St Johnston, 1991).

Mutations in the posterior group genes nanos, pumilio, tudor, valois and vasa have no effect on the localization of Staufen to the pole plasm of freshly laid embryos. In contrast, all cappuccino and spire mutations have dramatic effects on this localization (St Johnston, 1991).

Oskar protein interacts directly with Vasa and Staufen, in a yeast two-hybrid assay. These interactions also occur in vitro and are affected by mutations in oskar that abolish pole plasm formation in vivo. In the pole plasm, Oskar protein, like Vasa and Tudor, is a component of polar granules, the germ-line-specific RNP structures. Thus the Oskar-Vasa interaction constitutes an initial step in polar granule assembly. When the bait LexA-Osk is expressed together with the amino-terminal half of Stau protein fused to the activator, strong lacZ activity is observed, whereas little or no activity is detected when a carboxy-terminal Stau peptide is expressed. Whereas both Long Osk and Short Osk can interact with Vas and Stau, only Short Osk does so efficiently. It is thought that sequences between amino acids 451 and 606 are necessary but not sufficient for interactions of Osk with Vas and Stau. The biological significance of the direct interaction between Osk and Stau is unclear (Breitwieser, 1996).

It appears that there is an interaction between the actin and tubulin based cytoskeletons. Profilin, encoded by the gene chicakadee, a component of the actin based cytoskeleton, physically interacts with Cappuccino, involved in the microtubule based cytoskeleton. Mutants in chickadee resemble cappuccino in that they fail to localize Staufen protein and Oskar mRNA in the posterior pole of the developing oocyte. Also, a strong allele of cappuccino has multinucleate nurse cells, similar to those previously described in chickadee (Manseau, 1996).

In Drosophila, the localization of maternal determinants to the posterior pole of the oocyte is required for abdominal segmentation and germ cell formation. These processes are disrupted by maternal effect mutations in ten genes that constitute the posterior group. This paper presents the molecular analysis of one posterior group gene, mago nashi. Restriction fragment length polymorphisms and transcript alterations associated with mago nashi mutations were used to identify the mago nashi locus within a chromosomal walk. The mago nashi locus encodes a 147 amino acid protein with no similarity to proteins of known or suspected function. Nonsense mutations in mago nashi, as well as a deletion of the 5' coding sequences, result in zygotic lethality. The original mago nashi allele disrupts the localization of Oskar mRNA and Staufen protein to the posterior pole of the oocyte during oogenesis; anterior localization of Bicoid mRNA is unaffected by the mutation. These results demonstrate that mago nashi encodes an essential product necessary for the localization of germ plasm components to the posterior pole of the oocyte (Newmark, 1994).

cappuccino (capu) is a gene required for localization of molecular determinants within the developing Drosophila oocyte. The carboxy-terminal half of the Capu protein is closely related to that of the vertebrate limb deformity locus, which is known to function in polarity determination in the developing vertebrate limb. Capu shares both a proline-rich region and a 70-amino-acid domain with a number of other genes, two of which also function in pattern formation: the Saccharomyes cerevisiae BNI1 gene and the Aspergillus FigA gene. capu mutant oocytes have abnormal microtubule distributions and premature microtubule-based cytoplasmic streaming within the oocyte, but neither the speed nor the timing of the cytoplasmic streaming correlates with the strength of the mutant allele. This suggests that the premature cytoplasmic streaming in capu mutant oocytes does not suffice to explain the patterning defects. By inducing cytoplasmic streaming in wild-type oocytes during mid-oogenesis, it has been shown that premature cytoplasmic streaming can displace Staufen protein from the posterior pole, but not Gurken mRNA from around the oocyte nucleus (Emmons 1995).

The asymmetric localization of messenger RNA (mRNA) and protein determinants plays an important role in the establishment of complex body plans. In Drosophila oocytes, the anterior localization of Bicoid mRNA and the posterior localization of Oskar mRNA are key events in establishing the anterior-posterior axis. Although the mechanisms that drive Bicoid and Oskar localization have been elusive, oocyte microtubules are known to be essential. The plus end-directed microtubule motor kinesin I is required for the posterior localization of Oskar mRNA and an associated protein, Staufen, but not for the anterior-posterior localization of other asymmetric factors. Thus, a complex containing Oskar mRNA and Staufen may be transported along microtubules to the posterior pole by kinesin I (Brendza, 2000).

To determine if kinesin I is involved in oocyte patterning, mitotic recombination was used to generate mosaic female flies containing clones of homozygous Khc null germ line stem cells. The production of eggs and embryos by the mosaic females suggests that germ line stem cells can proliferate and proceed through oogenesis without kinesin I. However, embryogenesis fails, despite fertilization by wild-type males. Most embryos arrest before blastoderm formation, but a few proceed into early gastrulation stages. This maternal lethal effect is completely rescued by a wild-type Khc transgene. Thus, germ line expression of KHC is required for normal embryogenesis (Brendza, 2000).

Examination of embryos that reach the blastoderm stage reveals an absence of pole cells, the germ line precursors. To assay for earlier defects, the distributions of OSK and BCD mRNAs in Khc null oocytes were examined. The localization of BCD mRNA is normal, concentrated at the anterior during stages 8 to 10. In contrast, the localization of OSK mRNA is defective. It normally accumulates transiently at the anterior pole early in stage 8 and then moves to the posterior pole. In Khc null stage 8 to 10 egg chambers, OSK mRNA accumulates excessively at the anterior pole and is never concentrated at the posterior pole. This localization defect is completely rescued by a wild-type Khc transgene. Thus, although KHC is not required for anterior localization of either BCD or OSK mRNAs, it is required for the posterior localization of OSK. Perhaps kinesin I transports OSK mRNA along microtubules toward their plus ends and the posterior pole (Brendza, 2000).

Given that microtubule-disrupting drugs prevent the posterior localization of OSK mRNA during oogenesis, the possibility was considered that the absence of KHC blocks OSK localization indirectly by disturbing oocyte microtubules. The shift of the oocyte nucleus from posterior to anterior poles during stage 6, which is microtubule- dependent, appears normal in Khc null oocytes. Furthermore, the anterior localization of the MTOC component Centrosomin is normal (Brendza, 2000).

Microtubule organization was tested further by localizing a hybrid protein composed of the motor domain of KHC fused to a reporter enzyme, beta-galactosidase (beta-Gal). KHC::beta-Gal is thought to localize in regions of cells with high concentrations of microtubule plus ends. It is important to note that this chimeric protein does not rescue patterning defects in Khc null oocytes. In wild-type stage 9 to 10a oocytes, KHC::beta-Gal concentrates at the posterior pole. In Khc null oocytes, KHC::beta-Gal concentrates at the posterior pole in most instances. This suggests that in most of the stage 9 to 10a null oocytes with detectable amounts of KHC::beta-Gal, microtubule plus ends are concentrated at the posterior pole. This, and the indications that microtubules in the anterior end are normal, suggests that OSK mRNA mislocalization in Khc null oocytes is not due to a disruption of microtubule organization. In Khc null oocytes, KHC::beta-Gal staining is often not detected in oocytes, although it is visible in nurse cells. Perhaps efficient transport of the hybrid protein from nurse cells to oocyte requires the presence of native KHC (Brendza, 2000).

Posterior transport of OSK mRNA is thought to depend on Staufen protein. Staufen is transiently localized at the anterior end of the oocyte during stage 8 where it may form a complex with OSK mRNA. If kinesin I transports such OSK-Staufen complexes along microtubules to the posterior pole, then Staufen protein should be mislocalized in Khc null oocytes. Immunostaining with anti-Staufen confirms this prediction. In wild-type stage 8 to 10 oocytes, Staufen concentrates at the anterior end early, appears in granules along the cortex, and then concentrates at the posterior end. Granular Staufen distribution was detected in most oocytes observed. This is consistent with the hypothesis that Staufen and OSK mRNA form complexes at the anterior cortex that are transported to the posterior pole. In Khc mutant oocytes, Staufen protein overaccumulates in the anterior end during stage 8, is not detected in granules, and does not concentrate at the posterior pole. Normal Staufen distribution patterns are restored in Khc null oocytes by the addition of a wild-type Khc transgene (Brendza, 2000).

Thus, KHC, the force-generating component of the plus end-directed microtubule motor kinesin I, is required for the posterior localization of both OSK mRNA and Staufen protein. The participation of kinesin I in this mRNA motility process could be direct. It might attach specifically to osk-Staufen complexes at the anterior pole and transport them toward the posterior pole. However, initial tests for coimmunoprecipitation of KHC and Staufen from Drosophila ovary cytosol have not revealed any robust association, so perhaps the linkage is less direct. It is generally accepted that kinesin I transports membranous organelles toward microtubule plus ends. Thus, OSK and Staufen could localize to the posterior pole by virtue of association with mitochondria or other organelles carried by kinesin I. An alternative to these models is derived from the effect of a loss of KHC on the particulate staining pattern of Staufen. Before stages 7 to 8, while microtubules are still oriented with their plus ends toward the anterior, kinesin I might deliver, to the cortex, materials necessary for the assembly of transport-competent OSK-Staufen complexes. Thus, the lack of visible Staufen particles in Khc null oocytes may indicate that their assembly or persistence depends on kinesin I activity. New studies, using green fluorescent protein tags to follow the localization dynamics of OSK mRNA, Staufen, and organelles, may distinguish between these models and provide further insight into the mechanisms that drive the movements of maternal determinants for early developmental patterning (Brendza, 2000).

Assembly of endogenous oskar mRNA particles for motor-dependent transport in the Drosophila oocyte: Recruitment of Staufen and microtubule motors

oskar mRNA localization at the oocyte posterior pole is essential for correct patterning of the Drosophila embryo. This study shows, at the ultrastructural level, that endogenous oskar ribonucleoprotein complexes (RNPs) assemble sequentially with initial recruitment of Hrp48 (Heterogeneous nuclear ribonucleoprotein at 27C) and the exon junction complex (EJC) to oskar transcripts in the nurse cell nuclei, and subsequent recruitment of Staufen and microtubule motors, following transport to the cytoplasm. oskar particles are non-membrane-bound structures that coalesce as they move from the oocyte anterior to the posterior pole. This analysis uncovers a role for the EJC component Barentsz in recruiting Tropomyosin II (TmII) to oskar particles in the ooplasm and reveals that TmII is required for kinesin binding to the RNPs. Finally, it was shown that both kinesin and dynein associate with oskar particles and are the primary microtubule motors responsible for transport of the RNPs within the oocyte (Trucco, 2009).

This study shows that osk mRNA is synthesized in the nurse cell nuclei, where it assembles into small particles comprising Hrp48 and the Berentz-containing EJC, which are recruited independently to the RNA. The particles are then exported into the nurse cell cytoplasm where, upon loading of Stau, whose association is partially EJC dependent, they recruit both dynein and kinesin and associate with MTs. Upon transport into the oocyte, the small particles transiently reside at the anterior where they are remodeled, in a process requiring Hrp48, Btz, and Stau. During this process, the osk RNPs presumably lose their association with the MTs that mediated their transport from the nurse cells into the oocyte and bind to oocyte MTs for their subsequent transport (Trucco, 2009).

From the anterior, small osk particles are transported by kinesin toward the center of the oocyte, where a mixed population of small and more abundant large particles are observed. It is likely that these large particles arise through coalescence of small particles, as a result of their concentration in the center. Once formed, large particles do not appear to shuttle back to the anterior, as they were not observed in this area. Although a transient accumulation of osk mRNA in the oocyte center has been described as an important step prior to posterior transport, a recent live-cell imaging study of transgenic tagged osk mRNA argued against its existence. A probable explanation for this discrepancy lies in the different stages of oogenesis at which the analyses were performed. A central enrichment of osk mRNA is indeed detected at stage 8, whereas the in vivo analysis was restricted to stage 9 oocytes. It is possible that the transient central enrichment of osk particles reflects the dynamic organization of the MTs in the oocyte at stage 8 (Trucco, 2009).

From the center, large osk particles are transported along MTs toward the posterior pole where they form aggregates. Although a detailed understanding of MT organization will require more sophisticated ultrastructural approaches, such as EM tomography, this study has revealed that MTs are present throughout the oocyte, including the posterior cytoplasm. Indeed, osk RNPs are associated with MTs even in this region, suggesting that osk mRNA is actively transported on MT from the center of the oocyte to the posterior pole (Trucco, 2009).

At the posterior pole, osk aggregates form a continuum interspersed among abundant endocytic tubules where Long Osk has been shown to bind, suggesting that these membranous structures are involved in anchoring the mRNA at the posterior pole. However, endogenous osk RNPs detected throughout the egg chamber are non-membrane-bound structures that are not associated with any specific intracellular organelle, excluding a direct involvement of membrane traffic in osk localization (Trucco, 2009).

This analysis has shown that the loading of the shuttling proteins Hrp48 and Btz on osk mRNA is independent yet that both are required to generate mature particles that can associate with motor proteins and MTs. In particular, EJC components enhance the association between osk RNPs and TmII, which in its turn promotes the loading or stable association of Khc on osk transport particles. Moreover, in hrp48 and EJC mutants, osk is present in small particles uniformly distributed in the oocyte. It is likely that in these mutants the bulk of the mRNA in the oocyte is transported by cytoplasmic flows, explaining its homogeneous distribution and its failure to coalesce into larger particles. This also explains the 5-fold decrease in the number of actively moving particles observed in EJC mutant oocytes. The current findings demonstrate that the nuclear history of the mRNA is critical for the loading of motor proteins to RNPs (Trucco, 2009).

Surprisingly, the association of osk particles with motors and MTs is better preserved in the nurse cells than in the oocytes of hrp48 and EJC mutants, suggesting that the RNPs are remodeled upon entry into the oocyte. Indeed, RNP remodeling in the oocyte is probable, as incoming RNPs must switch from dynein-dependent to kinesin-dependent transport, and presumably detach from the MTs mediating their nurse cell-to-oocyte transport, to oocyte MTs mediating their transport to the posterior pole (Trucco, 2009).

The ultrastructural approach of this study has also provided insight into the function of the cytoplasmic actin-binding protein TmII in osk mRNA transport. TmII associates with osk particles mainly in the ooplasm, consistent with the idea that the RNPs are remodeled upon entry into the oocyte. Surprisingly, in TmII mutant oocytes the association of Khc with osk particles is reduced, suggesting that TmII promotes the recruitment or stability of Khc on osk transport particles (Trucco, 2009).

Ultrastructural analysis shows that Staufen is required for efficient recruitment or stabilization of the MT motor proteins on osk transport particles, explaining the reduced frequency of movements observed during in vivo imaging. The residual motors associated with osk RNPs may mediate their binding to the dense MT network at the oocyte anterior but not support their efficient transport, leading to the observed retention of the mRNA in this region. Alternatively, Staufen may have a role in the switching of osk RNPs from MTs mediating their transport from the nurse cells into the oocyte to the oocyte MTs responsible for the final transport of the mRNA to the posterior pole. A failure to dissociate from nurse cell-to-oocyte MTs would result in retention of the mRNA at the oocyte anterior. A similar, but less pronounced accumulation of osk mRNA is observed in btz mutant oocytes, where the loading of Staufen is affected (Trucco, 2009).

In WT egg chambers nearly 90% of osk RNPs are associated with both Khc and Dhc in the nurse cell cytoplasm and throughout the oocyte. This can explain why Exu-GFP particles, which are thought to contain osk mRNA, show accelerated movement in the nurse cell cytoplasm of khc null mutants. Indeed, it is likely that kinesin, which is present on the same osk particles as dynein, counteracts the dynein-mediated transport of osk RNPs from the nurse cells to the oocyte (Trucco, 2009).

This analysis of khc and TmII mutants has shown that when osk particles fail to associate with Khc, they also fail to accumulate in the oocyte center at stage 8, undergo only partial coalescence, and mostly accumulate around the oocyte cortex at stage 9. This indicates that Khc is a key motor transporting osk to the posterior pole. Interestingly, however, in both khc and TmII mutants, osk RNPs retain their association with Dhc and MTs, suggesting that dynein links osk RNPs to MTs also in the oocyte. Experiments involving live-cell imaging, antibody injection, and MT depolymerization confirm this and show that Khc and Dhc are the primary MT motors actively transporting osk particles. This analysis further suggests that during a single minute, Khc is responsible for active movement of nearly 75% of the particles, whereas dynein mediates the movement of the remaining 25% of particles in the oocyte. It is therefore concluded that in khc mutant oocytes, Dhc most likely transports osk particles to the minus ends of MTs at the lateral cortex, and that in WT oocytes, the activity of dynein in osk transport is masked by that of kinesin. Consistent with this, the speed of osk particle transport is greater in dhc hypomorphic than in WT oocytes, and previous studies have proposed a role of dynein in restricting kinesin activity (Trucco, 2009).

In situ hybridization coupled with immuno-EM is now an established technique that has been successfully used in the present study to visualize the assembly of osk transport particles in the Drosophila oocytes and to reveal the function of the different osk RNP components in this process. As a mechanism for localized protein expression, RNA localization is most powerful when tightly coupled to translational control. Future ultrastructural analysis combined with live-cell imaging is bound to provide new insight into the relationship between the RNA transport and translational control machineries (Trucco, 2009).

Bicoid mRNA localization and regulation

In the later vitellogenic stage in Bicoid mRNA localization, after transfer of the RNA from nurse cells to the oocyte, Staufen protein is required in order to anchor the Bicoid mRNA at the anterior pole of the Drosophila egg. Staufen protein colocalizes with BCD mRNA at the anterior. This localization depends upon Staufen's association with the Bicoid mRNA. Upon injection into the embryo, BCD transcripts specifically interact with Staufen. The required sequences have been mapped to three regions of the 3'UTR, each of which is predicted to form a long stem-loop. The resulting Staufen-BCD 3'UTR complexes form particles that show a microtubule-dependent localization. At the syncytial blastoderm stage, the microtubules associated with each nucleus are organized into a sheath that extends from the apical side around each interphase nucleus. These microtubules appear as a ring surrouding each nucleus: Staufen-containing particles lie either within these rings or in close proximity to them. At metaphase, the particles concentrate around both poles of the mitotic spindle, in close association with the astral microtubules that emanate from each spindle pole. In embryos treated with the microtubule-destabilizing drug colcemid, the Staufen-BCD 3'UTR particles show a random distribution (Ferrandon, 1994).

The formation of the anterior pattern of the Drosophila embryo is dependent on the localization of the mRNA of the morphogen Bicoid to the anterior pole of the egg cell. Staufen protein is required in a late step of the localization in order to anchor the BCD mRNA in the anterior cytoplasm. Stau protein contains five double-stranded RNA-binding motifs common to a family of dsRNA-binding proteins. Endogenous Stau protein associates specifically with injected BCD mRNA 3'-untranslated region (UTR), resulting in the formation of characteristic RNA-protein particles that are transported along microtubules of the mitotic spindles in a directed manner. The regions recognized by Stau in this in vivo assay are predicted to form three stem-loop structures involving large double-stranded stretches. The Stau interaction requires a double-stranded conformation of the stems within the RNA localization signal. Two loops contact one another through base pairing. One possibility is that this pairing occurs within the molecule, thus forming an element of tertiary structure called a pseudoknot. Attempts to model the putative pseudoknot in BCD mRNA were unsuccessful because it was impossible to bend the structure sufficiently to allow these two single-stranded loops to base-pair with one another. This suggests that the two loops base-pair with each other, but do so between two different mRNA molecules. Base pairing between two single-stranded loops plays a major role in particle formation. This loop-loop interaction is intermolecular, not intra-molecular; thus dimers or multimers of the RNA localization signal must be associated with Stau protein in these particles. The BCD mRNA 3' UTR can also dimerize in vitro in the absence of Stau. Thus, in addition to RNA-protein interactions, RNA-RNA interaction might be involved in the formation of ribonucleoprotein particles for transport and localization (Ferrandon, 1997).

The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, the third dsRBD from Drosophila Staufen has been studied. The domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and the amino acids required for this interaction have been identified. By mutating these residues in a staufen transgene, it has been shown that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of Bicoid and Oskar mRNAs. Using high-resolution NMR, the structure of the complex between dsRBD3 and an RNA stem-loop was determined. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins (Ramos, 2000).

Staufen and Neuroblast asymmetry

When neuroblasts divide, inscuteable acts to coordinate protein localization and mitotic spindle orientation, ensuring that asymmetrically localized determinants like Prospero partition into one progeny. staufen encodes a dsRNA-binding protein implicated in mRNA transport in oocytes. Prospero mRNA is also asymmetrically localized and partitioned during neuroblast cell divisions, a process requiring both inscuteable and staufen. Inscuteable and Staufen interact and colocalize with Prospero mRNA on the apical cortex of interphase neuroblasts. Staufen binds the 3'UTR of Prospero mRNA. These findings suggest that Inscuteable nucleates an apical complex and is required for protein localization, spindle orientation, and RNA localization. Stau, as one component of this complex, is required only for RNA localization. Hence staufen also acts zygotically, downstream of inscuteable, to effect aspects of neuroblast asymmetry (Li, 1997).

The Drosophila central nervous system develops from stem cell like precursors called neuroblasts, which divide unequally to bud off a series of smaller daughter cells, called ganglion mother cells. Neuroblasts show cell-cycle-specific asymmetric localization of both RNA and proteins: at late interphase, Prospero mRNA and Inscuteable, Prospero and Staufen proteins are all apically localized; at mitosis, Inscuteable protein remains apical, whereas Prospero mRNA, Prospero protein and Staufen protein form basal cortical crescents. In vitro culture of neuroblasts was used to investigate the role of intrinsic and extrinsic cues and the cytoskeleton in asymmetric localization of Inscuteable, Prospero and Staufen proteins. Neuroblast cytokinesis is normal in vitro, producing a larger neuroblast and a smaller ganglion mother cell. Apical localization of Inscuteable, Prospero and Staufen in interphase neuroblasts is reduced or eliminated in vitro, but all three proteins are localized normally during mitosis (apical Inscuteable, basal Prospero and Staufen). Microfilament inhibitors result in delocalization of all three proteins. Inscuteable becomes uniform at the cortex, whereas Prospero and Staufen become cytoplasmic; inhibitor washout leads to recovery of microfilaments and asymmetric localization of all three proteins. Microtubule disruption has no effect on protein localization, but disruption of both microtubules and microfilaments results in cytoplasmic localization of Inscuteable. It is concluded that both extrinsic and intrinsic cues regulate protein localization in neuroblasts. Microfilaments, but not microtubules, are essential for asymmetric protein anchoring (and possibly localization) in mitotic neuroblasts. These results highlight the similarity between Drosophila, Caenorhabditis elegans, vertebrates, plants and yeast: in all organisms, asymmetric protein or RNA localization and/or anchoring requires microfilaments (Broadus, 1997).

The generation of cellular diversity is essential in embryogenesis, especially in the central nervous system. During neurogenesis, cell interactions or asymmetric protein localization during mitosis can generate daughter cells with different fates. The asymmetric localization of a messenger RNA and an RNA-binding protein is described that creates molecular and developmental differences between Drosophila neural precursors (neuroblasts) and their daughter cells, ganglion mother cells (GMCs). The Prospero (Pros) mRNA and the RNA-binding protein Staufen (Stau) are asymmetrically localized in mitotic neuroblasts and are specifically partitioned into the GMC, as is Pros protein. Stau is required for localization of Pros RNA but not of Pros protein. Loss of localization of Stau or of Pros RNA alters GMC development, but only in embryos with reduced levels of Pros protein, suggesting that Pros mRNA and Pros protein act redundantly to specify GMC fate. GMCs do not transcribe the pros gene, showing that inheritance of Pros mRNA and/or Pros protein from the neuroblast is essential for GMC specification (Broadus, 1998).

Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs). During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda protein, which tethers it to the basal cortex of the dividing neuroblast. Interestingly, Prospero mRNA is similarly segregated by the dsRNA binding protein, Staufen. Staufen interacts in vivo with a segment of the Prospero 3' UTR. To assay RNA binding in vivo, the Prospero 3' UTR was injected into embryos expressing a green fluorescent protein (GFP)-Staufen protein fusion and the formation of Staufen ribonucleoprotein particles (RNP) was monitored. The full-length Prospero 3' UTR forms particles, as does the Bicoid 3' UTR, but not the coding region of the Prospero mRNA even though it is able to form an extended secondary structure. These RNPs are associated with the nuclei of the precellular embryo, and move with them to the cortex at stage 4. However, unlike the RNP particles formed between Staufen and the Bcd 3' UTR, the Staufen/Prospero 3' UTR particles do not associate with the astral microtubules. Similar results are observed when the Prospero 3' UTR is injected into embryos expressing wild-type Staufen (detected with anti-Staufen antibodies), rather than a GFP fusion. To further map the region of the Prospero 3' UTR with which Staufen interacts, either the 3' half of the UTR, or the 5' half were injected into embryos. Whereas the 3' segment recruits Staufen into RNPs within 5-10 min of injection, the 5' segment does so only slightly, if at all, after 20-30 min. Therefore, the region of the Prospero mRNA recognized by Staufen lies in the terminal 650 bases of the mRNA (Schuldt, 1998).

Staufen colocalizes with Prospero protein at all stages of the cell cycle. In embryos, Staufen is concentrated on the apical side of the neuroblast at interphase, then forms a crescent on the basal side of the cell in prophase, where it remains through mitosis before partitioning to the GMC at division. A similar subcellular distribution is seen in living embryos. This dynamic pattern of localization shows Staufen to be correctly placed to bind the Prospero mRNA throughout the cell cycle, and to mediate its segregation into the GMC (Schuldt, 1998).

Both the apical and basal localization of Staufen are abolished by the removal of a conserved domain from the carboxyl terminus of the protein, which interacts in a yeast two-hybrid screen with Miranda protein. Experiments in the oocyte have identified two regions of Staufen that are required for its function during oogenesis: a 99-amino-acid region in the middle of dRBD2, and the carboxy-terminal 157 amino acids that include dRBD5. As neither the dRBD2 insert nor the carboxy-terminal domain binds dsRNA in vitro, these two regions may be involved in some other aspect of Staufen function. When Staufen protein that lacks either the dRBD2 insert or the carboxy-terminal domain is expressed maternally, it can only partially rescue the abdominal defects caused by Staufen null mutations To test whether either of these domains is required for Staufen localization in neuroblasts, the subcellular distribution of Staufen mutants that lack the dRBD2 insert (dRBD2) or the carboxy-terminal domain of Staufen (dRBD5) were assayed. The removal of the dRBD2 insert has no effect on Staufen distribution in neuroblasts at any stage of the cell cycle. However, the loss of the carboxy-terminal 157 amino acids of Staufen eliminates both apical and basal localization. Staufen dRBD5 is distributed throughout the cytoplasm from interphase through mitosis. Therefore, the carboxyl terminus of Staufen is necessary to direct asymmetric distribution of the protein in neuroblasts. If the normal subcellular distribution of Staufen is mediated by a specific protein-protein interaction, then the site of the interaction may reside within the 157-amino-acid domain removed in Staufen dRBD5 (Schuldt, 1998).

Staufen binds directly to Miranda protein via the Staufen dRBD5 motif. Staufen dRBD5 does not interact with RNA in vitro but is required in vivo for Staufen protein localization, suggesting that it may interact with other proteins that anchor Staufen at the apical and basal cortex, or mediate Staufen's transport from one side of the neuroblast to the other. To identify proteins that might interact with dRBD5 to direct Staufen crescent formation, a yeast two-hybrid screen was carried out on a random-primed embryonic cDNA library using a LexA-dRBD5 fusion protein as bait. From 4,000,000 transformants, 10 positive clones were isolated, and the library plasmid could be recovered from 6 of these. All six clones contain the same insert, which encodes amino acids 506-776 of the Miranda protein. To test the specificity of the dRBD5/Miranda interaction, the Miranda clone was retransformed into yeast that contained either the original bait (lexA-dRBD5) or the control baits (lexA-lamin or lexA-BRCA2). Only yeast containing both lexA-dRBD5 and Miranda-VP16 expresses high levels of beta-galactosidase. Furthermore, this region of Miranda does not interact with all dRBDs, because no beta-galactosidase activity is observed when Miranda-VP16 is cotransformed with LexA-Staufen dRBD1. Thus, Miranda binds specifically to Staufen dRBD5, and does not interact with a closely related domain from the same protein (Schuldt, 1998).

To more precisely map the region of Miranda protein that interacts with Staufen-dRBD5, the fragment of Miranda identified in the yeast two-hybrid screen was divided into two parts (amino acids 506-638 and amino acids 639-776), and their interaction with dRBD5 was examined in a GST pull-down assay. 35S-labeled Staufen-dRBD5 coprecipitates with both the full-length fragment, GST-Miranda amino acids 506-776, and the amino-terminal segment of this region, GST-Miranda amino acids 506-638, but shows no interaction above background with the carboxy-terminal segment, GST-Miranda amino acids 639-776. This suggests that the Staufen binding site in Miranda corresponds to the predicted coiled-coil domain that extends from amino acids 526-593 (Schuldt, 1998).

Miranda colocalizes with Staufen protein and Prospero mRNA during neuroblast divisions, and neither Staufen nor Prospero RNA are localized in miranda mutants. Like Staufen, Miranda concentrates predominantly on the apical side of the cell at interphase. Interestingly, Miranda mRNA is also localized predominantly on the apical side of the neuroblast. Miranda protein then forms a crescent on the basal side of the neuroblast at prophase, where it remains until after cell division. Therefore, the subcellular distribution of Miranda suggests that it might interact with Staufen at all stages of the cell cycle (Schuldt, 1998).

It is concluded that Miranda binds to Prospero protein and to Staufen, which in turn binds Prospero mRNA, to form a complex on the apical side of the neuroblast. The complex may be anchored by Inscuteable at interphase, and then released as the cell cycle progresses. In mirandaRR127, Staufen accumulates on the apical side of the cell, suggesting that Miranda may regulate release from the apical cortex. Miranda, Prospero, Staufen, and Prospero mRNA then move as a group to the basal side of the cell during mitosis, a process that appears to require actin microfilaments. Staufen and Miranda also associate with the apical centrosome, although the significance of this interaction is unclear. Once at the basal cortex, the complex is anchored by factors that have not, as yet, been identified. However, as Miranda acts as the adapter between protein and RNA localization, these factors may be isolated in screens for other Miranda binding proteins. After cytokinesis, Miranda is rapidly degraded in the GMC, and Prospero is released and enters the nucleus. It may be important, therefore, to minimize translation of new Miranda protein in the GMC. Whereas Prospero mRNA is specifically segregated to the GMC, Miranda mRNA remains tightly anchored on the apical side of the neuroblast. By tethering Miranda mRNA in this way, Miranda protein, but not Miranda mRNA, is partitioned to the GMC at cell division (Schuldt, 1998).

Several interesting questions remain to be answered. What regulates the release of Miranda from the apical side of the cell? How are Miranda, Prospero, Staufen, and Prospero mRNA transported to the basal side of the neuroblast? Do they move as a complex, and how are they anchored at the basal cortex? Prospero and Staufen bind to the same region of Miranda, but it is not known whether they bind to the same molecule simultaneously. The answers to these questions may help to elucidate the mechanism of asymmetric protein and RNA localization not only in the nervous system, but also in other tissues, and in other organisms (Schuldt, 1998).

Neuroblasts in the developing Drosophila CNS asymmetrically localize the cell fate determinants Numb and Prospero as well as Prospero RNA to the basal cortex during mitosis. The localization of Miranda to the apical cortex, its interaction with Inscuteable in vitro and its role in localizing several downstream factors suggests that Miranda occupies a central link between Inscuteable at the apical cortex and the localization of Prospero, Staufen, and Prospero RNA to the basal cortex. How, early in mitosis, the apically localized Inscuteable dictates basal localization of intrinsic factors for asymmetric cell division may be elucidated by further studies on the genetic and cell biological mechanisms of the asymmetric localization of Miranda. The localization of Prospero requires the function of inscuteable and miranda, whereas Prospero RNA localization requires inscuteable and staufen function (Shen, 1998).

Miranda forms a crescent on the apical cortex of neuroblasts in late interphase. Later in mitosis, Miranda forms a crescent on the basal neuroblast cortex. Asymmetric localization of both Numb and Prospero has been shown to be dependent on the actin cytoskeleton. The actin dependence of Miranda localization was tested using the actin depolymerizing drug latrunculin A. After treatment of Drosophila embryos with 200 µM latrunculin A for 20 min, asymmetric localization of Miranda is completely disrupted, while membrane association is unperturbed. It is concluded that the asymmetric localization of Miranda during mitosis is an actin-dependent process. All Miranda fragments that contain the amino-terminal 298 amino acids exhibit the same asymmetric localization pattern as wild type Miranda. In contrast, a fragment containing amino acids 114-298 localizes to the cytoplasm and fails to segregate preferentially into the basal daughter cell, as does a fragment containing all residues carboxy-terminal to amino acid 300 (Shen, 1998).

The observation that Miranda protein fragments form an apical crescent that may coincide with the apical Inscuteable crescent led to a test of the possibility that Miranda interacts physically with Inscuteable. In an in vitro binding assay, Inscuteable coprecipitates with Miranda. An Inscuteable fragment from amino acids 252 to 615 also interacts with Miranda (Shen, 1998).

Miranda contains multiple functional domains: an amino-terminal asymmetric localization domain, which interacts with Inscuteable; a central Numb interaction domain, and a more carboxy-terminal Prospero interaction domain. Miranda and Staufen have similar subcellular localization patterns and interact in vitro. miranda function is required for the asymmetric localization of Staufen. Miranda localization is disrupted by the microfilament disrupting agent latrunculin A. These results suggest that Miranda directs the basal cortical localization of multiple molecules, including Staufen and Prospero mRNA, in mitotic neuroblasts in an actin-dependent manner (Shen, 1998).

When neuroblasts divide, Prospero protein and Pros mRNA segregate asymmetrically into the daughter neuroblast and sibling ganglion mother cell. Miranda is known to localize Prospero protein to the basal cell cortex of neuroblasts while the Staufen RNA-binding protein mediates Prospero mRNA localization. miranda is shown to be required for asymmetric Staufen localization in neuroblasts. Miranda thus acts to partition both Prospero protein and mRNA. Furthermore, Miranda localizes Prospero and Staufen to the basolateral cortex in dividing epithelial cells, which express the three proteins prior to neurogenesis. Analyses using miranda mutants reveal that Prospero and Staufen interact with Miranda under the same cell-cycle-dependent control. The wild-type Mira protein localizes predominantly to the cortex in interphase NBs, especially to the apical cortex along with Pros at late interphase. At the onset of prophase, the majority of the wild-type Mira becomes localized to the basal cortex as a crescent, while a fraction of the protein distributes to the apical region in a punctate manner. As the mitotic stage proceeds, an increasing proportion of Mira appears to be incorporated in the basal crescent. While some Mira protein is still observed apically during anaphase, most Mira protein segregates to the basally budding GMC. This pattern of subcellular localization is equally evident using polyclonal and monoclonal antibodies against a C-terminal polypeptide. mira mutations define three distinct functional regions along the mira sequence. The N-terminal 290 amino acids region acts in the basal localization of mira at mitosis in the NB and the epithelial cell. The region between amino acid 447 and 727 includes a domain necessary for the binding with Pros as well as the domain(s) required for the asymmetric localization of Stau in the NB. The C-terminal 103 amino acids region confers the cell cycle dependence on the interaction with Pros/Stau; the absence of this region results in the prolonged association with Pros/Stau during interphase without rapid proteolytic degradation in the GMC and NB. These observations suggest that the epithelial cell and neuroblast (both of epithelial origin) share the same molecular machinery for creating cellular asymmetry (Matsuzaki, 1998).

The mammalian Spinocerebellar Ataxia 8 noncoding RNA causes neurodegeneration and associates with Staufen in Drosophila

Spinocerebellar Ataxia 8 (SCA8) appears unique among triplet repeat expansion-induced neurodegenerative diseases because the predicted gene product is a noncoding RNA. Little is currently known about the normal function of SCA8 in neuronal survival or how repeat expansion contributes to neurodegeneration. To investigate the molecular context in which SCA8 operates, the human SCA8 noncoding RNA was expressed in Drosophila. SCA8 induces late-onset, progressive neurodegeneration in the Drosophila retina. Using this neurodegenerative phenotype as a sensitized background for a genetic modifier screen, mutations were identified in four genes: staufen, muscleblind, split ends, and CG3249. All four encode neuronally expressed RNA binding proteins that are conserved in Drosophila and humans. Although expression of both wild-type and repeat-expanded SCA8 induces neurodegeneration, the strength of interaction with certain modifiers differs between the two SCA8 backgrounds, suggesting that CUG expansions alter associations with specific RNA binding proteins. The demonstration that SCA8 can recruit Staufen and that the interaction domain maps to the portion of the SCA8 RNA that undergoes repeat expansion in the human disease suggests a specific mechanism for SCA8 function and disease. Genetic modifiers identified in the SCA8-based screens may provide candidates for designing therapeutic interventions to treat this disease (Mutsuddi, 2004).

The demonstration that Staufen, identified as an enhancer in the genetic screens, is recruited by SCA8, suggests that SCA8-Staufen associations, whether direct or indirect, are likely to be important for normal neuronal function and survival. The finding that the interaction is mediated by the CUG repeat containing the 200 nucleotide terminal portion of SCA8 RNA leads to the speculation that either the ability to interact with Staufen or the functional consequences of such an interaction could be compromised by CUG repeat expansion, leading to disease. Staufen has been shown to interact with the actin cytoskeleton and to be required for proper localization and targeting of RNAs. For example, rat staufen appears necessary for transport of RNAs to neuronal dendrites. In Drosophila, Staufen binds to the 3'UTR of Prosopero and mediates its proper localization in the larval neuroblasts. staufen has also been identified as a necessary component of long-term memory formation in Drosophila (Mutsuddi, 2004).

In conclusion, it is important to emphasize that the connection between repeat expansion and SCA8 ataxia remains a matter of contentious debate. Determining the clinical relevance of CTG repeat expansions to those patients who carry them will probably not be possible until the molecular context underlying SCA8 function in both normal and pathogenic contexts is more fully understood. The set of evolutionarily conserved and neuronally expressed RNA binding proteins identified in these experiments provides a critical molecular handle that once validated in mammalian model systems, should facilitate resolution of the controversy regarding the role of CTG repeat expansion in SCA8-associated neurodegeneration (Mutsuddi, 2004).

Miranda interacts with Staufen protein to couples oskar mRNA/Staufen complexes to the bicoid mRNA localization pathway

The double-stranded RNA binding protein Staufen is required for the microtubule-dependent localization of bicoid and oskar mRNAs to opposite poles of the Drosophila oocyte and also mediates the actin-dependent localization of prospero mRNA during the asymmetric neuroblast divisions. The posterior localization of oskar mRNA requires Staufen RNA binding domain 2, whereas prospero mRNA localization mediated the binding of Miranda to RNA binding domain 5, suggesting that different Staufen domains couple mRNAs to distinct localization pathways. This study shows that the expression of Miranda during mid-oogenesis targets Staufen/oskar mRNA complexes to the anterior of the oocyte, resulting in bicaudal embryos that develop an abdomen and pole cells instead of the head and thorax. Anterior Miranda localization requires microtubules, rather than actin, and depends on the function of Exuperantia and Swallow, indicating that Miranda links Staufen/oskar mRNA complexes to the bicoid mRNA localization pathway. Since Miranda is expressed in late oocytes and bicoid mRNA localization requires the Miranda-binding domain of Staufen, Miranda may play a redundant role in the final step of bicoid mRNA localization. These results demonstrate that different Staufen-interacting proteins couple Staufen/mRNA complexes to distinct localization pathways and reveal that Miranda mediates both actin- and microtubule-dependent mRNA localization (Irion, 2006).

Asymmetric localization of mRNAs is a common mechanism for targeting proteins to the regions of the cell where they are required. This process is particularly important in the developing oocytes of many organisms, where localized mRNAs function as cytoplasmic determinants. This has been best characterized in Drosophila, where the localization of bicoid (bcd) and oskar (osk) mRNAs to the anterior and posterior poles of the oocyte defines the primary axis of the embryo. bcd mRNA is translated after fertilization to produce a morphogen that patterns the head and thorax of the embryo, whereas osk mRNA is translated when it reaches the posterior of the oocyte, where Oskar protein nucleates the assembly of the pole plasm, which contains the abdominal determinant nanos mRNA, as well as the germ line determinants. Localized mRNAs can also function as determinants during asymmetric cell divisions. For example, the asymmetric inheritance of mating type switching in budding yeast is controlled by the localization of Ash1 mRNA to the bud tip, which segregates the repressor ASH1p into only the daughter cell at mitosis. Similarly, prospero (pros) mRNA localizes to the basal side of Drosophila embryonic neuroblasts and is inherited by only the smaller daughter cell of this asymmetric cell division, where Prospero protein acts as a determinant of ganglion mother cell fate (Irion, 2006).

To be localized, an mRNA must contain cis-acting localization elements that are recognized by RNA-binding proteins, which couple the mRNA to the localization machinery. This process is only well understood for ASH1 mRNA, which contains four localization elements that are recognized by She3p, which then links the mRNA to the myosin motor complex Myo4p/She2p so that it can be transported along actin cables to the bud tip. Biochemical and genetic approaches have led to the identification of a number of RNA-binding proteins that associate with localized mRNAs in higher eukaryotes, but it is not known how these interactions target the mRNA to the correct region of the cell (Irion, 2006).

One of the best candidates for an RNA-binding protein that plays a direct role in mRNA localization is the dsRNA-binding protein Staufen (Stau). Staufen was first identified because it is required for the localization of osk mRNA to the posterior of the oocyte and co-localizes with it at the posterior pole. This localization depends on the polarized microtubule cytoskeleton and the plus end-directed microtubule motor kinesin, suggesting that Staufen may play a role in coupling osk mRNA to kinesin, which then transports the osk mRNA complex along microtubules. The posterior localization of osk mRNA also requires the exon junction complex components Mago nashi (Mago), Y14, eIF4AIII and Barentsz (Btz), as well as HRP48, which is needed for the formation of Staufen/osk mRNA particles (Irion, 2006).

Staufen homologues seem to play a similar role in the microtubule-dependent localization in vertebrates. GFP-Stau particles have been observed to move along microtubules in cultured neurons, and the protein is a component of large ribonucleo-protein complexes that contain kinesin and dendritically localized mRNAs. In addition, a Xenopus Staufen homologue associates with Vg1 mRNA and is required for its microtubule-dependent localization to the vegetal pole of the oocyte, which is also thought to be mediated by a kinesin (Irion, 2006).

As well as this possible conserved role in kinesin-dependent transport, Drosophila Staufen is also required for the last phase of bcd mRNA localization and co-localizes with the mRNA at the anterior of the oocyte from stage 10B onwards. Furthermore, when the bcd 3′ UTR is injected into the early embryo, it recruits Staufen into particles that move in a microtubule-dependent manner to the poles of the mitotic spindles, consistent with minus end-directed microtubule transport (Irion, 2006).

Staufen also binds to prospero mRNA and is required for its localization to the basal side of the embryonic neuroblasts. In contrast to the other examples of Staufen-dependent mRNA localization, this process depends on the actin cytoskeleton and the adapter protein Miranda (Mira) (Irion, 2006).

The varied functions of Staufen raise the question of how the same protein can function in both actin- and microtubule-dependent mRNA localization, as well as in the targeting of osk and bcd mRNAs to opposite ends of the same cell. Some insight into this comes from the analysis of Staufen protein, which contains five conserved dsRNA-binding domains (dsRBDs). In all Staufen homologues, dsRBD2 is split by a proline-rich insertion in one of the RNA-binding loops, and deletion of this insertion disrupts the localization of osk mRNA, but not that of prospero mRNA, leading to the proposal that this domain couples Staufen/mRNA complexes to a kinesin-dependent posterior localization pathway. In contrast, removal of dsRBD5 prevents the localization of prospero mRNA, whereas osk mRNA localizes normally but is not translated at the posterior of the oocyte. Indeed, dsRBD5 binds directly to Miranda to couple Staufen/prospero mRNA complexes to the actin-based localization pathway. The localization of bcd mRNA also requires dsRBD5, although the loss of the insert in dsRBD2 also affects its localization slightly (Irion, 2006).

The results above suggest that different domains of Staufen couple mRNAs to distinct localization pathways, raising the possibility that the fate of Staufen mRNA complexes may depend on which Staufen-interacting proteins are present in the cell. To test this hypothesis, the effects of expressing Miranda during oogenesis were examined to determine whether it can influence the localization of bcd or osk mRNAs (Irion, 2006).

Although Miranda is not required during oogenesis, its ectopic expression causes a striking defect in anterior–posterior axis formation that reveals several important features of the mechanisms that control the targeting and translation of localized mRNAs. Firstly, these results provide strong support for the idea that the destination of Staufen/mRNA complexes is determined by the Stau-interacting factors that are present in the cell. During wild type oogenesis, Staufen associates with osk mRNA to mediate its kinesin-dependent localization to the posterior of the oocyte at stage 9, and this requires the insertion in Staufen dsRBD2, suggesting that this domain couples Staufen/osk mRNA complexes to the posterior localization pathway. However, the expression of Miranda is sufficient to target a proportion of these complexes to the anterior. This localization is mediated through the binding of Miranda to dsRBD5 of Staufen because deletion of this domain abolishes anterior localization without affecting the transport to the posterior pole. By contrast, deletion of the insert in dsRBD2 in the presence of Miranda results in the localization of all Staufen/osk mRNA complexes to the anterior pole. Thus, these two pathways act through different domains of Staufen to direct localization to opposite ends of the same cell. These pathways compete with each other, resulting in the partitioning of the Miranda/Staufen/osk mRNA complexes to the anterior and posterior poles, but each is capable of localizing all of the complexes when the other pathway is compromised. exu and swa mutants abolish the Miranda-dependent anterior localization, and osk mRNA now localizes exclusively to the posterior, whereas btz, mago and TmII mutants block the posterior localization pathway, resulting in the localization of all osk mRNA at the anterior cortex and the formation of reverse polarity embryos (Irion, 2006).

Since dsRBD5, which is not an RNA-binding domain, is necessary and sufficient for the interaction of Staufen with Miranda, the anterior localization of osk mRNA by Miranda provides a simple in vivo assay for the binding of Staufen to osk mRNA. This reveals that neither the insert of dsRBD2 nor the RNA-binding residues of dsRBD3 are required for the stable association of Staufen with the RNA. The lack of a requirement for the insert in dsRBD2 is consistent with the observation that dsRBD2Δloop binds dsRNA in vitro when expressed on its own, whereas the full-length dsRBD2 does not. It is more surprising, however, that the mutations in dsRBD3 have no effect on Staufen binding to osk mRNA since this domain binds to dsRNA with the highest affinity in vitro, and these mutations in the five key amino acids that contact the RNA abolish the domain's RNA-binding activity in vitro. The two other functional dsRNA-binding domains in Staufen (dsRBD1 and 4) must therefore be sufficient to form a stable complex with osk mRNA (Irion, 2006).

The specific effect of a quintuple mutant in dsRBD3 on posterior localization, but not on RNA binding of full-length Staufen, further suggests that these five amino acids play a role in coupling Staufen/osk mRNA complexes to the posterior localization pathway. Although it is possible that these residues are required for an interaction with a trans-acting factor, it seems more likely that it is the association of dsRBD3 with the RNA that is important because this affects either the folding of the RNA or the conformation of Staufen protein. For example, it has been suggested that the binding of Staufen dsRBDs1, 3 and 4 to osk mRNA presents a double-stranded region of the RNA to dsRBD2, which induces a conformational change in dsRBD2 that brings together the two RNA-binding regions of the domain and loops out the large insertion, which is then exposed to interact with the transport machinery. The effect of the point mutations in dRBD3 is consistent with this model and the idea that dsRBD2 functions as an RNA-binding sensor that couples Staufen/osk mRNA complexes to factors that target it to the posterior (Irion, 2006).

Although all mRNAs that accumulate in the oocyte localize at least transiently to the anterior, several lines of evidence indicate that Miranda links Staufen and osk mRNA specifically to the bcd localization pathway. (1) All other anterior mRNAs, except bcd and hu li tai shao (hts), localize to the anterior only during stages 9–10A and become delocalized at stage 10B when rapid cytoplasmic streaming begins. In contrast, Miranda maintains osk mRNA at the anterior throughout oogenesis, so that it is still localized in a tight anterior cap in the freshly laid egg. (2) Miranda, Staufen and oskar undergo the same change in their anterior localization at stage 10B as bcd mRNA: they initially localize as a ring around the anterior cortex and then move towards the middle of the anterior when the centripetal follicle cells start to migrate inwards. (3) Like bcd, the anterior localization of osk mRNA by Miranda requires Exu, Swallow and Staufen, whereas hts mRNA localization is independent of Exu and Staufen. Since the anterior localization does not require bcd mRNA itself, Miranda cannot simply hitchhike on the bcd mRNA localization complex, and it therefore presumably links osk mRNA to the same microtubule-dependent anterior transport pathway used by bcd mRNA (Irion, 2006).

In addition to its role in osk mRNA localization, Staufen associates with bcd mRNA during the late stages of oogenesis to mediate the final steps in its localization to the anterior cortex of the oocyte. Since this localization requires the Miranda-binding domain of Staufen and Miranda couples Staufen/mRNA complexes to the bcd localization pathway, it is attractive to propose that Miranda normally mediates the late anterior localization of bcd mRNA. mira mutants have no phenotype during oogenesis, however, although the protein is expressed in late oocytes. Thus, if Miranda does play a role in bcd mRNA localization, it must function redundantly with another unidentified factor. This is perhaps to be expected given the previous evidence for redundancy in the localization of bcd mRNA. For example, none of the small deletions within the bicoid localization signal abolishes its anterior localization, indicating that it contains redundant localization elements, and two distinct bcd mRNA recognition complexes have been purified biochemically from ovarian extracts (Irion, 2006).

The elucidation of the role of Miranda in bicoid mRNA localization will require the identification of other factors that couple Staufen/bicoid mRNA complexes to the anterior localization pathway, which may function redundantly with Miranda. There are no obvious candidates for these factors, however, since Staufen is the only known protein that is specifically required for the final step of bicoid mRNA localization. Indeed, one reason why such factors may have been missed in genetic screens for mutants that disrupt bicoid mRNA localization is because they are redundant with Miranda and have no phenotype on their own. For these reasons, it is hard to address the question of redundancy using a genetic approach, but further analysis of how Miranda targets Staufen/mRNA complexes to the anterior may help resolve this issue. For example, mapping the Miranda domains that direct anterior localization may provide a clue as to the molecular nature of the unidentified factors that also fulfil this function, while screens for proteins that interact with this domain could identify other components of the anterior localization pathway (Irion, 2006).

These results reveal that Miranda, like Staufen, has the capacity to mediate both microtubule- and actin-dependent localization, raising the question whether the former plays any role in its well-characterized function during the asymmetric divisions of the embryonic neuroblasts. The localization of Miranda to the basal side of the neuroblast is actin-dependent. However, the protein also accumulates at the apical centrosome during both embryonic and larval neuroblast divisions, and this localization is even more prominent in l(2)gl or dlg mutants. Furthermore, Miranda was independently identified as a component of the pericentriolar matrix and co-localizes with γ-tubulin on all of the centrosomes at syncytial blastoderm stage. Although the centrosomes disappear in the female germ line, the anterior cortex is the major site for microtubule nucleation and γ-tubulin localization in the oocyte. Thus, Miranda may localize to the anterior of the oocyte by the same mechanism as it localizes to centrosomes (Irion, 2006).

The phenotype of mira-GFP also provides insights into the translational control of osk mRNA. In wild type ovaries, osk mRNA is translationally repressed before it is localized, and this repression is then specifically relieved once the mRNA reaches the posterior pole. In principle, translational activation of osk mRNA could occur by a specific signal at the posterior, but it could also be due to some other consequence of localization, such as the concentration of the RNA in a small region or its association with the oocyte cortex. Evidence in favor of a specific posterior signal comes from an experiment in which a LacZ reporter gene under the control of the oskar 5′ region and the first 370 nt of the 3′ UTR was targeted to the anterior by the bcd localization element. Since this anterior RNA was not translated, concentration at the cortex appeared to be insufficient to relieve BRE mediated repression. However, it has recently emerged that this reporter RNA lacks the two clusters of insulin growth factor II mRNA-binding protein (IMP) binding elements in the distal oskar 3′ UTR that are essential for oskar translational activation at the posterior, making it hard to draw any conclusions from the lack of translation of this reporter RNA at the anterior. Mira-GFP provides an alternative way to test this hypothesis because it directs the anterior localization of wild type osk mRNA, with all of its translational control elements intact. This anterior mRNA is not translated during stages 9–13, despite being efficiently localized to the cortex, whereas the osk mRNA at the posterior of the same oocytes is translated normally. Thus, concentration at the cortex is not sufficient to de-repress translation, strongly supporting the idea that activation depends on a specific posterior signal (Irion, 2006).

Although the anterior osk mRNA is not translated at the normal time, the repression system breaks down at the very end of oogenesis, and the mRNA is very efficiently translated in mature oocytes. This suggests that some key component of the repression system disappears at this stage, and a good candidate is the BRE-binding protein Bruno. Bruno is highly expressed during oogenesis but is not detectable in embryos. Furthermore, the addition of Bruno is sufficient to cause the repression of exogenous osk mRNA in an embryonic translation system. These results indicate that Bruno is degraded at the end of oogenesis, whereas all other components necessary for translational repression of osk mRNA are still present in the embryo. Thus, the translation of anterior osk mRNA in mira-GFP oocytes is most probably triggered by the disappearance of Bruno (Irion, 2006).

Once it is translated at the posterior of the oocyte, Oskar protein nucleates the formation of the pole plasm with its characteristic electron-dense polar granules, which gradually assemble during stages 9–14 of oogenesis. This appears to be a stepwise process, in which Oskar protein recruits some polar granule components as soon as it is translated at stage 9, such as Vasa and Fat facets, while other components are added in sequence during the rest of oogenesis. For example, Tudor, Capsuleen and Valois are recruited during stage 10A, whereas nanos, Pgc and gcl mRNAs only become enriched at the posterior at stages 10B–11. It is therefore surprising that the anterior Oskar protein, which is only synthesized in stage 14 oocytes, can still nucleate fully functional pole plasm that induces the formation of anterior pole cells. Thus, although the pole plasm normally assembles in an ordered fashion over the last 5 stages of oogenesis, this whole process can still occur once oogenesis is complete. This indicates that the assembly of the pole plasm does not depend on the order of addition of its components, all of which must still be present and freely diffusible in mature oocytes (Irion, 2006).

Formation of the bicoid morphogen gradient: an mRNA gradient dictates the protein gradient: Transport of the Stau-bcd mRNA complex on microtubules

The Bicoid (Bcd) protein gradient is generally believed to be established in pre-blastoderm Drosophila embryos by the diffusion of Bcd protein after translation of maternal mRNA, which serves as a strictly localized source of Bcd at the anterior pole. However, evidence suggests the Bcd gradient is preceded by a bcd mRNA gradient. This study has revisited and extended this observation by showing that the bcd mRNA and Bcd protein gradient profiles are virtually identical at all times. This confirms a previous conclusion that the Bcd gradient is produced by a bcd mRNA gradient rather than by diffusion. Based on the observation that bcd mRNA colocalizes with Staufen (Stau), it is proposed that the bcd mRNA gradient forms by a novel mechanism involving quasi-random active transport of a Stau-bcd mRNA complex through a nonpolar microtubular network, which confines the bcd mRNA to the cortex of the embryo (Spirov, 2009).

Revisiting the formation of the morphogenetic bcd gradient, published results (Frigerio, 1986) have been corroborated and extended by demonstrating a gradient of bcd mRNA that is very similar to that of the Bcd protein. Therefore, the results contradict the SDD model (refering to the localized synthesis, diffusion and spatially uniform degradation of the Bcd protein). Although the SDD model correctly predicts an exponential Bcd protein gradient, the diffusion coefficient of Bcd, as measured in syncytial-blastoderm embryos, is two orders of magnitude too low to account for the fact that the steady state of its gradient is reached at syncytial blastoderm, a finding that is in serious conflict with this model. The results strongly suggest that the bcd mRNA gradient forms the protein gradient and is established by transport of the mRNA along the cortex of the embryo (Spirov, 2009).

The following model was used to explain the formation of the bcd mRNA gradient. The establishment of the bcd RNA gradient by nuclear cycle 10 and its disappearance during early nuclear cycle 14 occur in five phases. (1) The bcd RNA, which is associated with Stau and presumably many other proteins and anchored to the actin cytoskeleton at the anterior cortex of the mature oocyte, is released upon fertilization by calcium signaling. (2) This Stau-bcd mRNA complex is transported posteriorly along microtubules emanating from numerous microtubule-organizing centers (MTOCs) that are closely spaced and distributed throughout the cortex of the embryo. The posterior transport of bcd mRNA is driven by its concentration gradient. (3) This transport is arrested by the breakdown of the cortical microtubular network when the nuclei reach the cortex. At this time, a new microtubular network of astral microtubules forms. These extend from the centrosomes, located between the plasma membrane and each nucleus, and surround each nucleus with apical-basal polarity. Thus, the bcd mRNA gradient is established by nuclear cycle 10 and does not change until the end of nuclear cycle 13. (4) During syncytial blastoderm, the bcd mRNP particles are transported apically on astral microtubules by the minus end-directed dynein/dynactin motor complex, a process that depends on the maternal proteins Bicaudal D (BicD) and Egalitarian (Egl). (5) Whereas bcd mRNA is stable until nuclear cycle 13, it is rapidly degraded during early nuclear cycle 14. This degradation is assumed to be mediated by Stau and is triggered by apical factors and signals (Spirov, 2009).

This model combines the current results with those reported by others. The evidence that led to this ARTS (active RNA transport and protein synthesis) model will be discussed in detail. Despite its speculative aspects, it should serve as a useful hypothesis for future experiments that test its predictions. In addition, the model postulates a new principle to explain the formation of the bcd mRNA gradient: a quasi-random transport through a cortical microtubular network that is driven by a high initial concentration of bcd mRNA at the anterior pole (Spirov, 2009).

Stau protein binds to the 3'UTR of bcd mRNA in oocytes and colocalizes with bcd mRNA at the anterior pole of freshly laid eggs. Additional proteins probably stabilize the interaction of Stau with bcd mRNA in the embryo, as in oocytes. Localization of bcd mRNA to the anterior pole is established by continual active transport of the Stau-bcd mRNA complex on microtubules, mediated by the minus end-directed motor dynein, when nurse cells empty their content into stage 10B-13 oocytes. Subsequent anchoring of the Stau-bcd mRNA complex to the actin cytoskeleton stabilizes its anterior localization in mature oocytes. This anchoring step depends on swallow (swa), the product of which interacts with the dynein light chain and γTub37C, which is part of the MTOC at the anterior end of oocytes. Upon fertilization, calcium signaling releases the Stau-bcd mRNA complex from the actin cytoskeleton, which depends on the product of the sarah (sra) gene, an inhibitor of the calcium-dependent phosphatase calcineurin. Swa protein is no longer required and is degraded (Spirov, 2009).

A network of microtubules, in which the MTOCs are closely spaced (separated by a few microns), occupies the cortical region of embryos during nuclear cycles 1-9. Consistent with this observation is the pattern of cortical staining of early embryos for several Dgrips, proteins of the γ-tubulin ring complex that caps the minus ends of microtubules at MTOCs. Evidently, these microtubules nucleate from MTOCs that are established in late oocytes. No function has been reported previously for this cortical microtubular network, which breaks down at the end of nuclear cycle 9. It is proposed that its existence is crucial for the formation of the bcd mRNA gradient (Spirov, 2009).

A mechanism based on diffusion of the bcd mRNA cannot explain the gradient observed because bcd mRNA is restricted to the cortex of the embryo. Diffusion of bcd mRNA to the interior would dramatically reduce its concentration along the cortex, where its function is required, because unlike for Bcd protein in the SDD model, there is no source replenishing the lost bcd mRNA. Active transport of a Stau-bcd mRNA complex on microtubules, similar to that observed in late-stage oocytes, is suggested by the striking colocalization of Stau and bcd mRNA until the latter disappears. However, the microtubules with MTOCs located at the anterior pole are disassembled in late oocytes. Indeed, the cortical microtubular network in embryos at nuclear cycles 1-9 shows no sign of an overall polarity, but appears to be nonpolar, with its plus ends growing in all directions from MTOCs closely spaced throughout the cortex (Spirov, 2009).

How can such a nonpolar microtubular network establish a bcd mRNA gradient by active transport of the Stau-bcd mRNP particles? Because the network exhibits no polarity, it supports only random transport as would occur by diffusion. The only restriction to the random transport is its confinement to the cortex of the embryo. Like diffusion, it is driven by the concentration gradient of the transported molecules, here by the high initial concentration of bcd mRNA at the anterior pole. Average transport velocities of Stau-bcd mRNA complexes on microtubules, as measured in stage 10B-13 oocytes, range from 0.36 to 2.15 µm/second. Such a non-random transport in the embryo would move bcd mRNA molecules within minutes from the anterior to the posterior pole and thus destroy its function as an anterior morphogen. Therefore, it seems crucial that bcd mRNA transport in the embryo occurs through a nonpolar microtubular network. It is additionally important that the time of 90 minutes that is required to establish the bcd mRNA gradient at 25°C is tuned finely with the time required for the first nine nuclear divisions, after which the nuclei reach the cortex (Spirov, 2009).

The efficiency of a system employing random transport can be estimated from the average posterior drift velocity of bcd mRNAs along the cortex. When, 90 minutes after fertilization, syncytial blastoderm is reached, bcd mRNA has moved posteriorly on average by ~50 µm (from 5% EL at fertilization to 15% EL), which corresponds to an average drift velocity of ~0.01 µm/second. This is 100 times slower than the average transport velocity on a microtubule in the oocyte and is thus rather inefficient. Since this transport of bcd mRNA occurs on a microtubular network with randomly oriented microtubules, it is irrelevant whether transport is mediated by the minus end-directed dynein/dynactin or the plus end-directed kinesin motors. In the oocyte, Stau-bcd mRNP particles are transported exclusively by dynein in a process that depends on the presence of Exuperantia (Exu) in nurse cells. Since Exu disappears from late oocytes, this might permit Stau-bcd mRNPs to interact with dynein or kinesin upon their release from the actin cytoskeleton (Spirov, 2009).

Just before the present study was submitted, transport in oocytes through a microtubular network exhibiting only a slight directional bias (57% of plus ends oriented posteriorly) was shown to localize Stau-oskar (osk) mRNA particles to the posterior pole (Zimyanin, 2008). Although it is conceivable that the bcd mRNA gradient is established through such a biased microtubular network, the net average posterior velocity in oocytes of 0.03 µm/second (Zimyanin, 2008) would displace the bcd mRNA on average by 162 µm towards the posterior pole of the embryo by the time the bcd RNA gradient is established. This is more than twice the observed average posterior displacement of bcd mRNA. Nevertheless, the bcd mRNA gradient might be established through such a biased microtubular network if transport is mediated by both minus- and plus-end motors. In such a case, however, the average posterior drift velocity would also depend on the availability of both motors. If the probability of Stau-bcd mRNP interacting with either motor is the same, transport by the microtubular network becomes independent of its directional bias, and the network behaves like the nonpolar microtubular network. However, a nonpolar microtubular network is favored in the embryo because it seems more robust to disturbances (Spirov, 2009).

An intriguing feature of the bcd mRNA gradient during nuclear cycles 10-13 is the maintenance of a constant apical gradient similar to the basal gradient. It has been noted that bcd transcripts are localized to the narrow apical periplasm at late syncytial blastoderm, and that this localization depends on a signal in their 3'UTR. Apical transport of bcd mRNA becomes evident during nuclear cycle 14, when the excess of basal bcd mRNA disappears more rapidly than its apical counterpart (Spirov, 2009).

Although no net apical transport of bcd mRNA is apparent before its degradation during nuclear cycle 14, the establishment of an astral microtubular network during nuclear cycle 9 suggests that it might occur as early as nuclear cycle 10. Such a system, capable of transporting Stau-bcd mRNA particles to the apical periplasm, might be important to stabilize the bcd mRNA gradient against disturbances by the strong periplasmic flow that is observed in the cortex during nuclear cycles 10-13. Nevertheless, if Stau-bcd mRNA complexes detach when they reach the minus ends at the apical MTOCs, some apically localized bcd mRNAs might be subject to the periplasmic flow. Such a disturbance would be minor, as it would be corrected immediately by rapid apical transport of Stau-bcd mRNA particles, which occurs at a velocity of 0.5 µm/second (Spirov, 2009).

Why is it important to localize bcd mRNA not only to the basal, but also to the apical, nuclear periplasm? An answer is probably provided by elegant studies that have demonstrated that the nuclear concentration of Bcd protein remains approximately constant at a certain position along the anteroposterior axis during syncytial blastoderm (Gregor, 2007). This finding was surprising in view of the fact that the number of nuclei double after each nuclear division, their volume increases by 30% during interphase of each nuclear cycle, and the Bcd concentration drops fourfold when nuclear membranes disappear during mitosis. It was explained by measurements revealing that nuclear import of Bcd is sufficiently rapid to maintain a high and constant nuclear Bcd concentration. Hence, it might be crucial that Bcd can be imported through the entire nuclear surface (Gregor, 2007). Consistent with an accelerated nuclear import of Bcd by the product of the lesswright (lwr) gene (Epps, 1998), Lwr was found in cleavage-stage and syncytial-blastoderm nuclei (Spirov, 2009).

Whereas bcd mRNA is stable before nuclear cycle 14, basal bcd mRNA disappears owing to its degradation and transport to the apical periplasm within ~10 minutes of early nuclear cycle 14. Thus, the estimated half-life of basal bcd mRNA is ~2 minutes. Apical bcd mRNA decreases only when basal bcd mRNA becomes limiting. At this time, the estimated half-life of apical bcd mRNA is also ~2 minutes. Therefore, bcd mRNA is degraded in the basal and apical periplasm, or only in the latter. This degradation is presumably mediated by a bcd instability element (BIE) located within a 43-nucleotide sequence following the stop codon. In mammals, Stau may trigger the degradation of an mRNA by binding to its 3'UTR and to the nonsense-mediated decay (NMD) factor Upf1, in a process that is different from NMD and is called Staufen-mediated mRNA decay (SMD) (Kim, 2005). As the Drosophila genome encodes a Upf1 homolog, Stau might well function not only in the transport of bcd mRNA but also in its degradation (Spirov, 2009).

Since Bcd protein disappears ~25 minutes after bcd mRNA, a lag during which its level decreases at least tenfold, its half-life is less than 8 minutes at this time. The presence of a conserved PEST sequence in Bcd might be responsible for its short half-life, presumably also during earlier stages, a hypothesis that is consistent with the similarity between the slopes of the bcd mRNA and protein gradients (Spirov, 2009).

There are many ways to generate a morphogenetic gradient. The original proposal of how the Bcd protein gradient forms closely followed Wolpert's model of generating a morphogenetic gradient by a localized source synthesizing the morphogenetic molecules that are subject to diffusion and spatially uniform degradation. This model predicts a steady state at which the Bcd concentration decays exponentially along the anteroposterior axis. This study now shows that the Bcd protein gradient is generated by an entirely different mechanism. Since there is no source of bcd mRNA, its posterior transport from the anterior pole must be arrested when the optimal gradient is reached. This arrest is triggered by the breakdown of the cortical microtubular network and is well timed with the arrival of the nuclei at the cortex, when gap genes are activated by the Bcd protein. At this time, the gradient is established and remains constant until nuclear cycle 14, when bcd mRNA is rapidly degraded. Thus, the bcd mRNA gradient is not established as a steady state, but by a process that is terminated by the breakdown of the microtubular network required for its formation (Spirov, 2009).

Compared with the diffusion-based mechanism of the SDD model, a random active transport system has several advantages for the formation of the bcd mRNA gradient. The microtubular network is able to confine the movement of the bcd mRNA to the space where its function is required. The final shape of the gradient depends on several parameters: the initial concentration of bcd mRNA at the anterior pole, the transport velocity along microtubules, the average travel time per microtubule, the time between discharge from one and reloading onto another microtubule, and the time when transport is arrested by the breakdown of the microtubular network that supports the random transport. In addition, the availability of minus end- and plus end-directed motors might further influence the generation of the bcd mRNA gradient by random transport. Therefore, perhaps the greatest advantage of random active transport is that variations in these parameters during evolution permit the adaptation of the gradient to its optimal shape at the time when its function is required during development. For these reasons, it is suspected that random active transport represents a general mechanism that might have found wide application during evolution (Spirov, 2009).

The translational repressor Cup is required for germ cell development in Drosophila

In Drosophila, germ cell formation depends on inherited maternal factors localized in the posterior pole region of oocytes and early embryos, known as germ plasm. This study reports that heterozygous cup mutant ovaries and embryos have reduced levels of Staufen (Stau), Oskar (Osk) and Vasa (Vas) proteins at the posterior pole. Moreover, Cup was shown to interact with Osk and Vas to ensure anchoring and/or maintenance of germ plasm particles at the posterior pole of oocytes and early embryos. Homozygous cup mutant embryos have a reduced number of germ cells, compared to heterozygous cup mutants, which, in turn, have fewer germ cells than wild-type embryos. In addition, cup and osk were shown to interact genetically, because reducing cup copy number further decreases the total number of germ cells observed in heterozygous osk mutant embryos. Finally, cup mRNA and protein were detected within both early and late embryonic germ cells, suggesting a novel role of Cup during germ cell development in Drosophila (Ottone, 2012).

Germ plasm assembly is a stepwise process occurring during oogenesis. Accumulation of osk mRNA at the posterior of egg chambers is necessary for correct germ plasm assembly, which requires a polarized microtubule network, the plus-end motor kinesin I, and the activity of several genes (cappuccino, spire, par-1. mago nashi, barentz, stau, tsunagi, rab11, and valois. Localization of osk mRNA is strictly linked to the control of its translation, as unlocalized osk mRNA is silent. Upon localization at the posterior pole, the relieve of osk translational repression involves several factors, including Orb, Stau, and Aubergine. Localized Osk protein, in turn, triggers a cascade of events that result in the recruitment of all factors, such as Vas, Tud, and Stau proteins and nanos, germ less mRNAs (Mahowald, 2001), necessary for the establishment of functional germline structures. Posterior anchoring of Osk requires the functions of Vas Tud, as well as Osk itself, to direct proper germ plasm assembly. Misexpression of Osk at the anterior pole of oocytes causes ectopic pole plasm formation, indicating that Osk is the key organizer of pole plasm assembly. Moreover, it has been demonstrated that endocytic pathways acting downstream of Osk regulate F-actin dynamics, which in turn are necessary to attach pole plasm components to the oocyte cortex (Ottone, 2012).

As far as Cup is concerned, it has been demonstrated that Cup is engaged in translational repression of unlocalized mRNAs, such as osk, gurken, and cyclinA, during early oogenesis (Ottone, 2012).

The current results establish that Cup is also a novel germ plasm component. First, Cup colocalizes with Osk, Stau, and Vas at the posterior pole of stage 10B oocytes. Second, biochemical evidence indicates that Cup interacts with Stau, Osk, and Vas. Vas localization occurs not through its association with localized RNAs, but rather through the interaction with the Osk protein, which represents an essential step in polar granule assembly (Ottone, 2012).

As a consequence of these interactions, Cup protein is mislocalized in osk and vasmutant stage 10 oocytes, demonstrating that Osk and Vas are essential to achieve a correct localization of Cup at the posterior cortex of stage 10 oocytes. This study suggests that the presence of Cup, Osk, Stau, and Vas are required for a correct germ plasm assembly. Moreover, several immunoprecipitation experiments, using anti- Tud and anti-Vas antibodies, identified numerous P-body related proteins, including Cup, as novel polar granule components (Ottone, 2012).

All these results suggest that Cup plays at least an additional role at stage 10 of oogenesis. Cup, besides repressing translation of unlocalized osk mRNA, is necessary to anchor and/or maintain Stau, Osk, and Vas at the posterior cortex. This novel function of Cup is supported by the findings that, when cup gene dosage is reduced, Stau, Osk, and Vas are partially anchored and/or maintained at the posterior pole, even if these proteins are not degraded. Consequently, pole plasm assembly is disturbed and cup mutant females lay embryos with a reduced number of germ cells. Since the role of Cup, a known multi-functional protein during the different stages of egg chamber development, cannot be easily studied in homozygous cup ovaries, it is not surprising that the involvement of Cup in pole plasm assembly remained undiscovered until now (Ottone, 2012).

During embryogenesis, Cup exerts similar functions. In particular, Osk, Stau, and Vas proteins and osk mRNA are not properly maintained and/or anchored at the posterior pole of embryos laid by heterozygous cup mutant mothers. Surprisingly, osk mRNA is increased in heterozygous cup mutant embryos. Since osk mRNA requires sufficient Osk protein to remain tightly linked at the posterior cortex, the reduced amount of Osk protein observed in heterozygous cup embryos, should be not sufficient to maintain all osk mRNA at the embryonic pole and could stimulate, by positive feedback, de novo osk mRNA synthesis. Also, a direct/indirect involvement of Cup in osk mRNA degradation and/or deadenylation cannot be excluded (Ottone, 2012).

The findings that Cup has been found together with Osk, when Osk is ectopically localized to the anterior pole of the embryos, and that reducing cup copy number further decreases the total number of germ cells, observed in heterozygous osk mutant embryos, strengthen the idea that Cup is involved in germ cell formation and/or in maintenance of their identity (Ottone, 2012).

Unlike Osk protein, both cup mRNA and protein were detected within germ cells until the end of embryogenesis. These observations suggest that zygotic cup functions, during germ cell formation and maintenance, are not limited to those carried out in combination with Osk. The finding that homozygous cup mutant embryos display a further decrease of germ cell number, in comparison with heterozygous embryos, supports this hypothesis. Whether or not cup zygotic function is involved in the translational repression of specific mRNAs, different from osk, remains to be explored (Ottone, 2012).

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