Factors affecting Gurken mRNA localization and translation (part 2/2)

Bruno and Squid

The Drosophila gene squid (sqd) encodes a heterogeneous nuclear RNA binding protein (hnRNP), also known as hrp40. hnRNPs are a large family of proteins that have been implicated in the processing of nascent mRNA transcripts. Recent studies have demonstrated that a subset of hnRNPs, including human hnRNP A1 and A2, Saccharomyces cerevisiae Nplp3 and Hrp1, and Chironomus tentans Hrp36, rapidly shuttle between the cytoplasm and the nucleus. A specific motif, termed M9, has been shown to mediate this nucleocytoplasmic shuttling, and this motif is present in Sqd. Nuclear import of M9-containing hnRNPs is achieved by an association with the nuclear import protein Transportin. Studies of several of these hnRNPs have indicated that one of their major roles is the nuclear export of mRNAs, suggesting that Sqd may perform a similar function during Drosophila oogenesis (Norvell, 1999 and references).

Sqd protein is detected within the somatic follicle cells and the germ-line-derived nurse cells and oocyte. A germ-line mutation in a squid causes female sterility as a result of mislocalization of Gurken (GRK) mRNA during oogenesis. Alternative splicing produces three isoforms: SqdA, SqdB, and SqdS. These isoforms are not equivalent; SqdA and SqdS perform overlapping but nonidentical functions in GRK mRNA localization and protein accumulation, whereas SqdB cannot perform these functions. Furthermore, although all three Sqd isoforms are expressed in the germline cells of the ovary, they display distinct intracellular distributions. Both SqdB and SqdS are detected in germ-line nuclei, whereas SqdA is predominantly cytoplasmic. It is argued that SqdS is involved in the transport and localization of GRK mRNA. The ability of SqdA to prevent translation of ventrally localized GRK mRNA and its ability to provide peak levels of Grk protein required on the dorsal side of the egg chamber, strongly suggests that SqdA has a role in the accumulation of Grk protein. Moreover, the role of SqdA is both positive and negative, suggesting that SqdA may influence the association of GRK mRNA with appropriate translational regulators (Norvell, 1999).

Evidence is provided that GRK mRNA localization and translation are coupled by an interaction between Sqd and the translational repressor protein Bruno. Because Sqd protein binds GRK mRNA directly and belongs to the class of hnRNPs implicated in nuclear mRNA export, it seems likely that Sqd functions in the nuclear export of GRK mRNA. One complication to this model is that in the sqd mutant, GRK mRNA is still able to leave the nucleus and accumulate in the oocyte cytoplasm but not in the dorso-anterior corner. Therefore, the function of the Sqd protein appears to be in the regulated nuclear export of GRK mRNA, such that Sqd is responsible for delivering the GRK message to a cytoplasmic protein involved in its anchoring, possibly coupled to translation. Thus, one might expect that Sqd protein should interact with some cytoplasmic ovarian proteins (Norvell, 1999).

A number of proteins have been implicated in the translational regulation of GRK mRNA, both positively (e.g., Encore and Vasa) and negatively (e.g., Bruno). Therefore, an investigation was carried out to see whether Sqd protein could directly associate with any of these candidates. Using the in vitro association assay, interactions were sought between the Sqd isoforms and Encore, Vasa, or Bruno. Although a direct interaction between Sqd and Encore or Vasa could not be found, Sqd protein associates with Bruno protein in vitro. Although the Sqd-Bruno interaction observed in vitro is not extremely strong, it is very consistently observed over multiple experiments. These data show that Sqd protein and Bruno protein can associate with one another. Moreover, these data provide evidence for a link between GRK mRNA localization and translational regulation (Norvell, 1999).

In addition to its requirement in oogenesis, Sqd is also required somatically. A number of lethal alleles of sqd were generated and the ability of the individual isoforms to restore viability of sqd null alleles was investigated. Again, as with the ability of the specific isoforms to function during oogenesis, the three Sqd isoforms differ in their ability to rescue the viability of a lethal sqd allelic combination. Both SqdS and SqdB were capable of rescuing the essential somatic Sqd function: expression of either of these transgenes allows recovery of 11% and 19% of the expected number of mutant sqd adults, respectively. In contrast, however, SqdA is incapable of restoring the essential somatic function of Sqd, since less than 0.2% of the expected number of sqd adults were recovered. These data further demonstrate that the individual Sqd isoforms are not functionally equivalent (Norvell, 1999).

At least two lines of evidence indicate that Sqd protein must be present within the oocyte nucleus for GRK mRNA to be localized properly during oogenesis. (1) Oof the three Sqd isoforms, only SqdS is detected within the oocyte nucleus; among the Sqd transgenic females, only those expressing the SqdS isoform show properly localized GRK mRNA. The differential nuclear accumulation of the SqdS protein is associated with its ability to interact with Drosophila Transportin. SqdA does not possess a Transportin interaction motif. (2) The relationship between K10 and the distribution of Sqd protein also demonstrates the importance of Sqd accumulation within the oocyte nucleus. Mutations in both fs(1)K10 and sqd, consistently cause a mislocalization of GRK mRNA along the entire anterior cortex of the oocyte and lead to the production of strongly dorsalized eggs. Although the nuclear import of SqdS protein is most likely driven by its association with Transportin, K10 function is required for the stable accumulation of Sqd in the oocyte nucleus. This places K10 function upstream of Sqd in the germ line. Accumulation of Sqd protein in the nurse cells is not affected, and in addition Sqd is detectable within the oocyte cytoplasm of K10 mutants. Since the SqdS protein is the only Sqd isoform that is normally detected within the oocyte nucleus, the major effect of K10 must be on the nuclear retention of SqdS (Norvell, 1999 and references).

At this time it is unclear how K10 performs this function mechanistically. K10 protein will physically interact with the Sqd isoforms. The only known motif within the K10 protein is a potential helix-turn-helix domain in the carboxyl terminus, but site-directed mutagenesis of this domain has revealed that this motif is unnecessary for K10 function. K10 could be responsible for the modification of Sqd protein in such a manner as to promote nuclear retention, or alternatively, K10 could form a complex with Sqd protein that stabilizes its accumulation within the oocyte nucleus. In either case, the finding that Sqd protein requires the presence of K10 to accumulate in the oocyte nucleus further suggests that the phenotype of K10 mutant eggs is attributable to an effect on Sqd (Norvell, 1999 and references).

The ability to investigate the roles of the individual Sqd isoforms in the regulation of Grk during Drosophila oogenesis has revealed that there are two key aspects of Grk regulation: GRK mRNA localization and Grk protein accumulation. Both of these critical aspects of Grk regulation are accomplished by Sqd protein; however, these functions are performed differentially by the SqdS and SqdA isoforms. The severity effects of sqd mutation, therefore, reflect the loss of function of both of these proteins within the germ line, thus causing the mislocalization of GRK mRNA and the inappropriate accumulation of ectopic Grk protein. Restoration of either of these levels of regulation allows partial rescue of the D-V patterning defects of sqd mutants, but full rescue requires the function of both SqdS and SqdA. The data suggest that Sqd protein is a key regulator of both aspects of Grk regulation. The interaction of Sqd with the translational repressor protein Bruno provides a link between GRK mRNA localization and its translational regulation. Bruno has been shown directly to have a role in the translational repression of unlocalized Oskar mRNA. The interaction between Bruno and OSK mRNA is mediated by a specific sequence within the OSK message, which is termed BRE. As of yet, the molecular mechanism of Bruno action is not fully understood. However, correctly localized OSK mRNA must somehow be relieved from the Bruno-mediated repression by specific trans-acting factors localized to the posterior of the embryo (Norvell, 1999).

The interaction between Sqd and Bruno suggests that Bruno may play a role in the translational regulation of GRK mRNA. In support of this, GRK mRNA is known to contain a BRE within its 3' UTR, and Bruno has been shown to bind the GRK message. In addition, it has been demonstrated that during late stages of oogenesis, Bruno protein is concentrated at the anterior end of the oocyte, in a position that is coincident with localized GRK mRNA. On the basis of protein interaction data, it is suggested that Bruno may serve as a translational repressor of unlocalized GRK mRNA. As is the case for localized OSK mRNA, the appropriately localized GRK message would be relieved of its Bruno-mediated repression by other localized trans-acting factors. Moreover, the physical association between Sqd protein and Bruno protein suggests an appealing model to explain the similarity of the sqd and K10 mutant phenotypes. These two female sterile mutations represent the only cases in which mislocalized GRK mRNA is translated consistently and efficiently in all egg chambers. It is proposed that the role of Sqd protein is to take GRK mRNA from the oocyte nucleus, recruit Bruno in the cytoplasm, and deliver GRK mRNA to an anchor. In the absence of nuclear Sqd, in either sqd or K10 egg chambers, GRK RNA exits the nucleus by a generalized export mechanism, but does not associate efficiently with the repressor Bruno nor with the anchor. Because the interaction between GRK mRNA and Bruno does not occur, even the unlocalized GRK mRNA is translated efficiently. Using this model, Sqd protein provides the physical link between GRK mRNA transport, localization, and its appropriately regulated translation (Norvell, 1999).

The dorsalized eggs laid by hfp mutants are similar to those produced by females mutant for squid (sqd), which encodes an RRM-containing protein that has been shown to directly bind grk RNA. The sqd gene encodes three splice forms that contain the same N-terminal RNA binding domain but have divergent C termini and display distinct activities with respect to grk RNA localization and translational control.Are the dorsalized eggs of hfp caused by defects in the splicing of sqd? Using RT-PCR analysis of stage-dissected ovaries, it was found that, in wild-type oogenesis, sqd splicing is developmentally regulated. While the sqdA and sqdB transcripts are produced constitutively, the sqdS transcript is detected only in later stages. No alterations were detected in sqd splicing in hfp9 or in the stronger allelic combination hfp13/Df. Furthermore, no differences were detected in Sqd protein localization or levels in hfp mutants, and, thus, the defects in grk RNA localization seen in hfp are not likely due to alterations in sqd expression (Van Buskirk, 2002).

Females mutant for the squid (sqd) gene are sterile and lay eggs that display only dorsal structures. The resulting embryos are also dorsalized even if fertilized by wild-type sperm. The gene acts midway through oogenesis at about the time dorsoventral (D/V) axis is established within the growing egg chamber. The sqd gene encodes at least three distinct proteins generated by alternative RNA processing that are members of a well-characterized family of RNA-binding proteins. At least one SQD isoform is essential in somatic tissues. The ventralizing mutations gurken, torpedo, and cornichon are all epistatic to sqd. Strong alleles of grk and top can act as dominant suppressors of sqd dorsalization. One model of D/V axis formation postulates that squid is needed to organize a concentration gradient of a morphogen originating in the germinal vesicle (Kelley, 1993).

Translational regulation of localized transcripts is a powerful mechanism to control the precise timing and localization of protein expression within a cell. In the Drosophila germline, oskar transcript must be translationally repressed until its localization at the posterior pole of the oocyte, since ectopic production of Oskar causes severe patterning defects. Translational repression of oskar mRNA is mediated by the RNA-binding protein Bruno, which binds to specific motifs in the oskar 3'UTR. Bruno over-expression is shown to cause defects in antero-posterior and dorso-ventral patterning, consistent with a role of Bruno in both oskar and gurken mRNA regulation. Bruno and gurken interact genetically. Finally, Bruno is shown to bind specifically to the gurken 3'UTR; the dorso-ventral defects caused by Bruno over-expression are due to a reduction of Gurken levels in the oocyte. It is concluded that Bruno plays similar roles in translational regulation of gurken and oskar (Filardo, 2003).

Bru has mainly been studied with regard to its role in translational repression of the posterior determinant Osk. However, the most obvious effect of aret mutations in females is premature arrest of oogenesis, a phenotype unrelated to translational misregulation of osk mRNA. During early oogenesis, the cystoblast fails to develop into a 16-cell cyst in the presence of strong aret mutant alleles. In contrast, weak aret alleles produce apparently normal egg chambers, which then undergo degeneration at stage 9. Hence, Bru affects a number of cellular processes that take place in the germline, including osk translational regulation. By analogy to osk regulation, the aret phenotype might therefore be caused by misregulation of target RNAs which, in the wild-type, are tightly regulated by Bru. Another not mutually exclusive possibility is that lack of functional Bru impairs other processes in which the protein is involved and that are unrelated to its RNA-binding activity. The fact that Bru over-expression causes phenotypes similar to Bru loss-of-function, and the fact that these defects can be modulated by simultaneous over-expression of BRE-containing RNA, supports the hypothesis that at least some of the aret early oogenesis phenotypes are indeed the result of RNA mis-regulation (Filardo, 2003).

Bru over-production, like Bru loss-of-function, impairs ovarian development. Most remarkably, Bru-over-expressing egg chambers that develop beyond the earliest stages undergo an extra round of division with incomplete cytokinesis, suggesting a role of Bru in regulation of the cystocyte divisions. Another gene, encore (enc), has also been shown to be involved both in regulation of germline mitoses and in establishment of oocyte polarity, the latter due to its role in grk mRNA localization and translation. enc encodes a 210 kDa protein with one conserved R3H domain, a single-stranded nucleic acid-binding domain. Thus, Bruno is not the only RNA-binding protein to be involved in regulation of the cystoblast divisions and in establishment of polarity. Given the nature of the proteins, it is likely that both Enc and Bru mediate their oogenesis effects through RNA binding. In contrast to Enc, which is required for Grk accumulation, Bru appears to negatively regulate Grk levels, most likely at the level of translation (Filardo, 2003).

Bru also provides a new example of genes whose activity affects establishment of both the A/P and the D/V axis. Bru has previously been shown to repress osk mRNA translation and new results show that Bru negatively regulates grk as well. The grk 3'UTR contains a single BRE. The interaction between Bruno and grk is most likely responsible for the observed reduction in Grk signal in egg chambers in which Bru is over-expressed. Another protein involved in regulation of both osk and grk is the DEAD-box RNA helicase Vasa, although in this case mutant alleles show a reduction in Osk and Grk levels, suggesting a positive role for Vas in osk and grk mRNA translation. oo18 RNA binding (orb), encoding the Drosophila cytoplasmic polyadenylation element binding protein (CPEB) is also required for both osk and grk mRNA localization and translation. Thus, regulation of the mRNAs encoding the embryonic polarity determinants Osk and Grk appears to be intimately related, involving many of the same RNA regulatory proteins (Filardo, 2003).

Heterogeneous nuclear ribonucleoproteins, hnRNPs, are RNA-binding proteins that play crucial roles in controlling gene expression. In Drosophila oogenesis, the hnRNP Squid (Sqd) functions in the localization and translational regulation of gurken (grk) mRNA. Sqd interacts with Hrb27C, an hnRNP previously implicated in splicing. Like sqd, hrb27C mutants lay eggs with dorsoventral defects and Hrb27C can directly bind to grk RNA. These data demonstrate a novel role for Hrb27C in promoting grk localization. A direct physical interaction is also observed between Hrb27C and Ovarian tumor (Otu), a cytoplasmic protein implicated in RNA localization. Some otu alleles produce dorsalized eggs and it appears that Otu cooperates with Hrb27C and Sqd in the oocyte to mediate proper grk localization. All three mutants share another phenotype, persistent polytene nurse cell chromosomes. These analyses support dual cooperative roles for Sqd, Hrb27C and Otu during Drosophila oogenesis (Goodrich, 2004).

This study identifies a physical and genetic interaction between Hrb27C and Sqd. The two proteins were previously biochemically purified as part of an hnRNP complex from Drosophila cells, but it was not known if they interact directly nor had their RNA targets been isolated. The interaction was originally detected in a two-hybrid screen and it has been confirmed biochemically by co-immunoprecipitation. In vivo, this interaction requires the presence of RNA; this result was unexpected because the yeast two-hybrid constructs that originally revealed the interaction did not contain the RRMs and presumably cannot bind RNA. The interaction may require the full-length proteins to be in unique conformations that are achieved only when they are bound to RNA. The truncated proteins used in the yeast two-hybrid screen may be folded in such a manner that the protein-protein interaction domains are exposed even in the absence of RNA binding, making it possible for the interaction to occur in yeast (Goodrich, 2004).

A striking genetic interaction was detected between Sqd and Hrb27C; weak hrb27C mutants can strongly enhance the DV defects of weak sqd mutants. This suggests that the physical interaction has in vivo significance. The phenotypes of sqd and hrb27C place both proteins in a pathway required for proper grk localization and suggest a previously undetected role for Hrb27C. hrb27C mutants lay variably dorsalized eggs, and in situ hybridization analysis reveals that grk RNA is mislocalized. Since the mislocalized RNA in hrb27C mutants is translated, causing a dorsalized egg phenotype, it is likely that Hrb27C also functions with Sqd in regulating Grk protein accumulation. A role in the regulation of RNA localization and translation is novel for Hrb27C/Hrp48; it has been previously described as functioning in the inhibition of IVS3 splicing of P-element encoded transposase (Siebel, 1994; Hammond, 1997). Hrb27C has nuclear functions and has been observed in the nucleus and cytoplasm of somatic and germline cells in embryos (Siebel, 1995). Because Sqd is localized to the oocyte nucleus where it is thought to bind grk RNA, a model is favored where Hrb27C and Sqd bind grk RNA together and are exported possibly as a complex into the cytoplasm (Goodrich, 2004).

Two unpublished studies have implicated Hrb27C in osk RNA localization (J. Huynh, T. Munro, K. Litière-Smith and D. St Johnston, communication to Goodrich, 2004) and translational regulation (T. Yano, S. Lopez de Quinto, A. Shevenchenko, A. Shevenchenko, T. Matsui and A. Ephrussi, communication to Goodrich, 2004). Translational repression of osk RNA until it is properly localized is essential to prevent disruptions in the posterior patterning of the embryo. An analogous mechanism of co-regulation of mRNA localization and translation appears to control Grk expression. Certain parallels between osk and grk regulation are very striking. Both RNAs are tightly localized and subject to complex translational regulation; they also share certain factors that mediate this regulation. In addition to Hrb27C, the translational repressor Bruno appears to be a part of the regulatory complexes that are required for the proper expression of both RNAs. It will be interesting to determine if there are other shared partners that function in the regulation of both RNAs, as well as identify the factors that give each complex its localization specificity (Goodrich, 2004).

Otu is also involved in the localization of grk mRNA and interacts physically with Hrb27C. Though a definitive role of Otu in regulating Grk expression has not been previously described, Van Buskirk (2002) found that four copies of Otu104 (flies carry a transgene expressing only the 104 kDa isoform of Otu under the control of the otu promoter) are able to rescue the grk RNA mislocalization defect of half pint (hfp; poly U binding factor 68kD) mutants. Analysis of the hypomorphic alleles, otu7 and otu11, reveals a requirement for Otu in localizing grk RNA for proper DV patterning. Alternative splicing of the otu transcript produces two protein isoforms: a 98 kDa isoform and a 104 kDa isoform that differ by the inclusion of a 126 bp alternatively spliced exon (6a) in the 104 kDa isoform. This alternatively spliced exon encodes a tudor domain, a sequence element present in proteins with putative RNA-binding abilities. Interestingly, the otu11 allele, which contains a missense mutation in exon 6a, shows the grk localization and dorsalization defect, strongly suggesting that the tudor domain of Otu plays an important function in grk localization. Since otu mutants also show defects in osk localization, it appears that like several other factors, Otu is required for both grk and osk localization. Additionally, Otu has been isolated from cytoplasmic mRNP complexes (Goodrich, 2004).

Surprisingly, an additional shared phenotype of hrb27C, sqd and otu mutants was found. During early oogenesis, the endoreplicated nurse cell chromosomes are polytene. As they begin to disperse, the chromosomes are visible as distinct masses of blob-like chromatin that completely disperse by stage 6. The formation of polytene nurse cell chromosomes is due to the endocycling that occurs during early oogenesis. The mechanism of chromosome dispersal is hypothesized to involve the degradation of securin by the anaphase-promoting complex/cyclosome as a result of separase activity that cleaves cohesin. The significance of chromosome dispersion is not clear, but it has been suggested that it could facilitate rapid ribosome synthesis. A defect in the dispersal of polytene chromosomes has been previously described for otu mutants, and both sqd and hrb27C mutants also have nurse cell chromosomes that fail to disperse. Weak double transheterozygous allele combinations of sqd and hrb27C produce the persistent polytene nurse cell chromosome phenotype that is not observed in either of the weak mutants alone. Thus, genetic and physical interactions between these gene products suggest that they function together in regulation of this process (Goodrich, 2004).

The failure to disperse nurse cell chromosomes has also been observed in mutants of hfp where the nurse cell nuclear morphology defect is due to improper splicing of Otu104 (Van Buskirk, 2002). As was shown for hfp, expressing Otu104 in a sqd mutant background is able to rescue the polytene nurse cell chromosome phenotype. Although Hfp affects otu splicing, it is not clear how Sqd functions to mediate this phenotype, but evidence supports a role in Otu104 protein accumulation. The transcript that encodes Otu104 is clearly present in sqd mutants, as assayed by RT-PCR, but the level of Otu104 protein is decreased. These results suggest that the level of Otu104 is crucial to maintaining proper nurse cell chromosome morphology because the polytene phenotype of sqd mutants can be rescued by expressing only one extra copy of Otu104 and the majority of egg chambers from females heterozygous for otu13, otu11, or a deficiency lacking otu, have nurse cell chromosomes that fail to disperse (Goodrich, 2004).

The reduced level of Otu104 in sqd mutants raises the possibility that Sqd could translationally regulate otu RNA and that the polytene nurse cell phenotype may be a result of the decreased level of Otu104. Alternatively, Sqd and Hrb27C could form a complex that recruits and stabilizes Otu104 protein, and this complex in turn functions directly or indirectly to regulate chromosome dispersion. An indirect role seems more likely, given that Otu has never been observed in the nucleus. Since Otu104 can rescue the phenotype of sqd mutants and Otu and Hrb27C co-precipitate, it seems plausible that Sqd and Hrb27C interact with Otu in the nurse cell cytoplasm to affect an RNA target that could then mediate chromosome dispersion (Goodrich, 2004).

The genetic data support a model in which Sqd, Hrb27C, and Otu function together in a complex that affects at least two processes during oogenesis: DV patterning within the oocyte and mediation of nurse cell chromosome dispersion. Although the biochemical data are consistent with this model, it is also possible that Hrb27C and Sqd could form a complex that is distinct from a complex containing Hrb27C and Otu. However, the in vivo genetic interactions and mutant phenotypes reveal that all three proteins affect both processes and favor a model in which all three proteins function in one complex (Goodrich, 2004).

These results allow expansion upon the model proposed by Norvell (1999) for the regulation of grk RNA expression. Sqd and Hrb27C associate with grk RNA in the oocyte nucleus. Hrb27C and Sqd remain associated with grk in the cytoplasm where Otu and possibly other unidentified proteins associate with the complex as necessary to properly localize, anchor and translationally regulate grk RNA. A likely candidate to be recruited to the complex is Bruno, which interacts with Sqd (Norvell, 1999). Although the RNA target of Sqd, Hrb27C and Otu in the nurse cells is not known, the proteins may form a complex composed of different accessory factors to regulate localization and/or translation of RNAs encoding proteins that affect chromosome morphology (Goodrich, 2004).

Although Hrb27C, Sqd and Otu function together to affect different processes in the oocyte and the nurse cells, they probably function to mediate the precise spatial and temporal regulation of a target RNA that is unique to each cell type where the presence of additional factors would provide specificity to each complex. The importance of the precise localization and translational regulation of grk is well defined, and Hrb27C and Otu have not been identified as additional proteins that facilitate these processes. The transition from polytene to dispersed chromosomes in the nurse cells is less well characterized, but most probably requires precise spatial and temporal regulation as well. A complex containing Sqd, Hrb27C and Otu could function to ensure that the target RNA is properly localized and functional in the nurse cells only at the proper stage of development to promote chromosome dispersal (Goodrich, 2004).

arrest mutants have pleiotropic phenotypes, ranging from an early arrest of oogenesis to irregular embryonic segmentation defects. One function of arrest is in translational repression of oskar mRNA; this biochemical activity is presumed to be involved in other functions of arrest. To identify genes that could provide insight into how arrest contributes to translational repression or that may be targets for arrest-dependent translational control, deficiency mutants were screened for dominant modification of the arrest phenotype. Only four of the many deficiencies tested, which cover ~30% of the genome, modified the starting phenotype. One enhancer, identified fortuitously, is the Star gene. Star interaction with arrest results in excess Gurken protein, supporting the model that gurken is a target of repression. Two modifiers were mapped to individual genes. One is Lk6, which encodes a protein kinase predicted to regulate the rate-limiting initiation factor eIF4E. The second is Delta. The interaction between arrest and Delta mimics the phenotype of homozygous Delta mutants, suggesting that arrest could positively control Delta activity. Indeed, arrest mutants have significantly reduced levels of Delta protein at the interface of germline and follicle cells (Yan, 2004).

A screen of third chromosome deficiencies was screened for dominant modifiers of aret mutants. About three-quarters of the third chromosome was screened, corresponding to ~30% of the genome. Only four deficiencies dominantly modified the aret mutant phenotype, suggesting that the total number of genes in the genome with this property is small. For two of the four deficiencies the gene responsible for the interaction was identified, and a third interacting gene was fortuitously discovered while preparing for the screen. It was anticipated that two different types of modifiers might be detected by the screen: those in genes that act in the same process as Bru and those in genes that are themselves regulated by Bru or act in a process in which a limiting component is regulated by Bru. Characterization of the interacting genes suggests that examples of each type of modifier were discovered (Yan, 2004).

Bru has been proposed to translationally regulate grk mRNA. The supporting evidence includes (1) binding of Bru to grk mRNA in vitro and indirect evidence of binding in vivo; (2) rare dorsoventral patterning defects as a consequence of overexpression of Bru, and enhancement of this phenotype by reduction of grk gene dosage, and (3) evidence that localized Grk is present at reduced levels when Bru is overexpressed, although unlocalized Grk appears more abundant. However, there has been no evidence of excess Grk protein in aret mutants. Star is required for grk activity, and it acts post-translationally in either trafficking or secretion of Grk protein. When flies were both homozygous for aret and heterozygous for S1 they accumulated Grk protein in nurse cells, while ectopic accumulation could not be detected in either aret mutants or S1 heterozygotes alone. This synthetic effect on Grk protein accumulation is simple to rationalize. In aret mutants Grk protein is excessively translated, but an S-dependent delivery step could efficiently clear the protein from the nurse cells. When S activity is reduced, a detectable level of Grk remains in the nurse cells. The distribution of the ectopic Grk, both in cytoplasm and at the nurse cell boundaries, could correspond to the sites where the protein might stall during delivery. The actual site of S action is not known, and two different sites of S concentration, in endoplasmic reticulum or on the plasma membrane, have been reported. Although this explanation has some appeal, it is important to note that none of the evidence firmly establishes a role for Bru in translational repression of grk mRNA, and it remains possible that Bru could, for example, influence the site of translation rather than its efficiency (Yan, 2004).

Imp associates with Squid and Hrp48 and contributes to localized expression of Gurken and Oskar in the oocyte

Localization and translational control of Drosophila gurken and oskar mRNAs rely on the hnRNP proteins Squid and Hrp48, which are complexed with one another in the ovary. IGF-II mRNA-binding protein (Imp), the Drosophila homolog of proteins acting in localization of mRNAs in other species, is also associated with Squid and Hrp48. Notably, Imp is concentrated at sites of gurken and oskar mRNA localization in the oocyte, and alteration of gurken localization also alters Imp distribution. Imp binds gurken mRNA with high affinity in vitro; thus, the colocalization with gurken mRNA in vivo is likely to be the result of direct binding. Imp mutants support apparently normal regulation of gurken and oskar mRNAs. However, loss of Imp activity partially suppresses a gurken misexpression phenotype, indicating that Imp does act in control of gurken expression but has a largely redundant role that is only revealed when normal gurken expression is perturbed. Overexpression of Imp disrupts localization of gurken mRNA as well as localization and translational regulation of oskar mRNA. The opposing effects of reduced and elevated Imp activity on gurken mRNA expression indicate a role in gurken mRNA regulation (Geng, 2006).

Imp is the Drosophila homolog of a family of proteins that act in posttranscriptional regulation in a variety of animals. One of the founding members of the family, ZBP-1, binds to a localization element in the chicken beta-actin mRNA and appears to direct localization to the leading edge of embryonic fibroblasts. Another founding member, the Xenopus Vg1RBP/VERA protein, binds to signals directing localization of Vg1 and VegT mRNAs to the vegetal pole of the oocyte. Mammalian homologs, the Imp proteins, have been suggested to act in mRNA localization, mRNA stability, and translational regulation. A recent report (Munro, 2006) examined the RNA binding properties of Drosophila Imp protein, focusing specifically on the osk mRNA and its possible regulation by Imp. Although mutation of candidate Imp binding sites in the osk mRNA did block accumulation of Osk protein, loss of Imp activity did not cause a similar defect. This study shows that Imp interacts with Sqd and Hrp48, two proteins that regulate expression of osk and grk mRNAs. Mutation of the Imp gene does not substantially alter grk or osk expression. Nevertheless, the Imp mutant partially suppresses a grk misexpression phenotype, arguing that it does contribute to grk regulation but may act redundantly and does not have an essential role. Consistent with this interpretation, overexpression of Imp interferes with localization of grk mRNA (Geng, 2006).

Deployment of proteins that control patterning in the oocyte relies on coordinated programs of mRNA localization and translational control. Many RNA binding proteins contribute to these programs, and some interact with one another in regulatory RNPs. This study has shown that Imp is associated in an RNA-dependent manner with Sqd and Hrp48 and is thus part of a complex whose other members have clearly established roles in control of grk and osk expression. Imp does not have an essential role in regulation of either grk or osk mRNAs; both mRNAs are expressed with no obvious defects in Imp mutant ovaries. However, loss of Imp activity does partially suppress the grk misexpression defect in fs(1)K10 mutant oocytes, providing strong evidence that Imp contributes to regulation of grk. This view is reinforced by the colocalization of Imp with grk mRNA in vivo. Imp's role must be largely redundant, only becoming detectable when grk expression is perturbed. Overexpression of Imp has a much more dramatic effect, transiently blocking the dorsal localization of grk mRNA and disrupting localization and translational control of osk mRNA (Geng, 2006).

Squid/hnRNP helps Dynein switch from a gurken mRNA transport motor to an ultrastructural static anchor in sponge bodies

In Drosophila oocytes, gurken mRNA localization orientates the TGF-α signal to establish the anteroposterior and dorsoventral axes. This study has elucidated the path and mechanism of gurken mRNA localization by time-lapse cinematography of injected fluorescent transcripts in living oocytes. gurken RNA assembles into particles that move in two distinct steps, both requiring microtubules and cytoplasmic Dynein. gurken particles first move toward the anterior and then turn and move dorsally toward the oocyte nucleus. Evidence is presented suggesting that the two steps of gurken RNA transport occur on distinct arrays of microtubules. Such distinct microtubule networks could provide a general mechanism for one motor to transport different cargos to distinct subcellular destinations (Delanoue, 2007).

This study analyzed the molecular mechanism of grk mRNA transport and anchoring in the Drosophila oocyte using a number of novel methods, combining live cell imaging of oocytes with immunoelectron microscopy to covisualize grk RNA and transacting factors. grk mRNA is transported in particles containing many individual RNA molecules assembled with numerous molecules of Dynein motor components and Squid. Approximately two thirds of transport particles are in close association with MTs and are not consistently associated with membranes, such as ER or vesicles. This supports the idea that grk RNA particles are transported directly by motors on MTs. This notion is strengthened by the fact that the directed movement of the transport particles is disrupted very rapidly when MTs are depolymerized and Dhc, BicD, or Egl function is inhibited. Furthermore, the particles observed moving along MTs in live cell imaging experiments correspond well to the similar-sized grk RNA-rich particles that were visualized by EM. The direct movement of grk RNA particles along MTs is in stark contrast to the transport of yeast ASH1 RNA, which is thought to be cotransported with ER membrane (Delanoue, 2007).

Once delivered to its final destination at the oocyte dorso-anterior corner, many copies of both injected grk RNA and endogenous grk mRNA are anchored in large electron-dense structures previously described as sponge bodies, together with the same components present in the transport particles, including Dynein and Squid. Sponge bodies are distinct in appearance from transport particles and have been previously described in nurse cells and hypothesized to be RNA transport intermediates from the nurse cells to the oocyte. Although Exu-GFP was found in grk anchoring structures, these structures have been identified in the oocyte as functioning in anchoring, rather than transport, and containing components of the Dynein complex Dhc, Egl, and BicD. These data show that the endoplasmic reticulum is not involved in the transport and anchoring of grk mRNA (Delanoue, 2007).

Transport particles and sponge bodies are related to RNA particles (also termed germinal granules, P bodies, and neuronal granules) that display a large spectrum of sizes, composition, and morphology, reflecting several functions in RNA transport, storage, translational control, and processing. Nevertheless, it seems likely that the transport particles that were identified are related to bcd and osk mRNA granules as well as to neuronal RNA granules. The data demonstrate that sponge bodies play key roles in RNA anchoring, but it is not known whether they are also involved in translational control, degradation, and storage of mRNA (Delanoue, 2007).

Dynein is not only present in the sponge bodies but is also required for the static anchoring of grk RNA in the sponge bodies. However, anchoring does not require Egl or BicD, the motor cofactors that are required for RNA transport in the embryo and oocyte. While it is not certain whether Egl and BicD are required for cargo loading, transport initiation, or motor activity itself, the evidence shows that none of these functions are required for anchoring. grk RNA is therefore anchored by a similar mechanism to pair-rule and wingless transcripts in the syncytial blastoderm embryo. In both the embryo and oocyte, it is proposed that when the Dynein motor complex reaches its final destination, the motor becomes a static anchor that no longer depends on the transport activity of the motor (Delanoue, 2007).

Dynein (but not Egl and BicD) is not only a static anchor but is also required for the structural integrity of the grk RNA anchoring structures in the oocyte, the sponge bodies. Their rapid speed of disassembly upon Dynein inhibition (3-5 min) argues that Dynein has a direct role in anchoring and is required to form and maintain the large RNP complexes that constitute the sponge bodies. This evidence rules out that Dynein could indirectly be required for the delivery of anchoring components that are then used in anchoring grk when it is delivered to the sponge bodies. Dynein could also tether the cargo complex directly on the MTs when the transport particles reach their final destination, but the results show that MTs only flank the sponge bodies and are not, as predicted by this model, consistently interdigitated with most of the cargo and motor molecules that are detected in the sponge bodies. This is also consistent with the fact that disassembling MTs does not lead to a change in sponge body structure and only leads to a partial loss of endogenous grk mRNA anchoring (Delanoue, 2007).

In addition to its previously documented role in the second step of grk mRNA transport, a novel function was identified for hnRNP Squid, which plays an essential role in the anchoring of grk RNA at the dorso-anterior corner. Like Dynein, Sqd is also enriched at the site of anchoring upon injection of excess grk RNA. Inactivation of Sqd before transport begins leads to grk transport particles being present at the anterior of the oocyte in permanent anterior flux without anchoring, even for the particles that reach the dorso-anterior corner. Conversely, inactivation of Sqd after grk RNA arrives at the dorso-anterior corner leads to a breakdown of anchoring and the conversion of sponge bodies into anterior transport particles containing grk RNA. This suggests an active role for Sqd in keeping anchoring structures intact, and most likely a role for Sqd in promoting the conversion of transport particles into anchoring structures by facilitating their reorganization into anchoring complexes (Delanoue, 2007).

It is proposed that sponge bodies are assembled at the dorso-anterior corner by delivery of grk mRNA, Dynein motor component and Squid present together in transport particles. First, the same components are present in the transport particles and in the sponge bodies. Second, some transport particles containing endogenous grk mRNA are detected on dorso-anterior MTs. Third, injection of a large excess of grk RNA leads to an increase in the size and number of sponge bodies (Delanoue, 2007).

At the dorso-anterior corner, sponge bodies are maintained by both Dynein and Squid. When the Dynein motor complex reaches its final destination, the motor becomes a static anchor that no longer depends on the transport activity of the motor. Given the size of Dhc and the presence of many putative domains whose function remains elusive but that could play a central role in the switch from motor to anchor, it is proposed that Dhc can associate with many other cellular factors to form a large and immobile anchoring complex. Sqd is known to be involved in translational regulation, and it is proposed that association with this class of factors could help create a large and immobile anchoring complex. A strong link between molecular motors and hnRNPs has already been shown to control the localization of their associated mRNAs. For instance, She2 hnRNP acts as a linker between the Myosin motor complex and its mRNA cargo. Kinesin and hnRNP are present together in RNA-transporting granules in neurons. It is proposed that transport and anchoring of mRNA by molecular motors involve assembly into transport particles followed by reconfiguration of the same components into large electron-dense anchoring complexes at the final destination. Future work will establish how widely this model can be applied. It will also be interesting to determine whether the specificity of transport and anchoring of other RNA cargos in the Drosophila oocyte and embryo is also established by distinct combinations of RNA-binding proteins that influence the function of molecular motors. Time will tell what proportion of mRNA is anchored like pair-rule and grk transcripts by static functions of molecular motors, as opposed to other possible mechanisms of anchoring (Delanoue, 2007).

Factors affecting Gurken RNA localization and translation: the Spindle genes

During wild-type oogenesis, the two cells in each germline cyst appear to be equivalent: these are the progeny of the first division of the cystoblast, derived from asymmetric division of a germ-line stem cell. Both cells enter meiosis to become pro-oocytes in region 2a of the germarium. In region 2b, one of these two cells is selected to develop as the oocyte and remains in meiosis, while the other exits meiosis and reverts to the nurse cell pathway of development. The event gives rise to the first asymmetry in egg development, the selection of one of two cells to become the oocyte. Later in oogenesis, anterior-posterior polarity originates when the oocyte comes to lie posterior to the nurse cells and signals through the Gurken/Egfr pathway to induce the adjacent follicle cells to adopt a posterior fate. This directs the movement of the germinal vesicle and associated Gurken mRNA from the posterior to an anterior corner of the oocyte, where Gurken protein signals for a second time to induce the dorsal follicle cells, thereby polarizing the dorsal-ventral axis. A group of five genes, the spindle loci, is described which are required for each of these polarizing events. The five spindle genes were originally identified in a screen for maternal-effect mutants on the third chromosome because homozygous mutant females lay ventralized eggs. One spindle gene has been described in detail (Gonzalez-Reyes, 1997).

Mutations in spn-E (also known as homeless) give rise to an oocyte displacement phenotype, but also affect the oocyte cytoskeleton and mRNA localization, even when the oocyte is at the posterior of the egg chamber. spn-E encodes a DEAD box protein that is likely to function as an RNA helicase. Double spindle mutants cause both pro-oocytes to develop as oocytes, by delaying the choice between these two cells. spindle mutants inhibit the induction of both the posterior and dorsal follicle cells by disrupting the localisation and translation of Gurken mRNA. The transient mislocalization of Gurken mRNA to an anterior ring in spn mutant stage 9 egg chambers is very similar to the mislocalization of Gurken mRNA that is observed in fs(K10) mutants. However, K10 mutations produce a dorsalization of the egg chamber rather than a ventralisation, because the mislocalization of Gurken mRNA directs Gurken signaling to the follicle cells on all sides of the oocyte. In different spindle mutants, from 19% to 100% of egg chambers show a strong reduction or a complete absence of Gurken protein in the oocyte membrane. The oocyte often fails to reach the posterior of mutant egg chambers and differentiates abnormally. This analysis of spindle phenotypes suggests that these genes are likely to be involved in the localization and/or translation of Gurken mRNA without having any discernible effect on the Gurken mRNA level, but dramatically, reduced amounts of Gurken protein are produced. K10 mutants cause a similar mislocalization of Gurken mRNA without significantly affecting protein expression. Thus, spindle mutants reveal a novel link between oocyte selection, oocyte positioning and axis formation in Drosophila, leading to a proposal that the spindle genes act in a process that is common to several of these events (Gonzalez-Reyes, 1997).

The predominant phenotype produced by females mutant for okra, spindleB, and spindleD is a ventralization of the eggshell, reflected in a loss of dorsal appendage material that is similar to the phenotype produced by mutations in the grk-Egfr pathway. However, unlike grk and Egfr alleles, which produce fairly discrete ventralized phenotypes, all alleles of okr, spnB, and spnD produce a broad spectrum of ventralization. The mutant females also produce eggshell phenotypes that are not observed in grk-Egfr mutants, including dorsalized eggs with extra appendage material or multiple appendages as well as small eggs, although these phenotypes are comparatively rare. The majority of the eggs produced by okr, spnB, and spnD mutant females, including those that are only mildly ventralized, do not hatch and show no indication of embryonic development. In addition to the dorsal-ventral patterning defects observed in eggshells, okra mutants share another phenotype with mutants in the grk-Egfr signaling pathway: they produce eggs that often have a second micropyle at the posterior end. Thus okra mutation effects both dorsal-ventral and anterior-posterior patterning defects. These defects are accompanied by defective distribution of Gurken mRNA. GRK mRNA is normally localized to the oocyte during the early stages of oogenesis, and then, in mid-oogenesis, it is localized within the oocyte, first transiently in an anterior-cortical ring (stage 8), and then to a dorsal-anterior patch overlying the oocyte nucleus (stages 9 and 10). In okr mutant ovaries, GRK mRNA is correctly localized to the oocyte in early stages. However, in mid-oogenesis, instances of persistent localization of the RNA in an anterior-cortical ring are observed. The spnB and spnD mutant phenotypes are more severe. In okr mutant ovaries, levels of Grk are variably reduced throughout oogenesis. Thus defects in dorsal-ventral and anterior-posterior patterning can be explained by defective GRK mRNA distribution in okra, spnB and spnD mutants (Ghabrial, 1998).

The effects of okr, spnB, and spnD on Grk accumulation in the oocyte place these genes upstream of grk in the genetic hierarchy controlling dorsal-ventral patterning in the egg chamber. To assess the relationship between okr, spnB, and spnD and a different class of genes in the patterning hierarchy that are required for the localization of grk RNA and which produce dorsalizing phenotypes, the phenotypes produced by double mutants with K10 were examined. For all three double-mutant combinations, it was found that rather than producing all ventralized or all dorsalized eggs, the mutant females produce a broad spectrum of phenotypes ranging from completely dorsalized to completely ventralized. Given that these experiments do not reveal a simple epistatic relationship between okr, spnB, spnD, and K10, the three genes must affect Grk activity by a pathway that is at least partially independent of K10. Significantly, as this same spectrum of phenotypes is produced by K10 mutant females that have only one wild-type copy of grk, these results are consistent with a role for these genes in directly affecting the accumulation of Grk in the oocyte (Ghabrial, 1998).

During Drosophila oogenesis, signaling between the germline and the soma leads to the establishment of polarity in the egg and embryo. This process involves the interaction of Gurken, a TGFalpha-like protein, with the Egf receptor (Egfr). In early stage egg chambers, GRK mRNA is present predominantly along the posterior cortex of the oocyte, and in mid stage egg chambers, the GRK transcript becomes tightly localized to the future dorsal anterior corner of the oocyte. This localization of GRK mRNA restricts the distribution of Gurken protein and is critical in defining both the anterior/posterior and dorsal/ventral axes of the egg. The genomic sequence of the grk gene has been determined. By testing the requirement of various fragments of GRK mRNA in the localization process, localization signals have been found to be present in both the 5' and 3' regions of the gene. Sequences in the 5' noncoding region allow for accumulation of the transcript within the oocyte in early stage egg chambers, while signals in the coding region and the 3'UTR are necessary for localization in mid to late stage egg chambers. Active translation is not required for localization of the GRK mRNA. The mechanism of Gurken mRNA localization, therefore, differs from that of other localized RNAs studied to date (Thio, 2000).

The function of the GRK 3'UTR in GRK mRNA localization is likely mediated by the Sqd protein. sqd encodes an hnRNP protein and is required for the proper localization of GRK mRNA to the dorsal anterior corner. In females mutant for sqd, an anterior ring of GRK RNA is detected in the oocyte at stages 9 and 10. This expression pattern is the same as that seen with the transgenes that lack the 3'UTR. Interestingly, Sqd has been shown to bind to the GRK 3'UTR. In the absence of either Sqd protein or Sqd binding sites (i.e., the GRK 3'UTR), the Sqd-GRK mRNA complex is not formed, and the final step of GRK localization to the dorsal anterior corner fails to be completed. The formation of this complex has been proposed to be required for GRK mRNA to be properly transported and delivered to an anchor in the ooplasm at the dorsal anterior corner. The 3'UTR of GRK is necessary for Sqd binding and, consequently, for GRK mRNA localization in the late stages of oogenesis. GRK mRNA remains in a translationally repressed state until it is localized (Thio, 2000 and references therein).

After stage 7, all data are consistent with the oocyte nucleus being the primary site of gurken transcription. However, the G5U-L transcript (containing the 5'UTR fused to lac-Z and expressed under the control of the gurken promoter) could not be detected after stage 7; this may be due to its dispersal and/or instability in the rapidly growing oocyte after that stage. Only when additional domains corresponding to part of the coding region of the GRK mRNA were added could the transcript be detected after stage 7. It then accumulated in the oocyte. Interestingly, RNA made from a transgene containing the same additional domains of GRK but lacking sequences in the 5'UTR was also not detected after stage 7. It has been concluded that the 5'UTR contains a stability element. Therefore, regardless of whether GRK mRNA is transcribed in the follicle cells or the oocyte, GRK mRNA appears to contain at least two elements in its 5' region that affect accumulation of its transcript during the late stages of oogenesis: one in the 5'UTR and another in the 5' coding region. The presence of both regions ensures that detectable levels of the transcript are present in the oocyte after stage 7 (Thio, 2000 and references therein).

The particular element that was found within the coding sequences of GRK allows for the stable accumulation of GRK RNA in an anterior cortical ring (ACR element). Since the extracellular domain is one of the less conserved parts of TGFalpha proteins and has no significant conservation of particular residues, this region provides a convenient location for such a signal. It is presently unknown what particular cellular structure would provide an anchor for the GRK mRNA in a cortical ring. It is also not clear whether the association of such a cortical structure would occur through direct association with the ACR element or with a different 5' region of the transcript, where the ACR sequences would simply provide stability to the localized RNA. Further experiments moving the ACR region to a heterologous transcript may distinguish between these possibilities (Thio, 2000).

The COP9 signalosome (CSN) is linked to signaling pathways and ubiquitin-dependent protein degradation in yeast, plant and mammalian cells, but its roles in Drosophila development are just beginning to be understood. During oogenesis, one subunit of the CSN [COP9 complex homolog subunit 5 (CSN5/JAB1)], is required for meiotic progression and for establishment of both the AP and DV axes of the Drosophila oocyte. CSN5 mutations block the accumulation of the Egfr ligand Gurken in the oocyte, interfering with axis formation. CSN5 mutations also cause the modification of Vasa, which is known to be required for Gurken translation. This CSN5 phenotype (defective axis formation, reduced Gurken accumulation and modification of Vasa) is very similar to the phenotype of the spindle-class genes that are required for the repair of meiotic recombination-induced DNA double-strand breaks. When these breaks are not repaired, a DNA damage checkpoint mediated by mei-41 is activated. Accordingly, the CSN5 phenotype is suppressed by mutations in mei-41 or by mutations in mei-W68, which is required for double strand break formation. These results suggest that, like the spindle-class genes, CSN5 regulates axis formation by checkpoint-dependent, translational control of Gurken. They also reveal a link between DNA repair, axis formation and the COP9 signalosome, a protein complex that acts in multiple signaling pathways by regulating protein stability (Doronkin, 2002).

Establishment of both AP and DV polarity requires expression of the TGF-alpha homolog Gurken in the oocyte and activation of the Egf receptor and its downstream effectors in the adjacent follicle cells. CSN5 is required in the germline for these critical signaling events. Several results tie CSN5 to Grk-Egfr signaling: (1) CSN5 mutations affected both axes as shown by DV defects in the eggshell, mislocalization of bcd and osk RNAs in both the oocyte and embryo, and mislocalization of dpp, rho and twi expression in the embryo; (2) CSN5 germline clones affect the expression of follicle cell reporters for Grk-Egfr signaling: slbo and the PZ6356 enhancer trap in the posterior follicle cells, kek expression in the dorsal anterior follicle cells; (3) CSN5 alleles show strong genetic interactions with grk alleles, and (4) Grk protein is reduced in CSN5 germline clones, starting in region 2a of the germarium but still evident in stage 10 egg chambers or in ovary extracts (Doronkin, 2002).

Previous studies have shown that the accumulation of Grk protein can be affected by activation of a meiotic checkpoint in response to the persistence of DNA double-strand breaks. Mutations in several genes that play a role in DNA repair (okra, spn-B, spn-C and spn-D) activate this meiotic checkpoint and disrupt axial patterning in the oocyte. There is a remarkable similarity between the CSN5-mutant phenotype and defects caused by mutations in these spindle-class genes. In both cases mutant females produced eggs with a variety of partially penetrant eggshell defects: mild or strongly ventralized, dorsalized, or small eggs or eggs with multiple dorsal appendages. Embryonic patterning is also disrupted, and both axes are affected. As had been seen in spindle-class mutants, the oocyte of some CSN5-mutant egg chambers is positioned laterally or at the anterior end, and some have defects in karyosome morphology. There is also a similar, strong reduction in Grk protein, with one intriguing difference. At early stages of oogenesis in CSN5 mutants, the level of Grk protein is always strongly reduced, both in germline clones of the strong CSN5L4032 allele and in hypomorphic combinations of CSN5L4032 with viable excision mutants. Although Grk is also strongly reduced in CSN5L4032 germline clones at later stages, it often appears to be present at higher levels than in the germarium. With the hypomorphic combinations, it is often difficult to detect any reduction in Grk protein at later stages. By contrast, in spn-B and spn-D mutants, Grk accumulates normally in early oogenesis but then declines and is often undetectable by stage 9-10. In okr mutants, the amount of Grk protein varies from one egg chamber to the next in a single ovariole, but a bias towards lower levels at early stages has not been reported. Thus, there seem to be three different patterns of Grk accumulation in these mutants. CSN5 mutants appear to cause a more immediate response of Grk to DNA damage than do spn-B and spn-D mutants (Doronkin, 2002).

Because of the similarities between the phenotypes and because at least two of the spindle-class genes, okr and spn-B, encode components of the RAD52 DNA repair pathway, it seems likely that CSN5 directly or indirectly regulates DSB repair. The fact that mei-41 and mei-W68 mutations can suppress the CSN5 phenotypes reinforces this conclusion. Kinases in the ATM/ATR subfamily, which includes Mei-41, play a central role in checkpoint-mediated responses to DNA damage. These checkpoint kinases are thought to act as sensors of DNA damage, becoming activated on binding damaged DNA. Phosphorylation of several downstream effectors, including the Chk1 and Chk2 (Drosophila homolog: loki) kinases and p53, then restrains cell cycle progression until the DNA damage is repaired and the checkpoint kinases dissociate from the DNA. In Drosophila mei-41 mutants, the checkpoint cannot be activated, and oocytes with damaged DNA, such as those mutant for spindle-class genes, can proceed through oogenesis. Suppression of CSN5 phenotypes by mei-41 mutations demonstrate that the CSN5-mutant lesion acts upstream of the DNA damage checkpoint and suggest that DSBs arising during meiotic recombination cannot be efficiently repaired in CSN5-mutant cells (Doronkin, 2002).

Suppression by mei-W68 restricts the possible role of CSN5 further. mei-W68 encodes a topoisomerase II-like protein homologous to S. cerevisiae Spo11 and has been proposed to create the DSBs needed to initiate meiotic recombination. In flies mutant for mei-W68, DSBs are absent and meiotic recombination is eliminated. In double mutants of mei-W68 with either okr, spn-B or spn-C, Grk protein accumulation and eggshell patterning are normal and other spindle-class defects are suppressed. Heterozygosity for mei-W68 is sufficient to suppress hypomorphic CSN5-mutant phenotypes. Combination of this result with the mei-41 suppression result indicates that CSN5 acts in the recombination pathway to regulate the formation of DSBs or their successful repair (Doronkin, 2002).

During Drosophila oogenesis, unrepaired double-strand DNA breaks activate a mei-41-dependent meiotic checkpoint, which couples the progression through meiosis to specific developmental processes. This checkpoint affects the accumulation of Gurken protein, a transforming growth factor alpha-like signaling molecule, as well as the morphology of the oocyte nucleus. However, the components of this checkpoint in flies have not been completely elucidated. A mutation in the Drosophila Chk2 homolog (DmChk2/Mnk) has been shown to suppress the defects in the translation of gurken mRNA and also the defects in oocyte nuclear morphology. Drosophila Chk2 is phosphorylated in a mei-41-dependent pathway. Analysis of the meiotic cell cycle progression shows that the Drosophila Chk2 homolog is not required during early meiotic prophase, as has been observed for Chk2 in C. elegans. The activation of the meiotic checkpoint affects Wee localization and is associated with Chk2-dependent posttranslational modification of Wee. It is suggested that Wee has a role in the meiotic checkpoint that regulates the meiotic cell cycle, but not the translation of gurken mRNA. In addition, p53 and mus304, the Drosophila ATR-IP homolog, are not required for the patterning defects caused by the meiotic DNA repair mutations. It is concluded that Chk2 is a transducer of the meiotic checkpoint in flies that is activated by unrepaired double-strand DNA breaks. Activation of Chk2 in this specific checkpoint affects a cell cycle regulator as well as mRNA translation (Abdu, 2002).

Mutations in the spindle class of double-strand break (DSB) DNA repair enzymes, such as spn-B (DMC/RAD51-like) and okr (Dmrad54), affect meiosis and dorsal-ventral patterning in Drosophila oogenesis. These mutations activate a mei-41-dependent meiotic checkpoint. mei-41 encodes a member of the ATM/ATR subfamily of phosphatidylinositol-3-OH-kinase-like proteins. Activation of the mei-41-dependent checkpoint prevents efficient translation of gurken (grk) mRNA, which results in a ventralization of eggs and embryos. The effect on grk is presumably mediated through posttranslational modification of Vasa, an eIF4A-like translation initiation factor. In addition, the oocyte nuclear morphology is abnormal in the spindle mutants. In flies homozygous for mutations in a DSB repair enzyme and in mei-41, the patterning and the oocyte nuclear defects are suppressed, demonstrating that a defect in the nucleus affects Vasa and Grk in the cytoplasm via activity of mei-41. However, how activation of mei-41 causes patterning defects is not fully understood (Abdu, 2002).

To test whether the production of patterning defects by mutations in spindle-class genes is dependent on Chk2, double mutant flies were generated for spn-B, spn-D or okra, and Chk2. A null mutation in Chk2 completely suppressed the dorsal-ventral patterning defects. In double mutant flies, a dramatic increase is observed in the accumulation of Grk protein compared to spn-B single mutants, and an increase is observed in the restoration of dorsal-ventral patterning in the eggshell. A 100% suppression of oocyte nuclear defects is also observed. In wild-type egg chambers, the oocyte nuclear membrane is round and the DNA is located in the center of the nucleus. In contrast, in spn-B mutant egg chambers, the oocyte nuclear DNA is found in a variety of conformations, including wild-type shape (17%), oblong shape (34%), or smaller distinct clumps (49%). In about 90% of the egg chambers, the DNA seems to be attached to the oocyte nuclear membrane. In addition, the morphology of the oocyte nuclear membrane in the mutant egg chambers is also found in a variety of shapes, including round (59%), distorted (32%), or strongly distorted (9%). In egg chambers from the spn-B and Chk2 double mutant flies, the oocyte nuclear membrane and the appearance of the DNA within the oocyte are restored to wild-type (Abdu, 2002).

It was further observed that the posttranslational regulation of Vasa by mei-41 is also dependent on Chk2. Western blot analysis has demonstrated that the altered Vasa protein mobility seen in spn-B ovarian lysates is restored to wild-type in the double mutant flies for spn-B and Chk2. It is concluded that, in Drosophila, activation of Chk2 in the meiotic checkpoint regulates the translation of grk mRNA, apparently through the translational regulator Vasa. However, at this point, it is still not clear how Chk2 may affect translation via Vasa. It was recently reported that Chk2 physically interacts with the Drosophila RNA binding protein Orb (Iwai, 2002). Orb plays roles in localization and translation of several maternal mRNAs, including gurken mRNA. It is therefore possible that Chk2 can directly interact with the translation machinery and can lead to modification of Vasa (Abdu, 2002).

In summary, the results demonstrate that the Drosophila Chk2 homolog is a transducer of the meiotic checkpoint that is activated by unrepaired double-strand DNA breaks. Activation of Chk2 results in modification of two proteins, Vasa and Wee, which then affect progression of the meiotic cell cycle and translation of gurken mRNA. Wee is, however, not required for the patterning defects seen in the spindle mutations. Activation of the Chk2-dependent meiotic checkpoint may therefore control several cell cycle regulators which in turn may affect both meiosis and translation of gurken mRNA. In particular, it is likely that Wee1 activation regulates cell cycle progression, whereas Chk2 may utilize an independent target to regulate Vasa, which subsequently affects dorsal-ventral patterning as well as nuclear morphology of the oocyte. While dorsal-ventral signaling by Gurken is not a conserved feature of oogenesis found in other organisms, the fact that homologs of Drosophila Chk2 act during meiosis in other organisms raises the possibility that meiotic checkpoints in other species might also act through Chk2 to regulate translation during oogenesis and thus directly link the meiotic cell cycle to the development of the oocyte (Abdu, 2002).

The axial patterning defects displayed by maelstrom hypomorphs are fully penetrant in oocytes of the maelstrom null. AP axis determination in the Drosophila oocyte is a multistep process, the first known step of which is the establishment of microtubule-mediated cytoplasmic polarity in the stage 2 oocyte. This asymmetry, which is defective in spn mutants such as spn-A, spn-B and vasa, is a likely prerequisite for efficient Gurken signaling from the oocyte to the follicle cells overlying the posterior oocyte. A number of RNAs and proteins accumulate in the posterior of the wild-type oocyte during stages 2-6 in a distribution that both requires and reflects the oocyte polarity in this interval. Polarity was assayed in the oocyte indirectly by monitoring the localization of Bicaudal D [BicD and multiple RNAs including grk, osk, bicD and oo18 RNA binding (orb)]. In normal stage 5/6 oocytes, BicD forms a distinct gradient emanating from the posterior oocyte cortex. In maelstrom oocytes, although BicD is present at levels comparable with wild type, a wild-type gradient is not established. Instead, about half of stage 5/6 maelstrom oocytes show BicD in a diffuse or only vaguely polarized distribution. In the remaining oocytes, the marker forms a randomly localized focus within the ooplasm. Similarly, the normally polarized distribution of grk and other RNAs is lost in maelstrom oocytes. Gurken protein distribution in wild-type oocytes is comparable with that of BicD, albeit more punctate in appearance. In maelstrom oocytes, not only is the gradient lost, but Gurken levels are either highly reduced (~50%) or undetectable (~50%). The Gurken defect is probably sufficient to account for the observed polarity defects in mid- to late-stage maelstrom oocytes, in which variety of polarity markers, including multiple mRNAs (e.g. osk) and proteins (including Staufen, Oskar, Vasa), fail to accumulate in the posterior ooplasm. Dorsal appendages are also invariably vestigial or absent in the maelstrom null. The failure in establishing AP polarity in the early oocyte, together with reduction in Gurken accumulation, the DV phenotypes of the null and hypomorph, and failure to proceed to karyosome stage collectively puts maelstrom in the spindle-class of mutants (Findley, 2003).

spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila

The anteroposterior and dorsoventral axes of the Drosophila embryo are established during oogenesis through the activities of Gurken (Grk), a Tgfα-like protein, and the Epidermal growth factor receptor (Egfr). spn-F mutant females produce ventralized eggs similar to the phenotype produced by mutations in the grk-Egfr pathway. The ventralization of the eggshell in spn-F mutants is due to defects in the localization and translation of grk mRNA during mid-oogenesis. Analysis of the microtubule network revealed defects in the organization of the microtubules around the oocyte nucleus. In addition, spn-F mutants have defective bristles. spn-F was clond and found to encodes a novel coiled-coil protein that localizes to the minus end of microtubules in the oocyte, and this localization requires the microtubule network and a Dynein heavy chain gene. Spn-F interacts directly with the Dynein light chain Ddlc-1 (Cut up). These results show that this novel protein affects oocyte axis determination and the organization of microtubules during Drosophila oogenesis (Abdu, 2006; full text of article).

In a global two-hybrid screen, Spn-F (CG12114) was found to interact with the Ik2 (CG2615) protein. Mutations in Ik2 have been isolated and characterized, ik2 mutants share many phenotypes with spn-F, including a very similar bristle phenotype and specific effects on MT organization in oogenesis. However, ik2 mutants are lethal, whereas spn-F homozygotes survive. In addition, whereas spnF mutations have only mild effects on Oskar protein localization and a low frequency of bicaudal phenotypes, such effects are more pronounced in the ik2 mutants. Nevertheless, the striking similarities strongly suggest that the two genes function in a common pathway that affects certain types of MT more strongly than others. In normal mitotic cells, the minus ends of MTs are usually focused by the centrosomes in the interior of the cell, and plus ends contact the cortex. However, in specialized cells, such as the Drosophila oocyte, there are minus ends that make contact with the cortex. It is therefore possible that Spn-F and Ik2 are required for providing a stable connection between such cortical MT minus ends and cortical actin for subsets of MTs involved in specialized transport processes. Future experiments will address the interactions of Spn-F and Ik2 directly, and will determine whether, for instance, Spn-F might be a target of Ik2 (Abdu, 2006).

Vasa involvement in Gurken mRNA localization and translation

Localization of specific mRNAs to distinct sites within the Drosophila oocyte is an early and key step in establishing the anterior-posterior and dorsal-ventral axes. A new function is described for the RNA helicase encoded by the "posterior" group gene vasa in the control of the localization of Gurken mRNA, a "dorsal-ventral" patterning gene. Two new ethyl methane sulfonate-induced, female sterile alleles of vas have been isolated. In these mutants, GRK mRNA fails to become localized properly and Grk protein is barely detectable. These mutants result in the ventralization of the eggshell, resembling defects of hypomorphic grk and Egfr mutants. Surprisingly, fs(1)K10, a recessive female sterile mutation that results in mislocalization of GRK mRNA to the anterior end of the oocyte, is epistatic to these vas alleles. In other words, fs(1)K10:vas double-mutants are dorsalized, just like fs(1)K10 alone. This result demonstrates that Grk protein levels sufficient to dorsalize the egg chamber can accumulate in vas mutants, if fs(1)K10 is also mutant. Taken together these results suggest that regulation of GRK mRNA localization normally occurs, directly or indirectly, through the Vas RNA-dependent RNA helicase and suggest that accumulation of Grk protein may normally depend on GRK mRNA localization (Tinker, 1998).

Several vasa alleles exhibit a wide range of early oogenesis phenotypes. A detailed analysis of Vasa function during early oogenesis is reported using novel as well as previously identified hypomorphic vasa alleles. vasa is required for the establishment of both anterior-posterior and dorsal-ventral polarity of the oocyte. The polarity defects of vasa mutants appear to be caused by a reduction in the amount of Gurken protein at stages of oogenesis critical for the establishment of polarity. Vasa is required for translation of Gurken mRNA during early oogenesis and for achieving wild-type levels of Gurken mRNA expression later in oogenesis. A variety of early oogenesis phenotypes observed in vasa ovaries, which cannot be attributed to the defect in gurken expression, suggest that vasa also affects expression of other mRNAs (Tomancak, 1998).

While the activities of pole plasm components such as Vasa have been most thoroughly studied with respect to their function in pole cell formation and specification of the posterior soma, it is clear that some genes involved in pole plasm assembly also function in other stages of germline development. For instance, females homozygous for either of two strong nanos alleles exhibit defects in germ cell proliferation. Furthermore, pole cells lacking maternal nos function fail to complete migration and do not associate with the embryonic gonadal mesoderm, indicating a role for nos in the transition from pole cell to functional germ cell. Similarly, various vas alleles have defects in oogenesis and lay few or no eggs. Females trans-heterozygous for vas deficiency alleles, are blocked in early vitellogenic stages of oogenesis. Analysis of whether this phenotype is caused solely by loss of vas function has been confounded by the fact that these trans-heterozygous deficiency lines are haploid for a large number of genes; however, aside from large deficiencies, a clearly null allele of vas had not been identified. A new vas null allele, vas PH165, was generated by imprecise P-element excision, to investigate in detail the role of vas in oogenesis events prior to pole plasm assembly. Abrogation of vas function results in defects in many aspects of oogenesis, including control of cystocyte divisions, oocyte differentiation, and specification of posterior and dorsal follicle cell-derived structures. In vasa-null ovaries, germaria are atrophied, and contain far fewer developing cysts than do wild-type germaria. This is a phenotype similar to (but less severe than) that of a null nanos allele. vas PH165 oocytes only weakly concentrate many oocyte-localized RNAs, including Bicaudal-D, Orb, Oskar, and Nanos mRNA. Some oocyte-specific molecules, including Gurken RNA, remain concentrated in the oocyte in vas mutant ovaries. However, in the case of Grk, translation is severely reduced in the absence of vas function. This provides evidence that Vasa is involved in translational control mechanisms operating in the early stages of oogenesis (Styhler, 1998).

The vas PH165 mutant is null for vasa. At a low frequency (approx. 1% for each), defects are observed in germline differentiation and oocyte determination in vas PH165 mutant ovaries, including tumorous egg chambers, egg chambers with 16 nurse cells and no oocyte, others with two oocytes, and again others with a mislocalized oocyte. Far more frequently the normal 15 nurse cells and one oocyte are present; however, by at least two criteria, the oocytes produced in vas PH165 egg chambers are not fully differentiated. In wild-type development, the nurse cell nuclei endoreplicate during pre-vitellogenic oogenesis and become highly polyploid, whereas the oocyte nucleus remains diploid and condenses into a tight karyosome. However, in vas PH165 the oocyte nucleus appears more diffuse than does the wild-type oocyte nucleus. This would be consistent either with a failure to form the karyosome structure, perhaps involving a premature meiotic arrest in diplotene rather than in metaphase, or an increase in ploidy in vas PH165 oocytes. A very similar nuclear morphology has been observed in spindle (spn) mutant oocytes, which has been interpreted as resulting from a delay in oocyte determination (see Homeless). In addition, vas PH165 oocytes do not efficiently accumulate at least four oocyte-localized RNAs. In wild-type ovaries, the Bicaudal-D, ORB, OSK and NOS mRNAs all accumulate efficiently in the oocyte within the germarium and remain concentrated therein throughout the early stages of oogenesis. Oocyte accumulation of these RNAs is much less pronounced in vas PH165 egg chambers in which Bic-D RNA localization is essentially undetectable, and the concentration of ORB RNA in the oocyte is only slightly above the levels in the nurse cells. In vas PH165 egg chambers, OSK RNA is also not observed to accumulate in the oocyte in the germarium or first vitellarial stages of development. Rather, OSK RNA tends to concentrate in a somewhat diffuse manner in the center of the egg chamber. Finally, loss of vas function has the least pronounced effect on the accumulation of NOS RNA into the oocyte, but even in this case oocyte localization is incomplete and poorly maintained (Styhler, 1998).

Gurken translation is severely reduced in the absence of vas function. The phenotypes of late-stage vasa PH165 null mutant oocytes suggest that the Gurken signaling pathway may be inactive in these ovaries. This observation led to an investigation of the expression and distribution of GRK mRNA and protein in vas PH165. Unlike Bicaudal-D, ORB, OSK and NOS mRNA, localization of GRK mRNA to the oocyte remains highly efficient in vas PH165 egg chambers. However, while the distribution of GRK mRNA forms an obvious posterior crescent in wild-type oocytes from about stage 2-3, GRK mRNA remains tightly concentrated in an extremely small area in vas PH165 oocytes. Later in oogenesis, GRK RNA becomes anteriorly localized in both wild-type and vas PH165 oocytes, although in the mutant its distribution may extend further ventrally. Despite the relatively normal accumulation of GRK RNA in vas PH165 oocytes, the effects of a loss of vas activity are striking with regard to Grk protein accumulation: essentially no localized Grk is observed in vas PH165 oocytes. Furthermore, as measured on western blots, the level of Grk protein is greatly reduced in vas PH165 ovaries as compared with wild-type. Since grk is required for specification of dorsal and posterior follicle structures, the duplicated micropyles and dorsal appendage defects found in vas PH165 eggs (and in eggs produced by other vas alleles) are likely to be caused by the reduced level of Grk protein in vas mutants. These results also suggest that Vas may activate GRK translation in wild-type oocytes (Styhler, 1998).

The DEAD-box RNA helicase Vasa (Vas) is required for germ cell development and function, as well as for embryonic somatic posterior patterning. Vas interacts with the general translation initiation factor eIF5B (cIF2, also known as dIF2), and thus may regulate translation of specific mRNAs. In order to investigate which functions of Vas are related to translational control, the effects of site-directed vas mutations that reduce or eliminate interaction with eIF5B were examined. Reduction in Vas-eIF5B interaction during oogenesis leads to female sterility, with phenotypes similar to a vas null mutation. Accumulation of Gurken (Grk) protein is greatly reduced when Vas-eIF5B interaction is reduced, suggesting that this interaction is crucial for translational regulation of grk. In addition, reduction in Vas-eIF5B interaction virtually abolishes germ cell formation in embryos, while producing a less severe effect on somatic posterior patterning. It is concluded that interaction with the general translation factor eIF5B is essential for Vas function during development (Johnston, 2004).

A mutant form of Vas, VasDelta617, was analyzed that has greatly reduced ability to interact with eIF5B. Since residue 617 is not involved in binding to any known Vas-interacting protein other than eIF5B, and it is outside the region of Vas that contains the well-characterized catalytic domains that are present in all DEAD-box proteins, it is clear that VasDelta617 specifically disrupts the Vas-eIF5B interaction, and that this mutation can be used to identify developmental processes that are sensitive to an association between Vas and the general translational machinery. The VaseIF5B interaction is crucial for the progression of oogenesis, for correct dorsoventral patterning of the egg, and for expression of high levels of Grk in the developing oocyte. These results are most easily explained if grk is a target for Vas-mediated translational activation acting through its association with eIF5B (Johnston, 2004).

A role for Vas in positively regulating grk translation is consistent with previous work. Vas-mediated regulation of grk is in turn regulated in response to a meiotic checkpoint, activated when DNA double-strand break (DSB) repair is prevented during meiotic recombination. In response to this checkpoint, Vas is post-translationally modified, and Grk accumulation is reduced. It will be important to understand the nature of the DSB-dependent modification of Vas, and to determine whether it affects the Vas-eIF5B interaction, in order to gain insight into the mechanism connecting cell cycle regulation with oocyte patterning (Johnston, 2004).

The RNA-binding and unwinding activities of wild-type Vas and several mutant forms of Vas have been assessed through previous in vitro assays. Two mutant forms of Vas, encoded by the vasO14 and vasO11 alleles, are severely reduced for binding to an artificial RNA substrate, and a third form, encoded by vasD5, was defective for RNA unwinding but not for binding. Although vasD5 leads to defects in oogenesis, vasO11 phenotypically resembles vasPD, and vasO14 is a weak temperature-sensitive allele. In the light of the present results, it is surprising that a mutant form of Vas that cannot interact with RNA would nevertheless support oogenesis. Perhaps in vivo, the RNA-binding and helicase activities of Vas are stimulated or enhanced through a co-factor or through posttranslational modifications, and the in vitro assay used in an earlier study may not accurately reflect Vas activity in vivo (Johnston, 2004).

Are there target RNAs for Vas-eIF5B regulation in the pole plasm? Reduction of the Vas-eIF5B interaction by expressing VasDelta617 severely reduces pole cell formation. This happens despite the ability of vasPD;P{vasDelta617} oocytes to accumulate Osk, Vas and Tud at the posterior pole, demonstrating an essential role for Vas in pole cell specification that is dependent upon its association with eIF5B, and that cannot be substituted by Osk and Tud. The simplest interpretation of these results is that Vas derepresses translation of a localized RNA required for pole cell specification, in a manner analogous to what appears to be the case for grk (Johnston, 2004).

The possibility is considered that the Vas-eIF5B interaction could target osk mRNA. It has been shown that whereas Osk protein accumulates normally in vas mutant ovaries, Osk levels are severely reduced at the posterior of vas mutant embryos, suggesting a role for Vas in posterior accumulation of Osk after its initial recruitment, and/or in stabilizing Osk at the posterior. Comparable and substantial levels of Osk are observed at the posterior in vasPD;P{vasDelta617} and vasPD;P{vas+} embryos, arguing against a direct role for the VaseIF5B interaction in activating translation of osk mRNA. A requirement for Vas in Par1-mediated phosphorylation and stabilization of Osk has been suggested. Since VasDelta617 localizes normally and is able to interact with Osk, this mutation would not be expected to have any effect on this Osk modification pathway. Thus, the findings are consistent with a model whereby Vas influences Osk activity through effects on phosphorylation, anchoring and/or stability, perhaps through Par1, rather than directly regulating osk translation (Johnston, 2004).

Another candidate target for the Vas-eIF5B interaction is germ cell-less (gcl), the activity of which is important for pole cell specification but not for posterior patterning. Unfortunately, with current reagents Gcl protein cannot be detected even in wild-type embryos prior to pole bud formation, thus the effects on gcl translation of any mutation that abrogates pole cell formation cannot presently be addressed. In addition, effects on gcl cannot fully explain the severe consequences of the VasDelta617 mutation on pole cell formation, because the number of pole cells formed in maternal gcl-null embryos is somewhat higher than in vasPD;P{vasDelta617} embryos. This suggests that even if gcl is a target, the Vas-eIF5B interaction may regulate translation of more than one target RNA involved in pole cell formation (Johnston, 2004).

Is the Vas-eIF5B interaction required for posterior patterning? Although the Vas-eIF5B interaction is vital for pole cell specification, it is perhaps less so for posterior patterning and establishment of the Nos gradient. Previous analysis of hypomorphic mutations in posterior-group genes, including vas has indicated that a higher level of activity is required for pole cell specification than for posterior patterning. For example, all embryos produced by females homozygous for vasO14, osk301 and tudWC, lack pole cells, but some have normal posterior patterning and are able to hatch. The present results suggest two alternative explanations for these observations. One possibility is that the Vas-eIF5B interaction is required for posterior patterning, but that the residual activity present in VasDelta617 is sufficient to achieve the low activity level that is necessary. Alternatively, the Vas-eIF5B interaction may be dispensable for posterior patterning, and the fact that complete rescue of this phenotype is not observed with the vasDelta617 transgene may be due to an indirect effect of this mutation, resulting from a general destabilization of the pole plasm that occurs in embryos that do not form pole cells. In such embryos, pole plasm components localize initially but become fully delocalized by the blastoderm stage. Consistent with this idea, all of the pole plasm components examined that are downstream of Vas, including nos RNA, could be detected at the posterior of vasPD;P{vasDelta617} embryos, although to variable degrees (Johnston, 2004).

Previous work has suggested that nos may be a target for Vas-mediated translational regulation. Outside of the pole plasm, nos translation is repressed through the binding of Smg, and possibly other repressors, to its 3' UTR. Smg achieves this regulation at least in part through interaction with the eIF4E-binding protein Cup, thus influencing the cap-binding stage of translation. Within the pole plasm, in complexes with Osk and Vas, nos translational repression is overcome, potentially through a direct interaction between Osk and Smg that may displace Smg-Cup interaction. Analysis of VasDelta617 does not support an important role for the Vas-eIF5B interaction in activating nos translation in the pole plasm, since it is clear that translation of nos is far less sensitive to the level of this interaction than is translation of grk in early oocytes. The primary function of Vas in nos accumulation may therefore be in anchoring nos mRNA in complexes within the pole plasm, consistent with recent observations that nos mRNA is trapped at the posterior by complexes containing Vas. Of course it remains possible that the low level of residual eIF5B binding provided by VasDelta617 is sufficient to fulfill a role of Vas in activating translation of this transcript (Johnston, 2004).

How might Vas-eIF5B interaction regulate translation of grk and potentially other target mRNAs? Cap-dependent translation initiation in eukaryotes requires many translation initiation factors, and involves several main steps. Most known mechanisms of translational regulation impinge on the recruitment of the cap-binding complex eIF4F to the mRNA: this represents the rate-limiting first step of initiation. mRNA circularization through proteins such as Cup serves an important role in translational control by allowing 3' UTR-bound regulatory factors to influence translation initiation at the 5' end of the transcript (Johnston, 2004).

60S ribosomal subunit joining represents the interface between translation initiation and elongation, and the VaseIF5B interaction suggests a distinct mechanism of translational control occurring at this last stage of initiation. Although this step has not historically been considered a target for regulation, several examples have emerged to suggest that subunit joining may in fact be subject to regulation. Translational repression of mammalian 15-lipoxygenase (LOX) mRNA is mediated by hnRNP proteins that bind to a specific 3' UTR regulatory element, and which are thought to act by blocking the activity of either eIF5 or eIF5B. An additional link between mRNA 3' regulatory regions, and eIF5B activity, comes from analysis of two DEAD-box proteins in yeast, Ski2p and Slh1p. These proteins are required to achieve the selective translation of poly(A)+ mRNAs, relative to poly(A)- mRNAs, and genetic experiments suggest that they specifically repress the translation of poly(A)- mRNAs by acting through eIF5 and eIF5B (Johnston, 2004).

Together with these studies, the current work suggests that in the Drosophila germline, specific translational repression events may target eIF5B and the ribosomal subunit joining step of initiation. Vas, which potentially functions at the 3' UTR through interaction with specific repressor proteins, may act to alleviate a translation block occurring at this step. Such a model is consistent with what is known about translational regulation of grk. For example, grk translation is repressed by Bru, which binds to a Bruno-response element within its 3' UTR. Vas interacts with Bru, suggesting that Vas could function as a derepressor by overcoming Bru-mediated repression of grk translation. However, the inability of a vas transgene to ameliorate the phenotype of nosGAL4VP16-driven overexpression of Bru, might argue against this model. The mechanism by which Bru regulates grk remains unclear. Translational repression of osk by Bru relies on direct interaction with Cup, linking Bru with eIF4E. However, mutations in cup that prevent interaction with Bru do not appear to affect Grk expression, suggesting that Bru may operate through a distinct mechanism to regulate grk translation. In addition, in vitro translation assays have suggested that Bru can mediate translational repression through a cap-independent mechanism. Thus, Bru may be capable of regulating translation at more than one stage. Based on the observations for the mammalian hnRNP proteins on the LOX mRNA, and the Ski2p and Slh1p helicases in yeast, specific translational repressors such as Bru could target the subunit joining step of initiation (Johnston, 2004).

eIF5B is thought to form a molecular bridge between the two ribosomal subunits, and to play a fundamental role in stabilizing the initiator Met-tRNAiMet in the ribosomal P site. Inhibition of eIF5B activity could occur while the factor is bound to the initiation complex, at the start codon, and block its ability to link or stabilize the ribosomal subunits. Through circularization of the mRNA, this block could be achieved by trans-acting factors at the 3' UTR, and the Vas-eIF5B interaction may be involved in alleviating these specific repression events, potentially through displacement of a repressor protein. Alternatively, Vas could play a role in recruitment of eIF5B to specific transcripts. Since eIF5B is required for all cellular translation, a general mechanism must exist to recruit this factor to all transcripts. However, in a scenario where repressor proteins may be blocking the subunit joining step, either through a direct effect on eIF5B, or another mechanism, it is conceivable that eIF5B could become limiting for translation. In this situation, Vas could play a role in recruiting this factor to specific transcripts (Johnston, 2004).

Dorso-ventral axis formation of the Drosophila oocyte requires Cyclin G

In general, cyclins control the cell cycle. Not so the atypical cyclins, which are required for diverse cellular functions such as for genome stability or for the regulation of transcription and translation. The atypical Cyclin G (CycG) gene of Drosophila has been involved in the epigenetic regulation of abdominal segmentation, cell proliferation and growth, based on overexpression and RNAi studies, but detailed analyses were hampered by the lack of a cycG mutant. For further investigations, the cycG locus was subjected to a detailed molecular analysis. Moreover, a cycG null mutant was studied that was recently established. The mutant flies are homozygous viable, however, the mutant females are sterile and produce ventralized eggs. This study shows that this egg phenotype is primarily a consequence of a defective Epidermal Growth Factor Receptor (EGFR) signalling pathway. By using different read outs, it was demonstrated that cycG loss is tantamount to lowered EGFR signalling. Inferred from epistasis experiments, it is concluded that CycG promotes the Grk signal in the oocyte. Abnormal accumulation but regular secretion of the Grk protein suggests defects of Grk translation in cycG mutants rather than transcriptional regulation. Accordingly, protein accumulation of Vasa, which acts as an oocyte specific translational regulator of Grk in the oocyte is abnormal. A role is proposed of cycG in processes that regulate translation of Grk and hence, influence EGFR-mediated patterning processes during oogenesis (Nigel, 2012).

This study has shown the ventralized phenotype of cycG mutant eggs results from a downregulation of the EGFR signalling pathway. CycG is required for the translational rather than the transcriptional regulation of Grk within the oocyte. One may think of several mechanisms through which CycG might influence grk mRNA translation. DroID, a comprehensive resource for gene interactions in Drosophila, identified several protein interaction partners of CycG that may relate to its role as translational regulator of grk mRNA. Notably, CycG was identified in vivo in protein complexes together with RNA binding proteins, several potential splice factors and translational regulators, for example Eukaryotic translation initiation factor 4AIII (eIF4AIII), Bicoid stability factor (Bsf), Barentz (Btz), Cap binding protein 80 (Cbp80), and SC35. Moreover, a large scale yeast-two hybrid screen picked Gustavus (Gus) as a partner of CycG. This interaction gives a direct link to dorso-ventral axis formation, since Gus is required for the correct localization of Vasa in the Drosophila egg. Most interestingly, Vasa protein levels appear reduced in cycGHR7 mutant ovaries. This may suggest that CycG is a cofactor of Gus, which acts on Vasa stability in the oocyte. In the absence of CycG, Vasa may be degraded more rapidly. Since Vasa is required for efficient translation of Grk, downregulation of Vasa could affect Grk accumulation and result in ventralized eggs. Alternatively, CycG may affect Grk translation indirectly. It was shown that a meiotic checkpoint induced by unrepaired double-strand breaks affects efficient translation of Grk, thereby causing a ventralized eggshell phenotype. A typical example are mutants in the spindle-A (spn-A) gene, which encodes a homologue of the Rad51 recombinase and which is required for double-strand break repair. Spn-A and CycG were found as molecular partners in yeast-two hybrid screens, and hence, CycG may in fact be involved in meiotic recombination repair. Finally, mutants affecting the rasi-RNA pathway cause similar ventralized eggs: in these mutants DNA breaks accumulate due to defects in transposon silencing, effecting the meiotic checkpoint as well. Because CycG has been involved in radiation sensitivity in both Drosophila and mammals, it is tempting to speculate that it may be involved in double-strand break repair during meiosis, as well. Hence, in the absence of CycG, meiotic double-strand breaks would accumulate, thereby activating the meiotic checkpoint and indirectly affecting grk mRNA translation and axis formation of the oocyte (Nigel, 2012).

Tsunagi's involvement in Gurken mRNA localization

In Drosophila, formation of the axes and the primordial germ cells is regulated by interactions between the germ line-derived oocyte and the surrounding somatic follicle cells. This reciprocal signaling results in the asymmetric localization of mRNAs and proteins critical for these oogenic processes. Mago Nashi protein interprets the posterior follicle cell-to-oocyte signal to establish the major axes and to determine the fate of the primordial germ cells. Using the yeast two-hybrid system, an RNA-binding protein, Tsunagi, has been identified that interacts with Mago Nashi protein. The proteins coimmunoprecipitate and colocalize, indicating that they form a complex in vivo. Immunolocalization reveals that Tsunagi protein is localized within the posterior oocyte cytoplasm during stages 1-5 and 8-9, and this localization is dependent on wild-type mago nashi function. When tsunagi function is removed from the germ line, egg chambers develop in which the oocyte nucleus fails to migrate, Oskar mRNA is not localized within the posterior pole, and dorsal-ventral pattern abnormalities are observed. These results show that a Mago Nashi-Tsunagi protein complex is required for interpreting the posterior follicle cell-to-oocyte signal to define the major body axes and to localize components necessary for determination of the primordial germ cells (Mohr, 2001).

Database searches reveal that Tsunagi is significantly similar to the ribonucleoprotein (RNP) family of RNA-binding proteins with a single RNP domain and has been evolutionarily conserved. Homologs for Tsunagi exist from the fission yeast Schizosaccharomyces pombe to humans. As with Mago and its homologs, a Tsunagi-related protein is not detectable within the genome of the budding yeast Saccharomyces cerevisiae. The primary structure of Tsunagi is reminiscent of the SR family of splicing factors. Classic SR proteins contain at least one RNP domain followed by carboxy-terminal arginine/serine (RS) dipeptide residues. Each of the presumed Tsunagi homologs exhibits a series of basic residues at their carboxyl terminus, and within this region the vertebrate and yeast Tsunagi contain at least one RS-dipeptide repeat. However, the D. melanogaster and Caenorhabditis elegans proteins do not contain any RS sequences among the basic residues at their carboxyl termini (Mohr, 2001).

Drosophila Tsunagi is 60% identical and 72% similar to the human RNA-binding motif protein 8 (RBM8). RBM8 cDNAs have been used to recover clones encoding the Xenopus laevis protein Y14. Results from several laboratories suggest that Y14 is complexed with mRNAs and proteins to form ribonucleoprotein particles (mRNPs) that are preferentially exported from the nucleus to the cytoplasm. Therefore, in addition to having an RNA-binding motif, there is experimental evidence suggesting that Tsunagi-related proteins bind RNA (Mohr, 2001).

tsunagi is Japanese for 'connection' or 'link.' Hybridization of a probe from tsu cDNA to polytene chromosomes was used to ascertain the location of tsu in the genome. A single focus of hybridization on the right arm of chromosome 2, in polytene chromosome interval 45A4, was detected. Two genomic DNA contigs in the region, Dbp45A and hig, were examined by PCR for the presence of tsu sequence. A 30-40-kb P1 phagemid clone, DS02099 (BDGP), that maps to the distal end of Dbp45A was found to contain tsu (Mohr, 2001).

Given the role of the Mago-Tsunagi complex in the localization of OSK mRNA and Staufen protein, it is possible to envision a similar function for the complex during early oogenesis (stages 1-5). That is, during early oogenesis a Mago-Tsunagi complex is likely to be required for localizing mRNAs that encode components necessary to interpret the posterior follicle cell-to-oocyte signal (Mohr, 2001).

Aberrant AP axis formation during oogenesis in tsu mutant egg chambers is revealed by two phenotypes: (1) the migration of the oocyte nucleus from a posterior to an anterior position can be abnormal in tsu mutant egg chambers; (2) markers of microtubule organization such as Kinesin::ß-gal and Nod::ß-gal are found at ectopic sites within tsu mutant oocytes. These are phenotypes that are reminiscent of defects observed in mutants in which Gurken signaling is disrupted. When Gurken signaling is blocked, disassembly of the MTOC at the posterior pole is inhibited and the oocyte nucleus fails to migrate to the anterior pole. Anomalies in nuclear migration and in the distribution of markers used to assess the integrity of the microtubule network are also detected in mago mutant egg chambers. Disassembly of the MTOC has not been examined in tsu mutant egg chambers but is aberrant in mago mutant oocytes. The fact that Mago and Tsunagi proteins cooperate to establish the AP axis of the oocyte suggests that in oocytes from tsu mutant mothers the MTOC at the posterior pole may not disassemble (Mohr, 2001).

Mutations that disrupt Gurken signaling also alter the fates of follicle cells, causing posterior follicle cells to develop as anterior follicle cells. In egg chambers derived from tsu mutant females the accumulation and function of GRK mRNA and protein during stages 1-6 (the time when posterior follicle cell fates are specified) are apparently normal. This is also evident from the fact that follicle cell fates are properly specified in tsu mutant egg chambers, as determined by the formation of the aeropyle (a structure produced by the posterior follicle cells) and by monitoring egg chambers with follicle cell markers that reveal their fates. Importantly, germ-line and follicle-cell clonal analysis indicates that tsu+ function is necessary within the germ line but not in follicle cells. Therefore, it appears that the patterning abnormalities detected when egg chambers lack wild-type tsu function arise from a requirement for Tsunagi protein in mediating the response of the oocyte to the posterior follicle cell-to-oocyte signal (Mohr, 2001).

In situ hybridization and immunolocalization reveal that GRK mRNA and protein are altered during stages 9-10 in oocytes from tsu mutant females. The amount of detectable GRK mRNA and protein is often reduced relative to wild type or undetectable above background. In contrast, the amount of GRK mRNA and protein is indistinguishable from wild type in mago mutant egg chambers and they are both properly localized. The differences in GRK mRNA and protein in mago mutant and tsu mutant egg chambers during stages 9-10 suggest that the DV pattern abnormalities in tsu mutant egg chambers are not caused solely by the altered migration of the oocyte nucleus, and although the two proteins cooperate in specific developmental processes, Mago and Tsunagi proteins are likely to function independently in other aspects of oocyte development (Mohr, 2001).

DV patterning of the egg chamber occurs during midoogenesis from restriction of Gurken signaling to the future dorsal side of the egg chamber. The asymmetric localization of Gurken signaling is achieved by (1) migration of the oocyte nucleus to an anterior cortical position; (2) transport of GRK mRNA from the oocyte nucleus to the dorsal-anterior corner of the oocyte; (3) anchoring of GRK mRNA within the dorsal-anterior corner of the oocyte, and (4) translation of the spatially restricted GRK mRNA. Squid protein, a heterogeneous nuclear RNA-binding protein (hnRNP), has been implicated in the export of GRK mRNA from the nucleus and in its delivery to an anchor within the cytoplasm (Mohr, 2001).

The Tsunagi homolog Y14 was originally identified through its interaction in a yeast two-hybrid screen with Ran-binding protein 5 (RanBP5). RanBP5 is related to the nuclear transporter receptor proteins importin-ß and transportin, proteins that are part of the nuclear import/export machinery. Like Squid protein, RanBP5 has been shown to interact physically with transportin. Based on the association of Squid protein and Tsunagi homologs with cellular components required in nuclear import and export, biochemical evidence indicating that the two proteins function in RNA export and the genetic evidence provided here, it is reasonable to propose that Tsunagi protein and Squid protein may interact with the nucleocytoplasmic transport machinery to regulate export and/or anchoring of GRK mRNA within the dorsal-anterior corner of the oocyte (Mohr, 2001).

Several roles for Tsunagi protein in the export and/or anchoring of GRK mRNA are consistent with the data presented and the known biochemical roles of Tsunagi homologs. (1)Tsunagi protein may be necessary to maintain the stability of GRK mRNA during midoogenesis. (2) Splicing-dependent export of GRK mRNA from the oocyte nucleus may require Tsunagi+ protein. The RNA may not be detectable within the nucleus owing to rapid degradation of unspliced pre-mRNAs, as has been shown to occur in yeast, or it may be exported from the nucleus by a generalized export mechanism. (3)The protein may be required for the export and/or localization of GRK mRNA within the dorsal-anterior cortex. Further experimentation will be necessary to determine the molecular function of Tsunagi protein in the stability, export, and/or anchoring of GRK mRNA during midoogenesis (Mohr, 2001).

The molecular and genetic analysis of tsunagi has revealed that the encoded protein functions in at least three distinct processes during Drosophila oogenesis. In early oogenesis (stages 1-5), Tsunagi protein forms a complex with Mago protein that is critical for interpreting the posterior follicle cell-to-oocyte signal. During stages 8 and 9 of oogenesis, Tsunagi protein cooperates with Mago protein to localize components necessary for anchoring OSK mRNA within the posterior pole. Although other interpretations are possible, a simple model suggests that both during stages 1-5 and 8-9 the Mago-Tsunagi complex localizes RNAs encoding proteins that are essential for mediating axis formation and assembly of the germ plasm. At stages 9-10, Tsunagi protein has a function independent of Mago protein that is crucial for the export and/or localization of GRK mRNA. Tsunagi interacts with a component of the cellular localization machinery, suggesting that homologs of Tsunagi may also be involved in RNA localization (Mohr, 2001).

mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-golgi units involved in Gurken transport in Drosophila oocytes

The anteroposterior and dorsoventral axes of the future embryo are specified within Drosophila oocytes by localizing gurken mRNA, which targets the secreted Gurken transforming growth factor-alpha synthesis and transport to the same site. A key question is whether gurken mRNA is targeted to a specialized exocytic pathway to achieve the polar deposition of the protein. This study shows, by (immuno)electron microscopy that the exocytic pathway in stage 9-10 Drosophila oocytes comprises a thousand evenly distributed transitional endoplasmic reticulum (tER)-Golgi units. Using Drosophila mutants, it was shown that it is the localization of gurken mRNA coupled to efficient sorting of Gurken out of the ER that determines which of the numerous equivalent tER-Golgi units are used for the protein transport and processing. The choice of tER-Golgi units by mRNA localization makes them independent of each other and represents a nonconventional way by which the oocyte implements polarized deposition of transmembrane/secreted proteins. It is proposed that this pretranslational mechanism could be a general way for targeted secretion in polarized cells, such as neurons (Herpers, 2004).

To understand how Gurken, as a transmembrane protein, achieves its polar distribution, the organization of the exocytic pathway in Drosophila oocytes was elucidated. The exocytic pathway is similar to that found in other Drosophila cells observed so far. Namely, it contains a continuous ER that pervades the entire ooplasm, from which a multitude of tER-Golgi units arise. In the oocyte, as in S2 cells, the tER-Golgi units comprise an ER exit site (positive for dSec23p), closely apposed to a Golgi apparatus, either under the form of a cluster of vesicles and tubules, or a Golgi stack, both marked by the Golgi marker, dCOG5. S2 cells contain ~20 of these units, whereas the number is much greater in oocytes (~1000) but with an equivalent density (S2 cells have about a 60-100 times smaller volume than a stage 9 oocyte) (Herpers, 2004).

One way to explain Gurken deposition at the dorsal/anterior (D/A) corner is to argue for a concentration of the tER-Golgi units at this corner. It has been shown that cell migration in wound healing is accompanied by the redistribution and concentration of the cellular Golgi complex to the part of the cell facing the injury, thus sustaining a polarized secretion that helps in the healing. This study shows, using immunofluorescence and electron microscopy, that the thousand tER-Golgi units in the Drosophila oocyte are evenly distributed throughout the ooplasm. This concentration is therefore unlikely to be the underlying mechanism for Gurken polarity (Herpers, 2004).

Another way to explain that Gurken is only synthesized and transported in the tER-Golgi units localized at the D/A corner is to argue that they have a unique composition in regard to the three known proteins involved in its movement through the exocytic pathway: Star, Cornichon, and Brother of Rhomboid (Brho). Perhaps these three proteins only reside in the tER-Golgi units at the D/A corner, therefore rendering them, and only them, competent for Gurken transport. Star is thought to act as an ER chaperone helping in the exit from the ER. Cornichon is a potential Gurken receptor at the ER exit sites. Brho is a specific endoprotease located in the Golgi apparatus that cleaves Gurken just after its transmembrane domain, thus generating the lumenal active ligand of Torpedo, and a C-terminal membrane bound fragment whose fate is undetermined (Herpers, 2004).

Several lines of evidence, however, suggest that these proteins are not restricted to the tER-Golgi units of the D/A corner. (1) cornichon and brho mRNAs do not have a polarized localization, but rather occupy the entire oocyte. This suggests that the two proteins are expressed ubiquitously, as is Star protein in stage 6-10 oocytes. (2) In an S2 cell assay, Gurken is only cleaved and secreted when cells are transfected with both Brho and Star. When transfected with Brho alone, Gurken is cleaved but not secreted, and with Star alone, Gurken is neither processed nor secreted. Because in both squid and K10 mutants Gurken protein is transported to all tER-Golgi units along the anterior side and at the ventral/anterior corner. Gurken protein is found in the space between the oocyte and the nurse cells: this suggests that at least Star and Brho are present in all tER-Golgi units, including those away from the D/A corner, and they act in the processing and transport of Gurken. Similarly, Star, Cornichon, and Brho are also likely localized to the tER-Golgi units at the posterior pole, in WT stage 6-7 and stage 9-10 merlin oocytes where Gurken protein is synthesized, transported and processed, so Gurken signals to the posterior follicle cells. (3) It was recently published that in stage 10 germ line clones of the sec5 exocyst complex subunit, Gurken protein is synthesized in the middle and at the posterior pole of the Drosophila oocyte. This experiment, and those described in this study, shows that all tER-Golgi units potentially have the capacity of transporting and processing Gurken protein. Together, the tER-Golgi units at the D/A corner do not seem to contain a different set of transport and processing proteins from the others (Herpers, 2004).

This study shows, by using K10, squid, and merlin mutants, that what dictates the use of these numerous, seemingly identical and evenly distributed tER-Golgi units, is the restricted localization of gurken mRNA. This also could be the case for other transmembrane/secreted proteins (Herpers, 2004).

gurken mRNA is localized in a restricted manner at the D/A corner, where it is then anchored. The anchoring mechanism is not yet clear and is the subject of intense research, but it could be envisaged that an efficient recruitment of local tER-Golgi units would be achieved by anchoring the mRNA directly to the membrane. The ER has been suggested as such an anchor. Whatever the anchoring mechanism and wherever it is localized, gurken RNA diffuses locally, binds to ribosomes, is translated, and recognized by the signal recognition particle that targets it to the most proximal ER membrane, where the protein is synthesized and subsequently transported through the most adjacent tER-Golgi units (Herpers, 2004).

Additional mechanisms also ensure that local synthesis is followed by local polarized delivery at the D/A intercellular space, where the activity of Gurken is necessary but also needs to be restricted for proper oocyte development. In eukaryotic cells, the ER has been shown to be continuous throughout the entire cytoplasm. FRAP experiments on PDI-GFP-expressing oocytes as well as the use of a strong allelic combination of Cornichon have shown that the ER comprises a single lumen throughout the oocyte, including at the D/A corner. Partial diffusion of the newly synthesized Gurken in the membrane of the ER is therefore expected. Such a diffusion over long distance (>0.5 mm) within the ER has been shown for soluble proteins, such as the light and heavy chains of the immunoglobulins in frog oocytes. However, the maximum distance over which intracellular Gurken is found is 20 µm (Herpers, 2004).

Therefore, the diffusion of Gurken is likely to be prevented by efficient sorting mechanisms. Such mechanisms rely primarily on the transmembrane domain of Gurken and Cornichon. Gurken lacking its transmembrane domain diffuses in the ER in a very similar way as WT Gurken does in a strong cornichon mutant, except for the concentration observed at the D/A corner. This could be explained by the difference in diffusion between a transmembrane protein and a lumenal fragment. Nevertheless, this phenocopy suggests that Gurken binds Cornichon through its transmembrane domain. This interaction could mediate the efficient packaging of transmembrane Gurken in COPII transport vesicles. Cornichon presents homology to Erv14p, which is involved, in yeast, in the exit of the plasma membrane Axl2 transmembrane protein from the ER in COPII-coated vesicles. The interaction between Erv14p and Axl2p has been suggested to act via a novel mechanism that might be mediated by interactions of transmembrane segments. The binding of Gurken to Cornichon might rely on a similar mechanism, although it is not clear why a transmembrane cargo protein would need an extra transmembrane chaperone for its sorting and incorporation into COPII buds (Herpers, 2004).

This is particularly intriguing because efficient export from the ER also could be mediated by motifs found in the cytoplasmic domain of Gurken. In type I transmembrane proteins such as ERGIC53 and Emp46, a doublet of phenylalanine and leucine, respectively, is important for the exit from the ER. Both doublets are found in Gurken cytoplasmic domain (aa251 sfpvLLmlss lyvlfaavfm lrnvpdyrrk qqqlhlh kqr FFvrc). The removal of the cytoplasmic domain of Gurken does not seem, though, to affect to a great extent the efficient exit of the truncated protein from the ER, perhaps because it can still interact with Cornichon. The role of the cytoplasmic domain could perhaps be unraveled in a weak cornichon mutant background (Herpers, 2004).

Brefeldin A (BFA) treatment of the WT egg chambers was expected to lead to full retention/retrieval of Gurken in the ER followed by its diffusion. However, many oocytes remained seemingly unaffected, suggesting that the binding of Gurken to its sorting receptor Cornichon locked Gurken at tER sites. When the drug treatment was performed in a weak allele of cornichon, Gurken could be observed diffusing in the ER, suggesting that the locking mechanism was impaired. This diffusion, however, was not as extensive as the diffusion observed in the strong cornichon mutant under nontreated conditions. This partial diffusion could represent that of the complex CniCF5/Gurken (two transmembrane proteins) instead of Gurken alone. Why, under BFA treatment, CniCF5/Gurken complex diffuses more readily than Cornichon/Gurken is still not understood and further work is needed to elucidate the molecular details of the BFA effect in oocytes (Herpers, 2004).

Further work is needed to find out whether the polar synthesis and deposition relied upon by other transmembrane proteins stands alone as a pretranslational mechanism (through mRNA localization), or is coupled to efficient protein sorting events from the ER (Herpers, 2004).

All tER-Golgi units are able to work in synchrony for the transport of transmembrane proteins, of which the RNAs are not localized, such as Yolkless. However, a subset of tER-Golgi units can be recruited to perform the specific task of transporting a given transmembrane/secreted protein. This suggests that the different tER-Golgi units within a single cell can function in an uncoupled/nonsynchronous/independent manner, even though the ER is continuous. The restricted localization of transcripts is a necessary cue for imposing this uncoupling, as has been suggested for muscle heterokaryons and hybrid myotubes, although it is not clear in these systems whether the ER is continuous (Herpers, 2004).

This study has exemplified the functional uncoupling of the tER-Golgi units in Drosophila oocytes, and it is proposed that a similar mechanism also could take place for other types of highly polarized cells, such as neurons. This is suggested by a series of observations, showing that RNA encoding transmembrane proteins specific for the dendrites are translated in the dendrites themselves, and not exclusively in the cell body. It is also suggested by the immunofluorescence labeling of Golgi markers such as galactosyltransferase and GM130, in a dotty pattern along the dendrites, suggesting that perhaps Golgi-like structures could underlie this labeling. It is therefore possible that the mechanism identified here also occurs in neurons. mRNA encoding transmembrane/secreted proteins specific for the dendrites could be localized in these specialized domains and use dendritic Golgi-outposts to induce the local synthesis and transport of the proteins they encode. Whether in mammalian cells, the multiple ER exit sites and the dozens of Golgi stacks making up the Golgi ribbon also could function in an uncoupled manner and respond to a restricted mRNA localization remains to be elucidated (Herpers, 2004).

Drosophila Cornichon acts as cargo receptor for ER export of the TGF-like growth factor Gurken

Drosophila Cornichon (Cni) is the founding member of a conserved protein family that also includes Erv14p, an integral component of the COPII-coated vesicles that mediate cargo export from the yeast endoplasmic reticulum (ER). During Drosophila oogenesis, Cni is required for transport of the TGFalpha growth factor Gurken (Grk) to the oocyte surface. Cni, but not the second Drosophila Cni homologue Cni-related (Cnir), binds to the extracellular domain of Grk, and it is proposed that Cni acts as a cargo receptor, recruiting Grk into COPII vesicles. Consequently, in the absence of Cni function, Grk fails to leave the oocyte ER. Proteolytic processing of Grk still occurs in cni mutant ovaries, demonstrating that release of the active growth factor from its transmembrane precursor occurs earlier during secretory transport than described for the other Drosophila TGFalpha homologues. Massive overexpression of Grk in a cni mutant background can overcome the requirement of Grk signalling for cni activity, confirming that cni is not essential for the production of the functional Grk ligand. However, the rescued egg chambers lack dorsoventral polarity. This demonstrates that the generation of temporally and spatially precisely coordinated Grk signals cannot be achieved by bulk flow secretion, but instead has to rely on fast and efficient ER export through cargo receptor-mediated recruitment of Grk into the secretory pathway (Bökel, 2006).

Cni interacts with the membrane proximal part of the Grk extracellular domain. Tests were performed for potential corresponding protein interactions using a yeast two-hybrid system selecting for adenine and histidine autotrophy with bait constructs containing different portions of the Grk extracellular domain extending to the beginning of the transmembrane domain (amino acids 74-245, 179-245, 215-245). Introduction of a prey construct containing the Cni ORF into this background conferred the ability to grow under stringent selection conditions, indicating an interaction between the two proteins. Thus, the membrane proximal 31 residues of the Grk extracellular domain are sufficient to mediate binding to Cni in a yeast two-hybrid assay. Interaction was also observed with a prey construct encoding only the first 57 amino acids of Cni. By contrast, neither of the Grk bait constructs nor the CG18501 control were able to bind to a prey construct containing Cnir. These two hybrid data were confirmed by pull-down experiments, where a GST fusion protein containing amino-acids 197 to 245 of Grk could specifically co-purify a MBP fusion construct containing the N-terminal 57 amino acids of Cni, but not one with the corresponding Cnir domain or a lacZ control. This difference between Cni and Cnir in their ability to bind Grk may underlie the strict requirement for Cni during Grk secretion, even though the two Drosophila Cni-like proteins exhibit redundancy in other contexts (Bökel, 2006).

Yeast Emp24p is a cargo receptor cycling between the ER and the intermediate compartment (Schimmoller, 1995). Exit of Emp24p and associated cargo from the ER is in part mediated by binding of Emp24p to components of the COPII coat through diaromatic amino acid pairs in the C-terminal cytoplasmic tail. The intracellular domain of Grk was replaced with the short cytoplasmic tail of one of the D. melanogaster Emp24 homologues (CG3564 amino acids 194-208). A transgene expressing this fusion protein from the endogenous promotor (pGrk-EmpCyt) fully rescues the loss of grk, indicating that the transgene produced normal amounts of active Grk ligand. Interestingly, it also restores some Grk signalling activity in the absence of cni. Eggs laid by homozygous cni females containing one copy of pGrk-EmpCyt have normal anteroposterior polarity. Some eggs also possessed recognizable dorsal appendage material, indicating low to intermediate levels of Grk signalling in these egg chambers. Thus, fusing Grk to a domain known to mediate selective recruitment into COPII vesicles partially alleviates its dependence on Cni (Bökel, 2006).

Similar results were achieved using an analogous transgene replacing the Grk intracellular domains with a Cni fragment consisting of the C terminus after the second predicted transmembrane domain (Cni amino acids 100-145, pGrk-CniCyt). The transgene fully rescues the loss of grk and restores some signalling activity in the absence of cni. Eggs laid by cni mutant females carrying one copy of this transgene have normal anteroposterior polarity and show slight and variable rescue of the dorsoventral axis. The C-terminal domains of Cni-like and Emp24-like proteins may therefore be functionally equivalent (Bökel, 2006).

If Cni were only functioning as a cargo receptor for ER export of Grk, massive overexpression of Grk should result in bulk flow ER to Golgi transport and might thus overcome the requirement for cni. To test this assumption, the egg phenotypes produced by grk overexpression lines were analyzed. Expression of grk with the help of the maternal alpha-Tubulin Gal4 driver leads to a strong increase in the amount of Grk protein in stage 9 egg chambers when compared with endogenous Grk levels. When overexpressed in a wild-type background, the bulk of grk mRNA is still transported to the vicinity of the nucleus. Grk protein remains asymmetrically distributed, although the region with high Grk protein levels within the oocyte is more expanded when compared with wild type. This might be due to the saturation of the mechanisms normally responsible for retention of the protein near its site of translation and the subsequent secretion through a few local ER exit sites. The resulting egg chambers maintain DV polarity although they are severely dorsalized. The operculum, the dorsal-most chorion structure that is specified in follicle cells receiving maximal Grk levels, is expanded while the dorsal appendages normally specified at more lateral positions experiencing slightly lower Grk signalling are shifted to the ventral side of the egg (Bökel, 2006).

Overexpression of Grk in a cni mutant background results in uniform high levels of Grk protein within the oocyte. Interestingly, the resulting eggs possess variable amounts of dorsal appendage material, indicating restoration Grk signalling, albeit to lower levels than in the presence of Cni. However, the eggs lack DV and frequently even AP polarity, as can be seen by the patchy induction of dorsal appendage material around the entire egg circumference and the presence of a posterior micropyle, respectively (Bökel, 2006).

These observations show that the requirement for Cni can be overcome by Grk overexpression. Thus, cni function is not essential for the formation of an active ligand per se, but the Cni-mediated increase in the efficiency of Grk secretion is a prerequisite for the precise temporal and spatial control of Grk signalling (Bökel, 2006).

Ik2 is required for mRNA localization during oogenesis

In both Drosophila and mammals, IkappaB kinases (IKKs) regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. Mutations were identified in ik2, the gene that encodes one of two Drosophila IKKs; the gene is essential for viability. During oogenesis, ik2 is required in an NF-kappaB-independent process that is essential for the localization of oskar and gurken mRNAs; as a result, females that lack ik2 in the germline produce embryos that are both bicaudal and ventralized. The abnormal RNA localization in ik2 mutant oocytes can be attributed to defects in the organization of microtubule minus-ends. In addition, both mutant oocytes and mutant escaper adults have abnormalities in the organization of the actin cytoskeleton. These data suggest that this IkappaB kinase has an NF-kappaB-independent role in mRNA localization and helps to link microtubule minus-ends to the oocyte cortex, a novel function of the IKK family (Shapiro, 2006).

Protein kinases of the IkappaB kinase (IKK) family are known for their roles in innate immune response signaling pathways in both mammals and Drosophila. Mammalian IKKs all have roles in immune responses, but have a variety of targets. and IKKβ were identified in a protein complex that phosphorylates IkappaB and targets it for degradation, thereby allowing the nuclear localization and activation of NF-kappaB transcription factors. Gene targeting experiments in the mouse demonstrated that IKKβ, but not IKKalpha, is required for NF-kappaB activation by pro-inflammatory stimuli through receptors such as TLR4. IKKα activates the Rel/p52 transcription factor, because it activates proteolytic processing of the p100 precursor of p52 in an IkappaB-independent process. IKKepsilon and TANK binding kinase 1 (TBK1) are required to phosphorylate and activate the transcription factor Interferon regulatory factor 3 (IRF3) in response to viral infection. In addition to these immune response functions, IKKα has an NF-kappaB-independent role in epidermal differentiation and limb development (Shapiro, 2006 and references therein).

Dorsoventral patterning of the Drosophila embryo relies on the activation of Dorsal, a Rel-family transcription factor, by a signaling pathway that is homologous to mammalian TLR pathways. In response to activation of the receptor Toll, Cactus (the Drosophila IkappaB) is degraded, which allows Dorsal to move to embryonic nuclei and activate genes, such as twist, that are required for specification of ventral cell types. Phosphorylation of Cactus is required for its degradation, but the responsible kinase has not been identified. The Drosophila genome encodes two members of the IKK family. DmIkkβ (ird5 - FlyBase) is essential for the response to bacterial infection. DmIkkβ is required for proteolytic processing and activation of Relish, a p100-like Rel/ankyrin-repeat protein, like the role of mammalian IKKα in the activation of p100. The function of the second Drosophila protein kinase of the IKK family, ik2 (IkappaB kinase-like 2), has not been characterized, but it was a good candidate to control the phosphorylation and degradation of Cactus (Shapiro, 2006).

To test whether ik2 encodes a Cactus kinase, the phenotypes caused by loss of ik2 function were characterized. This study presents data showing that Ik2 is essential for dorsoventral and anteroposterior embryonic patterning of the Drosophila embryo. However, Ik2 does not act as a Cactus kinase, but exerts its effects on embryonic patterning through the localization of specific mRNAs during oogenesis. The data indicate that Drosophila Ik2 regulates RNA localization through regulation of the cytoskeleton and define a novel function for this protein family (Shapiro, 2006).

A saturation mutagenesis experiment had identified lethal complementation groups in polytene chromosome region 38E, the region that includes the ik2 gene. Missense mutations were identified in the ik2 kinase domain in all five alleles of the l(2)38Ea complementation group. A sixth allele, ik2alice, identified in a genetic mosaic screen for maternal effect mutations, had a missense mutation in the C-terminal end of the kinase domain. All six ik2 alleles caused recessive lethality, and the majority of the mutants die as first instar larvae. At low temperature and in uncrowded culture conditions, rare escaper adults (<1%) were observed, but they died shortly after eclosion (Shapiro, 2006).

If ik2 encoded the Cactus kinase, embryos produced by females that lack ik2 would not be able to degrade Cactus, so Dorsal would not enter embryonic nuclei to activate genes required for ventral cell fate specification and the embryos would be dorsalized. Because ik2 mutations were lethal, FRT/FLP recombination combined with the ovoD dominant female-sterile mutation were used to generate mutant clones in the female germline. More than 95% of the embryos laid by ik2alice and ik21 mutant females did not hatch; however, larval cuticle preparations showed that none of the embryos were dorsalized. Instead, the majority of embryos produced by ik2alice and ik21 mutants had a bicaudal phenotype, ranging from headless embryos to embryos with a duplicated abdomen in place of the head and thorax. In addition to this anteroposterior patterning defect, a large number of embryos from both ik2alice and ik21 germline clones had expanded ventral cuticular structures, the opposite of the expected phenotype. Some embryos were both ventralized and bicaudal, with expanded ventral denticle bands and filzkörper (a posterior structure) in both the tail and the anterior of the embryo. Both ik2 alleles produced bicaudal and ventralized embryos, but 89% of the embryos produced by ik2alice mutant females were bicaudal with no apparent dorsoventral abnormalities, whereas only 47% of embryos produced by ik21 mutant females were bicaudal, and the remainder of the embryos appeared to be too ventralized to score for ectopic posterior cuticular structures. A similar range of phenotypes was observed in embryos produced by ik22, ik23 and ik25 females with mutant germline clones (Shapiro, 2006).

Contrary to the prediction based on its sequence, ik2 does not act as a Cactus/IkappaB kinase in the Drosophila embryo. The embryonic ventralization caused by loss of ik2 is the opposite of the phenotype predicted for a Cactus kinase, and all effects of ik2 on the dorsoventral pattern of the embryo can be explained by a loss of activity of the Grk/Egfr pathway during oogenesis. Embryos that lack maternal activity of both Drosophila IKKs, Ik2 and DmIkkβ, are ventralized and are indistinguishable from ik2 single mutants, which rules out the possibility that the Drosophila IKKs act in both the Grk/Egfr and Toll pathways, and indicates that an unidentified kinase of another family is required to target Cactus for degradation. Additional experiments will be required to test whether ik2 plays other roles in the immune response (Shapiro, 2006).

It was found that instead of playing a role in Cactus degradation, Drosophila ik2 is required for the localization of specific mRNAs during oogenesis. Both the actin and microtubule cytoskeletons are disrupted in ik2 mutants, and defects in microtubule-based transport are sufficient to account for the defects in mRNA localization seen in ik2 mutants. Because Drosophila ik2 is specifically required for organization of the oocyte cytoskeleton, the results raise the possibility that some of the NF-kappaB-independent roles of the mammalian IKKs may act through the cytoskeleton (Shapiro, 2006).

The embryonic patterning defects caused by the loss of ik2 function are due to the failure to transport all osk mRNA to the posterior pole of ik2 mutant oocytes, which leads to bicaudal embryos, and failure to localize grk mRNA to the dorsal anterior of the oocyte, which leads to ventralized embryos. Loss of ik2 has a milder effect on bcd mRNA localization; bcd is correctly localized in most oocytes, but is not tightly restricted to the anterior pole in a minority of cases (Shapiro, 2006).

Many lines of evidence indicate that osk localization to the posterior pole depends on kinesin and gurken localization to the dorsoanterior corner depends on dynein. However, the kinesin and dynein motors in the oocyte are interdependent. For example, posterior localization of dynein and the anterodorsal localization of gurken are both disrupted in Khc mutants, and hypomorphic Dhc mutants have a reduced amount of Khc-β-gal at the posterior pole. Both motor systems are at least partially functional in ik2 mutants: most oskar is localized to the posterior pole of the oocyte (a kinesin-dependent process) and grk mRNA is localized anteriorly (a dynein-dependent process) (Shapiro, 2006).

Several lines of evidence suggest that the RNA localization defects seen in ik2 oocytes are associated with defects in a subset of dynein-mediated, minus-end-directed transport processes. The movement of grk mRNA to the dorsoanterior corner of the oocyte depends on two sequential dynein-based movements: grk mRNA moves first to the anterior of the oocyte along microtubules with plus-ends at the posterior pole and minus-ends at the anterior, and then moves dorsally on microtubules with minus-ends that form a cage around the oocyte nucleus. The dorsal movement of grk mRNA is specifically blocked in ik2 mutants, which would be consistent with a failure in this dynein-based movement. Restriction of bcd mRNA to the anterior margin of the oocyte, which is disrupted in some ik2 oocytes, depends on the swallow gene product, which binds dynein light chain. Overexpression of dynamitin disrupts dynein function and causes changes to the localization of grk and bcd mRNA that are similar to the phenotype of ik2 oocytes. In addition, BicD mutations produce a maternal effect phenotype similar to that of ik2. BicD is part of a protein complex with dynein light chain in early oocytes, neuroblasts and the early embryo, and has been proposed to link cargo to microtubules in both Drosophila and mammalian cells (Shapiro, 2006).

Although this evidence links ik2 to a dynein transport system, the most penetrant phenotype in ik2 mutants is osk mislocalization and subsequent production of bicaudal embryos, a kinesin-dependent process. However, loss of ik2 function, like the BicD gain-of-function mutations, does not eliminate kinesin function, because the majority of osk mRNA accumulates at the posterior pole. Because the kinesin and dynein motors in the oocyte are interdependent, osk mRNA mislocalization could be caused by a decreased kinesin activity that is secondary to dynein disruption (Shapiro, 2006).

In addition to defects in minus-end-directed transport, the organization of the microtubules is also perturbed in ik2 oocytes. The plus-ends of microtubules are localized correctly to the posterior pole of the oocyte. However, there are abnormal aggregates of microtubules around the oocyte nucleus, where a population of microtubule minus-ends is normally anchored, and the microtubule minus-end marker, Nod-β-gal, is not localized at the anterior of the oocyte. These defects suggest that abnormal organization of microtubule minus-ends during mid-oogenesis could be the basis of the defect in minus-end-directed transport (Shapiro, 2006).

The adult bristles and ovaries of ik2 mutants also displayed abnormalities in the actin cytoskeleton. The bristle defects are nearly identical to those caused by mutations in actin-associated proteins, or to bristles that were treated with F-actin-inhibitors. Bristles contain a central core of microtubules, but mutations in the dynein heavy chain gene Dhc64C or treatment with drugs that disrupt microtubule dynamics do not cause bristle phenotypes like the thick, branched bristles seen in ik2 mutants. The actin cytoskeleton of the oocyte is also disrupted in ik2 mutants, with ectopic sites of actin polymerization in the ooplasm. These actin defects are distinct from those caused by mutations that affect nurse cell ring canal actin, which suggests that actin organization is not globally disrupted in ik2 mutants and that the actin defects are restricted to the oocyte cortex (Shapiro, 2006).

Recent data have defined two sets of microtubules in the oocyte that are both nucleated from minus-ends at the centrosome associated with the oocyte nucleus; one set remains associated with the oocyte nucleus, whereas the remaining microtubules shift their minus-ends from the oocyte to the cortex. It was suggested that translocation of the minus-ends of the latter set of microtubules to the cortex could depend on actin and motor proteins. The current data suggest that anchoring of microtubule minus-ends to the oocyte cortex depends upon an Ik2-dependent interaction of microtubule minus-ends with the F-actin network, analogous to the interaction of microtubule plus-ends with the actin cytoskeleton through microtubule tip proteins (Shapiro, 2006).

The phenotypes of ik2 in the ovary and adult bristles are very similar to those caused by mutations in spn-F. Like ik2 mutations, null mutations in spn-F affect the localization of osk and grk mRNAs during oogenesis, and cause bicaudal and ventralized embryos. spn-F mutant oocytes have ectopic sites of F-actin polymerization, and spn-F bristles are similar to ik2 mutant bristles. Ik2 and Spn-F have been shown to interact in a yeast two-hybrid screen, which suggests that these proteins can form a complex. Spn-F associates specifically with microtubule minus-ends. It is therefore proposed that Ik2 and Spn-F act together to regulate interactions between the minus-ends of microtubules and the actin-rich cortex (Shapiro, 2006).

cutoff and aubergine mutations result in retrotransposon upregulation and checkpoint activation in Drosophila; checkpoint activation causes a loss of germ cells and a reduction of Gurken protein

Gametogenesis is a highly regulated process in all organisms. In Drosophila, a meiotic checkpoint which monitors double-stranded DNA breaks and involves Drosophila ATR and Chk2 coordinates the meiotic cell cycle with signaling events that establish the axis of the egg and embryo. Checkpoint activity regulates translation of the transforming growth-factor-alpha-like Gurken signaling molecule which induces dorsal cell fates in the follicle cells. Mutations in the Drosophila gene cutoff (cuff) affect germline cyst development and result in ventralized eggs as a result of reduced Grk protein expression. Surprisingly, cuff mutations lead to a marked increase in the transcript levels of two retrotransposable elements, Het-A and Tart. Small interfering RNAs against the roo element are still produced in cuff mutant ovaries. These results indicate that Cuff is involved in the rasiRNA pathway and most likely acts downstream of siRNA biogenesis. The eggshell and egg-laying defects of cuff mutants are suppressed by a mutation in chk2. Mutations in aubergine (aub), another gene implicated in the rasiRNA pathway, are significantly suppressed by chk2 mutation. These results indicate that mutants in rasiRNA pathways lead to elevated transposition incidents in the germline, and that this elevation activates a checkpoint that causes a loss of germ cells and a reduction of Gurken protein in the remaining egg chambers (Chen, 2007).

cutoff (cuff) mutations were isolated in a large-scale female-sterile screen of Drosophila, and one additional allele was identified in a screen for P element insertions. Females transheterozygous for cuff alleles lay eggs with various degrees of ventralization. The dorsoventral polarity of the egg and embryo depends on the levels of the Gurken (Grk) ligand, which is produced and secreted by the germline and activates the EGF receptor (Egfr) in the overlying follicle cells. To determine whether Grk-Egfr signaling was affected, the grk expression pattern was analyzed in a strong cuff mutant background. In wild-type egg chambers at stage 9 of oogenesis, grk RNA becomes restricted to the future dorsal-anterior side of the oocyte and forms a cap around the oocyte nucleus. Grk protein is translated from the tightly localized RNA and is also spatially restricted to the membrane overlying the oocyte nucleus. cuff mutants do not significantly disrupt grk RNA localization. However, in many mid-stage egg chambers, the Grk protein level is greatly reduced, such that between 10% and 40% of the egg chambers contain no detectable Gurken protein at all, consistent with defects in grk translation. In wild-type egg chambers by stage 3 of oogenesis, the oocyte nucleus forms a compact structure termed the karyosome. In cuff mutants, karyosome formation is affected in 10%–20% of the egg chambers, in which the DNA assumes various shapes and is often found in separate clumps (Chen, 2007).

Genomic database searches identified the yeast gene Rai1 as a homolog of cuff. This gene has been shown to interact with a nuclear 5′–3′ exoribonuclease (Rat1) that is involved in rRNA processing and transcriptional termination. A cytoplasmic homolog of Rat1, Xrn1, has also been described in yeast and vertebrates and has been implicated in mRNA regulation that is localized to cytoplasmic processing bodies. An HA-tagged Drosophila Rat1 (CG10354) construct was generated and overexpressed with a fully functional FLAG-tagged Cuff in the ovary. Using immunoprecipitation (IP), no any interaction between the exoribonuclease and Cuff was detected. It is therefore possible that Drosophila Rat1 is not the correct partner for Cuff. This is also supported by the observation that overexpressed Rat1, as expected, localizes to the nucleus, whereas overexpressed Cuff localizes to the cytoplasm. It was not possible to to detect endogenous Cuff protein with an anti-Cuff antibody, presumably because of low levels of protein expression. However, overexpressed HA-tagged Cuff partially colocalizes with perinuclear puncta in the nurse cells in younger egg chambers. A similar localization pattern has been described for the helicase Vasa, and it was found that Cuff partially colocalizes with Vasa in the cytoplasm. The perinuclear localization pattern, also designated as nuage in the germ cells and related to mammalian P bodies, has been described for components of the RNAi machinery and for genes involved in RNA degradation (Chen, 2007).

Given the eggshell ventralization and the karyosome defect, cuff has mutant phenotypes similar to those of a group of mutants known as the spindle-class genes. Several members of this group encode DNA-repair genes, for instance, spindle(spn) B (XRCC3) and okra (DmRad54). In these mutants, the DSBs that are created during recombination persist and thus activate Chk2 through the Drosophila ATR homolog mei-41. The activity of these kinases negatively regulates the translation of Grk, possibly through a posttranslational modification of Vasa; this modification in turn leads to ventralization of the eggs laid by mutant females. Inactivation of the checkpoint, for instance through mutations in chk2 or mei-41, suppresses the eggshell defects of the spindle-class DNA-repair mutants. In addition, in double mutants of the DNA-repair genes and the genes required for initiating the DSBs, such as c(3)g, mei-W68, or mei-P22, DSBs are not generated; therefore, the checkpoint is not activated, and the eggshell morphology is normal, even in the presence of the repair mutants. To check whether Cuff is involved in the repair of DSBs initiated in prophase of meiosis I, mei41;cuff and cuff;c(3)g double mutants were generated. Although both mutations suppress the eggshell defect of spnB or okra to wild-type morphology, neither suppresses the eggshell defect of cuff, indicating that Cuff does not function in the meiotic repair pathway. Surprisingly, however, a mutation in chk2 partially suppresses the eggshell defect of cuff as well as the defects in cyst development. chk2 cuff double mutants lay mostly wild-type-looking eggs, and have cysts with highly branched fusomes in the germaria, and the females lay more eggs than cuff single mutants, although the rescue is not 100%. In certain allelic combinations, it was possible to observe a dominant effect in the chk2 suppression of the cuff eggshell defect (Chen, 2007).

Previous work has suggested the DNA-repair checkpoint, upon activation, regulates Grk translation through a posttranslational modification of Vas, and that this modification results in slower Vas electrophoretic mobility. To address whether the checkpoint acts in the same manner in cuff mutants, Vas mobility was assayed in cuff mutant combinations. In cuff mutants, Vas migrates slightly more slowly than wild-type control, consistent with the modification seen in the DNA-repair mutants. The mobility is not changed in mei41;cuff double-mutant background, which is consistent with the fact that mei41 mutants do not significantly suppress the eggshell phenotype of cuff. However, Vasa mobility is restored to wild-type in the chk2 cuff double mutant. This suggests that although the checkpoint is activated through a different sensing mechanism in cuff mutants, upon activation the checkpoint involves Chk2 and acts through similar pathways to affect Gurken translation in the egg chambers that escape the early arrest (Chen, 2007).

Several of the spindle-class genes, such as spnE and aub, have been shown to be essential components of the RNAi machinery. Because overexpressed Cuff has a perinuclear localization, whether Cuff might also be required in RNAi pathways was tested. Recently, a specific branch of the RNAi pathways, that involving the repeat-associated small interfering RNA (rasiRNA), has been implicated in the control of retrotransposable elements in the Drosophila germline. Using qRT-PCR, the level of Het-A and Tart, two of the retrotransposable elements responsible for maintaining the telomere in Drosophila, was studied. Previously, it has been shown that in spnE and aub mutants, Het-A and Tart transcripts are derepressed and that this derepression results in a marked elevation in the transcripts level. Compared with heterozygous controls, spnE homozygous mutant females have Het-A and Tart transcript levels that are upregulated by approximately 10-fold, whereas in aub mutants only Het-A is significantly upregulated. In cuff mutant females, the elevation for both transcripts is even more pronounced. Compared with Het-A levels in the heterozygous control, those in cuff mutants are elevated more than 800-fold, and Tart transcript levels increase by more than 20-fold. Transposable elements are normally silenced in the Drosophila germline by the rasiRNA pathway; this silencing process appears to be strongly impaired in the cuff mutants. Whether the upregulation of the transposable elements in cuff mutants could be due to a reduction in the level of rasiRNAs was further tested. However, it was found that the levels of the 25-nt-long roo interfering RNA are not reduced in cuff mutant ovaries, in contrast to ovaries mutant for aub. This indicates that Cuff is not involved in the biogenesis of the rasiRNAs and points to a function for Cuff in the actual silencing process. Because high transcript levels of the retrotransposable elements in the germline are correlated with elevated transposition incidents, which in turn lead to decreased chromosomal integrity, it is possible that such chromosomal defects activate the checkpoint involving chk2. In addition, because transposable elements are involved in the regulation of chromatin structure, the existence of a chromatin checkpoint that involves Chk2 activity is also possible. Once Chk2 is activated, either by the mutants in DNA-repair pathways or by RNAi components such as Cuff and Aub, Chk2 activity leads to posttranslational Vas modification and a negative regulation of Grk translation. However, unlike DNA repair mutations, cuff and aub mutations are not suppressed to wild-type morphology and fecundity by mutations in mei41, suggesting that they activate the checkpoint through a different, or additional, sensing mechanism. Furthermore, most of the mutants in DNA-repair pathways do not cause defects in cyst development or germline stem cell maintenance. These additional defects seen in cuff mutants could be due to the timing of checkpoint activation. DNA-repair mutants activate the meiotic checkpoint during meiotic prophase, which initiates after the formation of the 16 cell cyst, whereas cuff and aub mutants appear to act earlier in oogenesis, given that they already have effects during the mitotic cycles preceding the onset of meiosis. The transposon-activated checkpoint leads not only to translational arrest of Grk, but also to mitotic cell-cycle arrest. Many of the arrested germline cells and cysts eventually undergo apoptosis, leading to gradual loss of both germline stem cells and developing cysts in cuff mutants. However, germ cells that escape the early arrest encounter the second checkpoint effect, which leads to a reduction in Gurken translation (Chen, 2007).

It was recently discovered that there are a large number of different small RNAs generated in the germline of both mammals and flies. Many of them are associated with Piwi family proteins, and most have no known functions. Because the germline represents a special cell type that will pass its DNA on to future progeny, it is possible that selfish elements have developed a high propensity to remobilize in the germline. Furthermore, it is very plausible that in most organisms the germline has evolved sophisticated mechanisms to defend itself against such transposable elements. Many of the small RNAs found in the germline may be involved in the defense against transposable elements, as well as in the regulation of transcription and translation. When the machinery to generate these small silencing RNAs or the effector complexes that are responsible for transcript degradation are disrupted, chromosomal integrity might be at risk. This study has found that a checkpoint involving the conserved Chk2 kinase monitors the RNAi-mediated events in the Drosophila germline and ensures the genomic integrity of the progeny. Chk2 therefore acts as a surveillance factor for both transposon-generated problems as well as DNA-repair problems in the germline. Whether Chk2 has a similar role in the mammalian germline will be interesting to investigate in the future (Chen, 2007).

zucchini and squash encode two putative nucleases required for rasiRNA production in the Drosophila germline: zucchini and squash are required early during oogenesis for the translational silencing of osk mRNA and at later stages for proper expression of the Grk protein

RNAi is a widespread mechanism by which organisms regulate gene expression and defend their genomes against viruses and transposable elements. This study reports the identification of Drosophila zucchini (zuc) and squash (squ), which function in germline RNAi processes. Zuc and Squ contain domains with homologies to nucleases. Mutant females are sterile and show dorsoventral patterning defects during oogenesis. In addition, Oskar protein is ectopically expressed in early oocytes, where it is normally silenced by RNAi mechanisms. Zuc and Squ localize to the perinuclear nuage and interact with Aubergine, a PIWI class protein. Mutations in zuc and squ induce the upregulation of Het-A and Tart, two telomere-specific transposable elements, and the expression of Stellate protein in the Drosophila germline. These defects are due to the inability of zuc and squ mutants to produce repeat-associated small interfering RNAs (Pane, 2007).

RNAi has been shown to be involved in axial polarization in the Drosophila germline. In this species, establishment of dorsal-ventral (DV) and anterior-posterior (AP) axes is achieved through the localized translation of specific mRNAs. The protein products of gurken (grk) and oskar (osk) genes are essential for this process. Early during oogenesis, grk RNA encoding a TGFα-like molecule is localized to the posterior of the oocyte, where it signals the posterior fate to the adjacent follicle cells. Following the reorganization of the microtubule cytoskeleton at stage 8, the oocyte nucleus and grk RNA are relocalized to the dorsal-anterior corner of the oocyte. Grk protein now induces dorsal cell fates in the surrounding epithelial cells. In contrast to Grk, which is expressed throughout oogenesis, osk mRNA is kept silenced early during oocyte development. At later stages, Osk protein is found at the posterior of the oocytes, where it directs the organization of the germ plasm as well as abdomen formation of the future embryo. The silencing of oskar translation from stage 1 to 6 is controlled by a set of genes, including armitage (armi), maelstrom (mael), spindle-E (spn-E), and aubergine (aub), which have been shown to be required for RNAi phenomena. Mutations in these genes induce ectopic expression of Osk at early stages of oocyte development. This observation revealed a connection between the RNAi machinery and the establishment of the AP axis during Drosophila oogenesis. armi encodes the homolog of Arabidopsis SDE-3 helicase, which plays a role in post-transcriptional gene silencing (PTGS), a mechanism closely related to RNAi. mael encodes an evolutionarily conserved protein that is required for the proper localization of Ago2 and Dicer, two components of the RNAi machinery. aub and spn-E encode a member of the PIWI class of Argonaute proteins and a DExH RNA helicase, respectively. Aub and spn-E are involved in the silencing of some classes of transposable elements and tandem repeats in the germline, in heterochromatin formation, in double-stranded RNA (dsRNA)-mediated RNAi in embryos, and in the defense against viruses. Interestingly, spn-E and aub are also involved in telomere regulation. In most eukaryotes, the telomeres are maintained through the action of telomerase, the enzyme that ensures the addition of six- to eight-nucleotide arrays to the chromosome ends. However, in Drosophila, telomere elongation occurs after the transposition of non-long-terminal repeat (non-LTR) HeT-A, TAHRE, and TART retrotransposons. Mutations in spn-E and aub cause the upregulation of Het-A and TART expression in the germline, which, in turn, increases the frequency of telomeric element attachments to chromosome ends (Pane, 2007).

This study shows that the genes zucchini and squash are required early during oogenesis for the translational silencing of osk mRNA and at later stages for proper expression of the Grk protein. It is proposed that insufficient levels of Grk protein in zuc and squ mutants are at least partially due to activity of a checkpoint that affects Grk translation, similar to the effects of DNA repair mutants in meiotic oocytes. zuc encodes a member of the phospholipase-D/nuclease family, while squ encodes a protein with limited similarity to RNAase HII. Like Aub, Mael, and Armi proteins, Zuc and Squ localize to nuage, an electron-dense structure surrounding the nurse cell nuclei implicated in RNAi and RNA processing and transport. Zuc and Squ physically interact with Aub, thus pointing to a direct role for these proteins in the RNAi mechanisms. In further support of this conclusion, it has been demonstrated that zuc and squ are required for the biogenesis of rasiRNAs in ovaries and testes. Accordingly, mutations in these genes abolish the production of this class of siRNAs and lead to the deregulation of transposable elements and tandem repeats in the Drosophila germline (Pane, 2007).

zucchini and squash cause dorso-ventral patterning defects and egg chamber abnormalities during oogenesis: zuc and squ were identified in a screen for female sterile mutations on chromosome II of Drosophila. zuc and squ mutant females are viable, but produce eggs with a range of DV patterning defects. Flies with the most severe allele of zuc, zucHM27, lay few eggs, all of which are completely ventralized and often collapsed, whereas those with the weaker alleles, zucSG63 and zucRS49, produce some eggs with a more normal eggshell phenotype in addition to the ventralized eggs). In addition, a P element insertion in the coding region of the gene also acts as a strong loss-of-function allele with ventralized eggshell phenotypes. Three independent alleles of squ were recovered from the screen, namely squHE47, squPP32, and squHK3, and these alleles also generate a range of ventralized eggshell phenotypes (Pane, 2007).

Similar eggshell phenotypes have been described for mutations in other spindle class genes, which include both DNA repair enzymes such as spindle-B (spn-B) or okra (okr), as well as the RNAi components spn-E, aub, and mael. Similar to the spindle class mutants, several additional developmental defects can be observed in the zuc and squ mutants during oogenesis. In the wild-type oocyte, the nucleus condenses in a compact sphere, known as the karyosome. In contrast, the DNA in the nuclei of zuc and squ oocytes appears dispersed or in separate structures. Since compaction of chromatin in the karyosome occurs at stage 3, the defects observed in zuc and squ egg chambers indicate a function for the genes in the early development of the oocyte. Similar to spnE mutants, in a small number of zuc and squ egg chambers the oocyte is not positioned at the posterior as in wild-type, but is found in the middle of the egg chamber. Finally, fusion of egg chambers can also be observed in zuc mutants, resulting in egg chambers with 30 nurse cells and two oocytes. Many egg chambers in the zuc mutant undergo degeneration at different stages (Pane, 2007).

Grk expression is affected in zuc and squ mutants: The DV patterning defects suggested that the Gurken protein is not properly expressed in the mutant egg chambers. In earlier stages of oogenesis, Grk protein is detected in the oocyte similar to the wild-type egg chambers. At stage 9 in wild-type oocytes, Grk is localized in a cap above the oocyte nucleus, where it specifies the dorsal fate of the adjacent follicle cells. In zuc mutants, the amount of Grk protein found in the dorsal-anterior corner of the oocyte is strongly reduced or absent, suggesting that zuc controls the expression of Grk during mid-oogenesis. To further address this question, the distribution pattern of the grk transcript was analyzed in wild-type and zuc mutant egg chambers. In wild-type, grk mRNA localization mirrors the distribution of the protein and is found in the dorsal-anterior corner of the oocyte. Similarly, in zuc mutant egg chambers, grk mRNA is properly localized during mid-oogenesis. zuc therefore affects accumulation of the Grk protein in mid-oogenesis, most likely affecting the translation of the transcripts. This phenotype is also characteristic of the spindle class mutants in general (Pane, 2007).

In squ mutants, Grk protein also fails to accumulate properly in the oocyte at stage 9. Similar to zuc, analysis of grk transcripts in these mutants revealed that the grk mRNA is correctly localized in the majority of the squ egg chambers in mid-oogenesis. This result suggests that squ is also required for Grk translation (Pane, 2007).

Squid, Cup, and PABP55B function together to regulate gurken translation in Drosophila

During Drosophila oogenesis, the proper localization of gurken (grk) mRNA and protein is required for the establishment of the dorsal–ventral axis of the egg and future embryo. Squid (Sqd) is an RNA-binding protein that is required for the correct localization and translational regulation of the grk message. Cup and polyA-binding protein (PABP) interact physically with Sqd and with each other in ovaries. cup mutants lay dorsalized eggs, enhance dorsalization of weak sqd alleles, and display defects in grk mRNA localization and Grk protein accumulation. In contrast, pAbp mutants lay ventralized eggs and enhance grk haploinsufficiency. PABP also interacts genetically and biochemically with Encore. These data predict a model in which Cup and Sqd mediate translational repression of unlocalized grk mRNA, and PABP and Enc facilitate translational activation of the message once it is fully localized to the dorsal–anterior region of the oocyte. These data also provide the first evidence of a link between the complex of commonly used trans-acting factors and Enc, a factor that is required for grk translation (Clouse, 2008).

This study has taken a direct approach to identify proteins that interact with Sqd protein in ovaries. Using an Sqd antibody, immunoprecipitations out of ovarian extracts were performed, proteins were isolated that specifically interacted with Sqd, and those proteins were identified by mass spectrometry. Four of the Sqd-interacting proteins were positively identified in the mass spectrometry analysis: Cup, PABP55B, Imp, and Hrb27C/Hrp48. The remaining bands were not identified with certainty. Imp and Hrb27C/Hrp48 are two factors that have previously been shown to be involved in RNA localization, and both Hrb27C/Hrp48 and Imp bind to grk mRNA. The identification of these two factors confirmed that the immunoprecipitation method could successfully identify functional Sqd interactors (Clouse, 2008).

One of the Sqd interactors identified in the mass spectrometry analysis was the novel 150-kDa protein Cup. cup mutants display egg chambers with nurse cell nuclear morphology defects and eggs with open chorions. Cup interacts with several factors known to be required for osk localization and translation, such as Exu, Yps, eIF4E, Me31B, and Bruno and independent studies have shown that osk mRNA is prematurely translated in cup mutants. Cup co-localizes with the cap-binding protein, eIF4E, and eIF4E is not properly localized to the oocyte posterior pole in cup mutants. Cup competes away eIF4G, another translation initiation factor, for binding to eIF4E, thereby repressing translation. Together, these data are consistent with the following model for Cup-mediated translational repression; Cup represses the translation of RNAs containing BREs through interactions with Bruno. In this complex, Cup binds directly to eIF4E and interferes with eIF4G binding to eIF4E. Because eIF4G binding to eIF4E is a prerequisite for translation initiation, Cup represses translation by blocking this interaction. Direct biochemical data supporting this model have recently been obtained (Chekulaeva, 2006). It is proposed that Cup represses grk translation by a similar mechanism prior to its localization to the dorsal–anterior of the oocyte (Clouse, 2008).

Cup activity is used by several transcript-specific factors to mediate translational repression of that RNA in a developmentally appropriate context. For instance, Cup is required to mediate the translational repression of the nanos (nos) transcript. Cup has been shown to interact with Nos protein and co-localizes with Nos in the germarium. cup and nos also interact genetically, as heterozygosity for cup suppresses nos-induced phenotypes in early oogenesis. Later in development, Cup binds to Smaug, a factor that specifically binds to nos RNA and is required for its translational repression in embryos. In this example, Cup is required for Smaug to interact with eIF4E and mediate nos repression. Consistent with this biochemical model, Smaug-mediated translational repression is less efficient in cup mutants (Clouse, 2008).

This study as shown that Cup is also required for grk translational repression. This contrasts with previous reports that grk expression is normal in cup mutants, but these earlier reports used relatively weak cup alleles and monitored Grk levels by immunofluorescence. In contrast, in this study alleles were used that allowed assessment of the eggshell phenotype in cup mutants, providing the most sensitive assay for defects in Grk levels. These analyses showed that the different cup alleles vary greatly in phenotypic strength and range of phenotypes (Clouse, 2008).

Using two different alleles of cup from two distinct genetic backgrounds, it was shown that cup mutants lay dorsalized eggs, display defects in Grk protein accumulation, and display less efficient grk mRNA localization. Furthermore, Cup interacts biochemically with Sqd and Hrb27C/Hrp48 in ovarian extracts. Finally, heterozygosity for cup is able to enhance the moderate dorsalization observed in weak allelic combinations of sqd. Together, these data strongly support a model in which Cup functions with Sqd and Hrb27C/Hrp48 to mediate the translational repression of the grk message (Clouse, 2008).

Once grk mRNA is properly localized to the future dorsal/anterior of the oocyte, translational control must be switched from repressive to promoting. In many cellular situations, this activation is accomplished by binding of PABP to polyA tails of transcripts. In fact, PABP55B contains four RNA-recognition motifs (RRMs) that directly bind to polyA tails. PABP55B also has a C-terminal polyA domain that is used for oligomerization of PABP55B on polyA tails. Once PABP55B is bound to RNA, it binds to eIF4G, and this interaction helps to increase the affinity of eIF4G for eIF4E. With this increased affinity, eIF4G is able to effectively compete with Cup for binding to eIF4E, and translation is able to begin (Clouse, 2008).

There are at least three polyA-binding proteins in the Drosophila genome (CG5119 at 55B, CG4612 at 60D, and CG2163 at 44B), which are predicted to function as general translation factors, so it is conceivable that PABP55B could regulate a subset of RNAs. CG2163 has also been designated as PABP2 and has been shown to have essential roles in germ line development and in early embryogenesis (Benoit, 2005). This study has shown that PABP55B mediates the translational activation of fully localized grk mRNA. Specifically, heterozygous pAbp55B mutants lay ventralized eggs in certain genetic combinations, and heterozygosity for pAbp55B also enhances the weakly ventralized phenotype of grk heterozygotes, consistent with a role in translational activation of grk (Clouse, 2008).

PABP55B binds to Enc in ovarian extracts, and that this interaction may be direct and not bridged by an RNA molecule. Furthermore, heterozygosity for pAbp55B is able to enhance the weakly ventralized phenotype of enc mutants raised at 25 °C. Taken together, the biochemical and genetic interactions suggest that PABP55B and Enc function together to mediate the translational activation of grk mRNA once it is localized to the dorsal–anterior of the oocyte (Clouse, 2008).

Previously, Enc has been shown to be required for activation of grk translation in mid-oogenesis. An effect on osk mRNA localization has also been previously observed in enc mutants, but it is unclear at what level this process is affected, or whether this effect is direct. In addition, Enc has been shown to interact with subunits of the proteasome early in oogenesis. Because of its large size and its ability to interact with several different proteins, Enc may play multiple roles during oogenesis. Considering the function of Enc in grk translational activation and its localization to the dorsal–anterior region of the oocyte, It is hypothesized that Enc could function as a scaffolding protein that helps to mediate the transition from translational repression to activation of grk mRNA (Clouse, 2008).

Cup functions with Sqd in a protein complex that mediates the translational repression of grk mRNA before it is properly localized. It is clear from the analysis of mutants such as spn-F and encore, in which mislocalized grk mRNA is translationally silent, that these two steps can be uncoupled. It is proposed that once the RNA has reached the future dorsal–anterior region of the oocyte, PABP, Sqd, and Enc facilitate the translational activation of grk mRNA, PABP is shown associating with the complex once it is fully localized; however, it is possible that PABP associates with the grk transport complex in an inactive form that is remodeled following its anchorage at the dorsal–anterior of the oocyte (Clouse, 2008).

Previous studies have shown that Bruno (Bru) binds directly to Cup protein and is required for the translational repression of osk. Bru binds to specific sequence elements in the osk 3′ UTR called Bruno Response Elements (BREs), and mutations in these BREs have been shown to reduce Bru binding and result in ectopic Osk accumulation in the oocyte. Similarly, Bru has also been shown to bind to grk mRNA and to Sqd protein. Overexpression of bru cDNA leads to ventralization of the eggshell, consistent with reduced Grk protein expression in the oocyte. Furthermore, disrupting bru expression in certain genetic contexts has been shown to result in excess Grk protein in the oocyte, consistent with Bru being required to mediate grk translational repression. In light of the results presented in this study, it is proposed that this phenotype is the result of Bru-mediated repression of grk translation by Cup (Clouse, 2008).

The mechanism of grk translation and the trans-acting factors required for translational control largely parallel the mechanism employed by osk RNA, so an important question to be answered is how these two different RNAs are differentially transported and translationally regulated in distinct parts of the oocyte at the appropriate stage in oogenesis. Since the same group of trans-acting factors is involved in the expression of both RNAs, the specificity could be provided by cis-acting sequences within the RNA molecules themselves that affect the activity of common trans-acting factors. Alternatively, RNA-specificity could be generated by as-yet unidentified trans-acting factors. Given that Enc functions in grk translational activation, but is not required for osk translational activation, it is possible that Enc is providing some degree of specificity to the commonly used machinery that mediates translational control of multiple, unrelated transcripts. Currently, Enc is the only factor known to function uniquely in the translational activation of grk mRNA, and these results provide the first evidence of a link between this factor and the general translational control machinery that is used by multiple RNAs in oogenesis (Clouse, 2008).

UAP56 RNA helicase is required for axis specification and cytoplasmic mRNA localization in Drosophila

mRNA export from the nucleus requires the RNA helicase UAP56 (Helicase at 25E) and involves remodeling of ribonucleo-protein complexes in the nucleus. This study shows that UAP56 is required for bulk mRNA export from the nurse cell nuclei that supply most of the material to the growing Drosophila oocyte and for the organization of chromatin in the oocyte nucleus. Loss of UAP56 function leads to patterning defects that identify uap56 as a spindle-class gene similar to the RNA helicase Vasa. UAP56 is required for the localization of gurken, bicoid and oskar mRNA as well as post-translational modification of Osk protein. By injecting grk RNA into the oocyte cytoplasm, this study shows that UAP56 plays a role in cytoplasmic mRNA localization. It is proposed that UAP56 has two independent functions in the remodeling of ribonucleo-protein complexes. The first is in the nucleus for mRNA export of most transcripts from the nucleus. The second is in the cytoplasm for remodeling the transacting factors that decorate mRNA and dictate its cytoplasmic destination (Meignin, 2008).

UAP56 is a conserved member of the DExH/D RNA helicase superfamily implicated in many aspects of RNA metabolism including general mRNA export from the nucleus. In human cells, UAP56 is preferentially associated with spliced mRNA and has a major role in bulk mRNA export (Gatfield, 2001). Furthermore, Xenopus UAP56 and its yeast homologue Sub2p are thought to be required co-transcriptionally for the recruitment of the mRNA export factor Aly/REF (see Drosophila Aly) to mRNA. In yeast, the THO complex, which functions in transcription elongation, interacts with mRNA export factors to form the TREX (TRanscription/EXport) complex, linking transcription and export. The human THO complex becomes associated with spliced mRNA during the course of splicing. In Drosophila cells, UAP56 has been shown to be essential for the export of both spliced and intronless poly(A)+ mRNAs. Drosophila uap56 is an essential gene that was first identified as an enhancer of position effect variegation and encodes a nuclear protein named Hel25E/UAP56. UAP56 is proposed to promote an open chromatin structure by unwinding or releasing the mRNA from the site of transcription. It is also thought to be involved in regulating the spread of heterochromatin (Meignin, 2008).

During Drosophila oogenesis, the antero-posterior and dorso-ventral axes of the future embryo are specified through the cytoplasmic localization and translational regulation of a large number of specific transcripts. The most extensively studied mRNAs are gurken (grk) that encodes a TGFβ signal, bicoid (bcd) that encodes the anterior morphogen, and oskar (osk), which specifies posterior structures and the future germ line. All transcripts in the oocyte are thought to be transcribed in the nurse cell nuclei and transported through actin-rich ring canals into the oocyte, where they are selectively localized by transport on microtubules (MTs) by molecular motors. Some of these mRNAs are then localized within the oocyte cytoplasm. During their complex path of localization, these transcripts are thought to be present in large RNP complexes that contain a variety of RNA binding proteins. The composition of these complexes is thought to vary during the different steps of the biosynthesis, export and localization of the transcripts, and RNA helicases play essential roles in remodeling the RNPs during RNA processing, transport, localization, anchoring and translation (Meignin, 2008).

RNA helicases are also likely to be involved in remodeling a diverse range of trans-acting factors required for mRNA localization. These include cytoplasmic determinants and motor cofactors such as BicaudalD (BicD) and Egalitarian (Egl) as well as nuclear components such as hnRNPs or splicing factors. Such trans-acting factors are thought to dictate which molecular motor the transcripts associate with and therefore their cytoplasmic destination. For example, in the Drosophila oocyte, oskar (osk) mRNA requires Kinesin 1 to transport it to the plus ends of MTs while grk mRNA requires cytoplasmic Dynein (Dynein) to transport it to the minus ends of MTs (Meignin, 2008).

In some cases, factors recruited to the RNA in the nucleus remain with the RNA and function in the cytoplasm. For example, the nuclear exon-exon junction components (EJC) Mago nashi, Y14 and eIF4AIII are recruited by osk transcripts in the nucleus and then play a role in its cytoplasmic localization. Once at its final destination at the posterior pole, osk mRNA is translated using two distinct initiation codons, resulting in two different proteins. The short form of Osk promotes the assembly of polar granules by recruiting Vasa, a member of DExH/D-box family of putative RNA helicases at the posterior pole of the oocyte. Vasa is also required for promoting translation of grk through an interaction with the translation factor eIF5B/dIF2. In contrast, grk transcripts are able to recruit in the cytoplasm all the factors required for their localization (Meignin, 2008).

In Drosophila, at least 12 genes are members of the DExH/D RNA helicase superfamily. One of the best described, vasa, was originally identified as a member of the posterior class of maternal effect genes. Vasa is required for the translation of osk and nos mRNAs during the assembly of the pole plasm and for the localization and translation of grk mRNA during oogenesis. Other RNA helicases play a role during Drosophila oogenesis; belle (bel), hel25E or uap56, spindle E (spn-E) and eIF-4AIII. In addition, the small repeat-associated siRNAs (rasiRNAs) pathway, containing spn-E, aubergine and armitage, is required for axis specification. In many of these cases, the intracellular localization and translational regulation of bcd, osk and grk mRNA are affected to varying degrees (Meignin, 2008).

This study tested whether the RNA helicase UAP56 is required in the cytoplasm for mRNA localization and post-translational modification in addition to its well-studied roles in the nucleus in splicing and mRNA export. By creating new alleles of the gene, it was shown that uap56 is a spindle-class gene. uap56 mutants have strong Dorso-ventral egg shell defects caused by a mislocalization of grk mRNA and its incorrect translational regulation. grk mRNA injected into the oocyte cytoplasm of uap56 mutants fails to localize correctly, suggesting that the uap56 phenotype is due to a lack of a factor required in the cytoplasm for mRNA localization. It was also shown that UAP56 plays a role in osk and bcd mRNA localization and Osk post-translational modification. Thus UAP56 plays multiple roles in mRNA localization and post-translational modification in the oocyte cytoplasm. It is proposed that UAP56 is required for remodeling of cytoplasmic RNP complexes required for mRNA localization and post-translational modification (Meignin, 2008).

UAP56 is an RNA helicase that has been shown to play important roles in mRNA metabolism in the nucleus. In Drosophila, UAP56 is a component of the Exon Junction Complex (EJC) (Gatfield, 2001) and is required in tissue culture cells for bulk mRNA export from the nucleus. Using new mutations in the gene, this study has shown that UAP56 is also required for bulk mRNA export during Drosophila oogenesis. UAP56 also has a novel and unexpected role in mRNA localization and post-translational modification in the cytoplasm since uap56 mutants have defects in grk, osk and bcd mRNA localization and Osk post-translational modification. uap56 mutants show strong dorso-ventral egg shell defects due to disruption in cytoplasmic transport of grk mRNA, in addition to other phenotypes that display phenotypes that define uap56 as a spindle-class gene, like the RNA helicase and posterior group gene vasa. Therefore, these data have uncovered a new cytoplasmic role for UAP56 in mRNA transport and post-translational modification. It is proposed that UAP56 is required in the oocyte cytoplasm to remodel RNP complexes involved in mRNA localization and post-translational modification (Meignin, 2008).

The analysis of new alleles of the uap56 gene has revealed that UAP56 is required for general nuclear mRNA export from the nurse cell nuclei. This explains why it was impossible to study the uap56 null mutants during oogenesis since a total block in mRNA export causes cell lethality. The current observations are in good agreement with the previous work showing that UAP56 is essential for bulk mRNA export in Drosophila tissue culture cells and other model systems. In wild-type, the intracellular distribution of bulk polyadenylated RNAs reveals a cytoplasmic localization with an accumulation in the Nuage. In contrast, this distribution is disrupted in uap56 mutants. During Drosophila oogenesis, the Nuage is characterized by electron dense germ line specific structures that form around the nurse cell nuclei. It is suggested that the Nuage someway facilitates the assembly of mRNP particles that mediate the transport, translational regulation and storage of specific transcripts. The Nuage contains Vasa, Maelstrom, Aubergine and Belle, all thought to be required for axis specification in the oocyte. Most components of the Nuage are also localized at the posterior pole of the Drosophila oocyte. This study found that poly(A)+ RNA is present at high levels in particles in the Nuage and that UAP56 is involved in the formation of the Nuage since Vasa protein is absent from the Nuage in uap56 mutants. However, UAP56 is not specifically localized in the Nuage nor at the posterior pole, so UAP56 is neither a posterior group gene nor a component of the Nuage. These observations are interpreted as indicating that UAP56 is required upstream of Nuage and pole plasm formation. It is proposed that UAP56 is required to facilitate the correct assembly of different mRNAs, including grk and osk with the appropriate RNA binding proteins in RNPs, before, during and possibly after export from the nurse cell nuclei. This assembly is crucial for the downstream events responsible for the transport and assembly of the RNPs into the Nuage (Meignin, 2008).

Interestingly, a small fraction of poly(A)+ RNA was found in the oocyte nucleus, an observation that could be explained in two possible ways. Either a sub-population of RNA is transcribed in the oocyte nucleus or a sub-population of poly(A)+ RNA is transported from nurse cells to oocyte and then imported into the oocyte nucleus. The second explanation is favored for the following reasons. First, it is thought that the oocyte nucleus is transcriptionally inactive. Second, the I factor mRNA is known to be synthesized in the nurse cells, transported into the oocyte and then imported into the oocyte nucleus. While the genetic background that was studied is not very active for I factor transcription, it is possible that the poly(A)+ RNA detected in the oocyte nucleus represents transcripts of other transposable elements or some endogenous transcripts that follow the same pattern of biogenesis and import into the oocyte nucleus (Meignin, 2008).

During oogenesis and early embryogenesis, the majority of UAP56 protein is present in the nucleus and is probably associated with DNA. These results are consistent with previous work showing that UAP56 is closely associated with salivary gland chromosomes and localized to the nuclei of Drosophila embryos and ovaries as well as acting as an enhancer of position affect variegation in Drosophila and affecting heterochromatic gene expression in yeast. These data together with the conclusion that uap56 is a spindle-group gene strongly suggest that the gene is involved in chromatin organization (Meignin, 2008).

In yeast, Sub2p associates with the TREX (TRansport-EXport) complex, which is required directly for transcription elongation, splicing and export and is recruited during transcription. In contrast, the mammalian TREX complex is recruited during splicing. It is proposed that, in Drosophila, UAP56 has an intermediate role between yeast and human cells. It is likely to bind mRNA during transcription and be involved in splicing in a similar manner to nonsense-mediated mRNA decay (NMD) (Meignin, 2008).

The new alleles of uap56 reveal a role of UAP56 in mRNA localization and translational modification in the cytoplasm, key processes in axis specification. A reduction in the intensity of tau GFP staining in the oocyte was found, although the antero-posterior gradient, which is essential for mRNA localization, was unaffected. While the possibility cannot be excluded that the reduction in efficiency of localization of injected grk mRNA is due to a lower density of MTs in the oocyte, the alternative interpretation is favored, in which UAP56 plays a more direct cytoplasmic role in promoting remodeling of factors required for grk RNA localization (Meignin, 2008).

The observations in the context of the previous work on UAP56 suggest that, as well as being a general RNA export factor in Drosophila, the protein has a specific role in the localization and post-translational modification of a sub-population of key transcripts. In this respect, UAP56 is similar to some other components of the EJC, such as Mago nashi, Y14 and eIF4AIII, which are involved in mRNA localization and post-translation control of a subset of Drosophila transcripts. Interestingly, mutants in the small repeat-associated siRNAs (rasiRNAs) pathway also show similar defects in axis specification to uap56 mutants, raising the possibility that UAP56 is a component of the rasiRNA pathway. Nevertheless, the results are surprising, given that UAP56 is thought of as a ubiquitous house keeping gene with an essential function in the export of all mRNAs. A partial loss-of-function of an equivalent essential general mRNA export factor, NXF1, does not give rise to specific developmental defects, such as the ones observed with uap56 alleles. rasiRNA mutants show an accumulation of double strand breaks in egg chambers. However, no differences were found in the accumulation of H2A staining of double strand breaks between wild type and mutant germaria, in contrast to armi, aub and spn-D mutants. It is concluded that the defects observe in axis specification in uap56 mutants are not due to double strand breaks perturbing signaling in the germ line. Therefore, it is proposed that, unlike NXF1, UAP56 is likely to remain on the RNA after export and has a role in the remodeling of RNP complexes in the cytoplasm. This idea is supported by the fact that UAP56 is detected in the cytoplasm as well as the nucleus. However, no UAP56 colocalized is observed with mRNA in the cytoplasm or recruited by injected RNA. These observation are interpreted as indicating that UAP56 may only associate with RNA transiently in the cytoplasm, while acting as a cytoplasmic RNA remodeling factor. Such a role is novel for UAP56, and it remains to be discovered whether it is a general feature of this RNA dependent helicase in a variety of model systems (Meignin, 2008).

The HP1 homolog Rhino is required for transposon silencing and affects chromosome structure and egg polarity

Piwi-interacting RNAs (piRNAs) silence transposons and maintain genome integrity during germline development. In Drosophila, transposon-rich heterochromatic clusters encode piRNAs either on both genomic strands (dual-strand clusters) or predominantly one genomic strand (uni-strand clusters). Primary piRNAs derived from these clusters are proposed to drive a ping-pong amplification cycle catalyzed by proteins that localize to the perinuclear nuage. This study shows that the HP1 homolog Rhino is required for nuage organization, transposon silencing, and ping-pong amplification of piRNAs. rhi mutations virtually eliminate piRNAs from the dual-strand clusters and block production of putative precursor RNAs from both strands of the major 42AB dual-strand cluster, but not of transcripts or piRNAs from the uni-strand clusters. Furthermore, Rhino protein associates with the 42AB dual-strand cluster, but does not bind to uni-strand cluster 2 or flamenco. Rhino thus appears to promote transcription of dual-strand clusters, leading to production of piRNAs that drive the ping-pong amplification cycle (Klattenhoff, 2009).

Drosophila piRNA pathway mutations lead to germline DNA damage and disrupt axis specification through activation of Chk2 and ATR kinases, which function in DNA damage signaling. Mutations in the rhi locus lead to very similar patterning defects (Volpe, 2001). The mei-41 and mnk genes encode ATR and Chk2, respectively. To determine whether the axis specification defects associated with rhi result from damage signaling, double mutants were generated with mnk and mei-41 and axis specification was quantified by scoring for assembly of dorsal appendages, which are egg shell structures that form in response to dorsal signaling during oocyte development. Only 17% of embryos from rhiKG/rhi2 females had two wild-type appendages. However, 80% of embryos from mnk;rhiKG/rhi2 double-mutant females had two appendages. In addition, 33% of embryos from mei-41;rhiKG/rhi2 double-mutant females had two appendages. Consistent with these observations, rhi mutations disrupt dorsal localization of Gurken and posterior localization of Vasa in the oocyte, and localization of both proteins is restored in mnk; rhiKG/rhi2 double mutants (Klattenhoff, 2009).

Drosophila javelin-like encodes a novel microtubule-associated protein and is required for mRNA localization during oogenesis

Asymmetrical localization of mRNA transcripts during Drosophila oogenesis determines the anteroposterior and dorsoventral axes of the Drosophila embryo. Correct localization of these mRNAs requires both microtubule (MT) and actin networks. This study identified a novel gene, CG43162, that regulates mRNA localization during oogenesis and also affects bristle development. The Drosophila gene javelin-like, which was identified based on its bristle phenotype, is an allele of the CG43162 gene. Temale mutants for jvl produce ventralized eggs owing to the defects in the localization and translation of gurken mRNA during mid-oogenesis. Mutations in jvl also affect oskar and bicoid mRNA localization. Analysis of cytoskeleton organization in the mutants reveal defects in both MT and actin networks. Jvl protein colocalizes with MT network in Schneider cells, in mammalian cells and in the Drosophila oocyte. Both in the oocyte and in the bristle cells, the protein localizes to a region where MT minus-ends are enriched. Jvl physically interacts with SpnF and is required for its localization. Overexpression of Jvl in the germline affects MT-dependent processes: oocyte growth and oocyte nucleus anchoring. Thus, these results show that a novel MT-associated protein affects mRNA localization in the oocyte by regulating MT organization (Dubin-Bar, 2011).

In order to investigate further the role of Spn-F in MT organization, new proteins that interact with Spn-F or Ik2 were sought. This study led to identification of the gene CG43162 as a novel MT-associated protein, which is part of this complex. Moreover, the study showed that CG43162 encodes the javelin-like (jvl) gene. Several lines of evidence suggest that that jvl encodes the CG43162 gene: (1) Using fine deficiency mapping of jvl mutants showed that jvl is found in CG43162 region; (2) it was shown that downregulation of CG43162 specifically in the bristles led to defects in bristle morphology, similar to the defects found in jvl mutants; 3) furthermore, a mutation in CG43162 (CG43162D590) failed to complement jvl in both ovarian and bristle phenotypes, suggesting that CG43162D590 and jvl are two different alleles of the same gene; and (4) expression of CG43162 protein in oocytes was found to rescue jvl female sterility. Considering all of these results, it is concluded that the CG43162 gene encodes jvl (Dubin-Bar, 2011).

Moreover, it is suggested that Jvl is part of the Spn-F and Ik2 complex, based on the following evidence: (1) Spn-F physically interacts with Jvl (yeast two hybrid and GST pull-down assays); (2) Spn-F physically interacts with Ik2 (Dubin-Bar, 2008); (3) jvl shares similar mRNA localization and bristle defects to spn-F and ik2; (4) Spn-F and Ik2 colocalize with Jvl to MT, where Jvl determines this localization pattern (Dubin-Bar, 2011).

For further analysis of the jvl gene, Jvl protein localization was characterized. For this purpose, the localization of Jvl protein in S2R+ cells and human cells was analyzed. GFP-Jvl fusion protein was localized to the MT network. Next, the localization pattern of Jvl during oogenesis was analyzed. Using an antibody raised against the Jvl protein, it was found that Jvl is localized to the region where the MT minus-ends reside. At early stages of oogenesis, Jvl protein localizes as a tight crescent in the posterior pole of the oocytes. During mid-oogenesis, Jvl protein is localized all around the cortex, with enrichment at the anterior pole. It was also demonstrated that GFP-Jvl colocalizes with MTs in the nurse cells. Moreover, in the bristles, GFP-Jvl is localized asymmetrically, accumulating at the bristle tip, where other MT minus-end markers are found. Considering these results, indicating that Jvl localizes with the MT network in S2R+ and human cells along with its localization in the egg chamber and developing bristle, it is concluded that Jvl protein is associated with the MT network, specifically with the MT minus-ends (Dubin-Bar, 2011).

jvl1 mutants are female fertile. However, flies hemizygous for jvl1 and flies transheterozygous for jvl (jvl1/jvl2) are female sterile. Beside sterility, it was noticed that the jvl mutant females laid eggs with dorsal-ventral defects. Determination of dorsal-ventral polarity of the eggshell depends on Grk protein signaling. In the hemizygous mutants, grk mRNA localizes in the anterior margins of the oocyte and in ectopic sites inside the oocyte. It has been suggested that grk mRNA moves in two distinct steps, both of which require MT and the motor protein Dynein. Each step depends on a different MT network. First grk mRNA moves towards the anterior of the oocyte, where it localizes transiently, and then to its final localization in the dorsal anterior corner of the oocyte. In jvl mutants, grk mRNA does not reach its final localization in the dorsal anterior corner of the oocyte, suggesting that the MT network upon which this step depends might be impaired in jvl mutants. This MT network is specifically associated with the oocyte nucleus and the minus-end in the dorsal-anterior corner of the oocyte. Next, it was found that Grk protein in jvl mutants is also mislocalized. Grk protein is colocalized with ectopic actin puncta close to the anterior of the oocyte. This localization pattern is also observed in Bicaudal-C and trailer-hitch mutants. It has been suggested that the sequestration of Grk in the actin cages interfered with the signaling to the follicle cells; therefore, it is suggested that sequestration of Grk in the actin cages in jvl mutant females similarly led to the dorsal-ventral polarity defects of the eggshell. In addition to the effect on grk mRNA and protein localization, jvl also affects bcd and osk mRNA localization. In wild-type, bcd mRNA is localized to the anterior pole of the oocyte facing the nurse cells, whereas osk mRNA is localized to the opposite posterior pole. The polar localization of these two mRNAs is maintained throughout the rest of oogenesis and well into early embryogenesis. The anterior localization of bcd requires both intact MTs and dynein motor protein function. osk localization to the posterior pole is achieved by two phases of transport: long-range MT-dependent transport by kinesin to the posterior, followed by actomyosin V-dependent positioning at the oocyte cortex (Dubin-Bar, 2011).

What could be the function of Jvl protein during oogenesis? The effects of jvl on grk and bcd mRNA localization, along with the particular changes affecting cytoskeletal organization close to the oocyte nuclear membrane as evident for Nod:KHC:β-gal localization and Tau mislocalization, suggest that jvl might be involved in either transport to the minus-end of MTs or in the organization of the minus-ends of the microtubule around the oocyte nucleus, as been suggested for its interactor, Spn-F (Abdu, 2006). However, it was also noticed that in jvl mutants, osk mRNA and protein are mislocalized. These phenotypes are probably not due to defects in either transport or organization of the MT plus-end, as the plus-end motor protein Kinesin I was properly localized as in the wild type. Examination of the cytoskeleton components of the oocyte shows that both actin and MTs are misorganized in jvl mutants. The MT levels along the anterior cortex of the oocyte were reduced with specific effects on the MT that surrounds the oocyte nucleus. However, ectopic aggregations of the actin cages were found in the middle of the oocyte. The defects in the organization of both actin and MT network, together with the defects in osk mRNA and protein localization, suggest that jvl could provide a connection between the actin and MT network. In summary, these results suggest that jvl plays a role in organization of the MT in the oocyte or in the stabilization of the connection between MT and actin cytoskeleton in the oocyte (Dubin-Bar, 2011).

This study also examined the effects of overexpression of Jvl in the germline. Overexpression of Jvl with different germline-specific Gal 4 affects oocyte growth, oocyte localization and, in later stages, oocyte nucleus localization. Interestingly enough, all of these phenotypes could arise from effects on MT network function (Dubin-Bar, 2011).

Oocyte growth depends on several processes: early in oogenesis, until stage 7, the oocyte grows at approximately the same rate as a single nurse cell. At these stages, oocyte growth is due to the transport of mRNAs and proteins, including products of early pattern-formation genes from the nurse cells to the oocyte. This transport is a microtubule-dependent process. Later in oogenesis, after stage 7, oocyte growth depends on the transport of components such as lipid droplets, mitochondria and other single particles from the nurse cells into the oocyte. This transport is an actin-dependent process. Beginning in stage 8, the oocyte expands through the uptake of yolk from the surrounding follicle cells and hemolymph. Consequently, oocyte growth is more rapid than nurse cell growth. During stage 11, the remaining nurse cell cytoplasm is rapidly transferred to the oocyte, resulting in doubling the oocyte volume. Overexpression of Jvl affects oocyte growth during stage 6 to stage 8, although the egg chamber size seems to be similar to that of wild-type stage 6 to 8 egg chambers. In these stages, oocyte growth depends on the transport of nutrients from the nurse cells to the oocyte, suggesting that overexpression of Jvl disrupted this transport. The fact that Orb protein is not detected in Jvl-overexpressing small oocytes strengthens this possibility (Dubin-Bar, 2011).

Another phenotype that was obtained in moderate overexpression of Jvl is mislocalization of the oocyte nucleus in 15% of stage 9 egg chambers. During early stages of oogenesis, the oocyte nucleus localizes to the posterior pole of the oocyte. After stage 7, following Grk signal and reorganization of the MT network, the nucleus migrates towards the anterodorsal corner of the oocyte. Positioning of the oocyte nucleus involves two anchoring steps: first anchoring to the lateral membrane, which requires dynein but not kinesin motor protein; and, second, after it localizes to the anterodorsal corner, anchoring to the anterior cortex of the oocyte, which requires both dynein and kinesin motor proteins. Moreover, nucleus anchoring also requires correct organization of the MT scaffold that surrounds the oocyte nucleus. Moderate expression of Jvl did not affect nucleus position in stage 8 egg chambers. At this stage, the nucleus was always at the dorsal anterior corner, as in the wild type. This finding implies that anchoring to the lateral cortex and migration of the oocyte nucleus is not affected in Jvl-overexpressing ovaries. However, the anchoring of the nucleus to the anterior membrane was affected. This could be due to misorganization of the MT scaffold that surrounds the nucleus. Thus, these results demonstrate that overexpression of Jvl protein affects MT-dependent processes such as transport of determinants from the nurse cells to the oocyte, and anchoring of oocyte nucleus to the anterior cortex of the oocyte. Taking into account the phenotypes detected in jvl mutants, the finding that Jvl is an MT-associated protein, together with the effects of Jvl overexpression on MT-dependent processes during oogenesis, it seems likely that jvl has a role in MT organization during oogenesis (Dubin-Bar, 2011).

Most importantly, although jvl encodes for a protein with no homology beside insects, its association with MT network in mammalian cells, along with its effect on MT network in Drosophila, may suggest the existence of mammalian protein(s) with a function analogous to Jvl (Dubin-Bar, 2011).

Drosophila Syncrip binds the gurken mRNA localisation signal and regulates localised transcripts during axis specification

In the Drosophila oocyte, mRNA transport and localised translation play a fundamental role in axis determination and germline formation of the future embryo. gurken mRNA encodes a secreted TGF-alpha signal that specifies dorsal structures, and is localised to the dorso-anterior corner of the oocyte via a cis-acting 64 nucleotide gurken localisation signal. Using GRNA chromatography, this study characterised the biochemical composition of the ribonucleoprotein complexes that form around the gurken mRNA localisation signal in the oocyte. A number of the factors already known to be involved in gurken localisation and translational regulation, such as Squid and Imp, were identified, in addition to a number of factors with known links to mRNA localisation, such as Me31B and Exu. Previously uncharacterised Drosophila proteins were identified, including the fly homologue of mammalian SYNCRIP/hnRNPQ, a component of RNA transport granules in the dendrites of mammalian hippocampal neurons. It was shown that Drosophila Syncrip binds specifically to gurken and oskar, but not bicoid transcripts. The loss-of-function and overexpression phenotypes of syncrip in Drosophila egg chambers show that the protein is required for correct grk and osk mRNA localisation and translational regulation. It is concluded that Drosophila Syncrip is a new factor required for localisation and translational regulation of oskar and gurken mRNA in the oocyte. It is proposed that Syncrip/SYNCRIP is part of a conserved complex associated with localised transcripts and required for their correct translational regulation in flies and mammals (McDermott, 2012).

This study has identified Drosophila Syp as a novel conserved component of localised RNP granules. Syp associates specifically with grk and osk and is required for their localisation and translational regulation in the Drosophila germline. Although SYNCRIP has been studied in mammalian cells using biochemical approaches, this study is the first to address the function of Syp in vivo, and particularly in generating cellular asymmetry in the germline. Despite a number of genetic screens that have been carried out to identify the genes required for axis specification in flies, Syp was not previously identified as being required for axis determination. This work together with a number of other biochemical based studies illustrates that there are many other essential factors that are still to be identified as having a role in axis specification (McDermott, 2012).

Evidence is presented in this study that Syp is required for axis specification and germline formation by affecting the localisation and translation of grk and osk mRNAs. The phenotypes of loss of function mutations in the gene, and overexpression of the protein support an interesting role for Syp in regulating grk and osk mRNAs. It is noted that the loss of function phenotypes are of low penetrance and are such that further studies are required to uncover the precise mechanism of Syp function. However, Syp is not the only component of grk and osk mRNPs that has a partially penetrant loss of function phenotype. Indeed, others such as IGF-II mRNA-binding protein (Imp) give a stronger phenotype only when in combination with other mutations. Unexpectedly, overexpression of Syp gives the same phenotype as its loss of function, but at a higher penetrance. These results are potentially interesting, but difficult to interpret with certainty. The interpretation is favoured that a certain stoichiometry is necessary within the RNP complexes in which Syp is found. Overexpression of Syp may cause the displacement of certain translational repressors, allowing grk mRNA to be prematurely translated in the nurse cells. In contrast, with loss of function of Syp, grk mRNA localisation and translational regulation may be disrupted in different ways because loss of Syp could lead to decreased stability of the RNP complex. This could in turn lead to the loss of certain components necessary for localization and translational regulation. The eggshell phenotypes observed in the syp germline clones also include some defects that are not typical of a disruption of grk mRNA localisation and translation. These results are interpreted as indicating that Syp has additional target mRNAs in the germline whose localisation and/or translation are affected in the syp mutant. On the basis of the morphological defects observed in a number of eggs these targets may include mRNAs involved in follicle cell migration or actin organisation during oogenesis, such as bullwinkle (bwk) or chickadee (chic). Mislocalisation and/or altered translation of these mRNAs could result in the shorter eggs and the bwk-like dorsal appendage defects that are observed (McDermott, 2012).

The physical association of Syp with these mRNAs further supports a function for Syp in the regulation of grk and osk mRNAs, although it is unclear whether this is through a direct or indirect interaction. Syp does not appear to colocalise with localised grk or osk in the oocyte, and so Syp may function before the mRNAs reach their final destination in the oocyte in order to influence localisation and translation. Syp was also identified biochemically with a number of the factors already known to be required for grk and osk mRNA localisation and translational regulation. These include Sqd, Imp, PTB, PABP, Me31B and BicC. The homologues of these factors were also identified in biochemical studies of SYNCRIP interactors in mammalian cells. Therefore, it is proposed that the current results have uncovered a conserved module of RNA binding proteins that are required for both mRNA transport and translational regulation. The Syp associated complex that this study has uncovered binds to grk via a relatively small stem loop sequence, the GLS. While Syp also associates with osk mRNA, the minimal region necessary and sufficient for osk mRNA localisation has not yet been defined. Therefore, it is unknown whether a small region is required in this case, and if so whether it is in any way similar to the GLS, structurally or in primary sequence (McDermott, 2012).

Taking the results of this study in the context of other data and the previous publications on SYNCRIP and its associated proteins in mammalian hippocampal neurons, it is proposed that Syp may be present at neuronal synapses in a complex with at least some of the same proteins that are required for grk mRNA localisation. This idea is supported by the fact that at least some of these factors, namely Sqd, Imp, PTB, PABP and Me31B are also present in the Drosophila nervous system. Our expression studies show that Syp is absent from embryos but is highly expressed in the larval nervous system. Given the role of Syp in mRNA localization and translational regulation in the oocyte, and its presence in larval brains, it is proposed that Syp could have a similar function in the nervous system. In comparison with the oocyte, much less is known about localised transcripts and their translational regulation in the nervous system, and it remains to be determined to what extent this proposal is valid and in which neuronal tissues Syp is required in larvae. Nevertheless, this work is the first demonstration that Syp functions in mRNA localisation and translational control in the oocyte and coupled with the work on mammalian SYNCRIP showing association with RNP granules in the dendrites of hippocampal neurons it is attractive to propose that Syp protein also has a conserved function in regulation of neuronal mRNAs (McDermott, 2012).

asunder is required for dynein localization and dorsal fate determination during Drosophila oogenesis

asunder (asun) is a critical regulator of dynein localization during Drosophila spermatogenesis. Because the expression of asun is much higher in Drosophila ovaries and early embryos than in testes, this study sought to determine whether ASUN plays roles in oogenesis and/or embryogenesis. The female germline phenotypes were characterized of flies homozygous for a null allele of asun asund93. asund93 females were found to lay very few eggs and contain smaller ovaries with a highly disorganized arrangement of ovarioles in comparison to wild-type females. asund93 ovaries also contain a significant number of egg chambers with structural defects. A majority of the eggs laid by asund93 females are ventralized to varying degrees, from mild to severe; this ventralization phenotype may be secondary to defective localization of gurken transcripts, a dynein-regulated step, within asund93 oocytes. Dynein localization was found to be aberrant in asund93 oocytes, indicating that ASUN is required for this process in both male and female germ cells. In addition to the loss of gurken mRNA localization, asund93 ovaries exhibit defects in other dynein-mediated processes such as migration of nurse cell centrosomes into the oocyte during the early mitotic divisions, maintenance of the oocyte nucleus in the anterior-dorsal region of the oocyte in late-stage egg chambers, and coupling between the oocyte nucleus and centrosomes. Taken together, these data indicate that asun is a critical regulator of dynein localization and dynein-mediated processes during Drosophila oogenesis (Sitaram, 2014).

A Genetic Screen Based on In Vivo RNA Imaging Reveals Centrosome-Independent Mechanisms for Localizing gurken Transcripts in Drosophila

Chromosome arm 3L of Drosophila was screened for EMS-induced mutations that disrupt localization of fluorescently-labeled gurken (grk) mRNA, whose transport along MTs establishes both major body axes of the developing Drosophila oocyte. Rapid identification of causative mutations by SNP recombinational mapping and whole genomic sequencing resulted in the definition of nine complementation groups affecting grk mRNA localization and other aspects of oogenesis, including alleles of elg1, scaf6, quemao, nudE, Tsc2/gigas, rasp and Chd5/Wrb, and several null alleles of the armitage Piwi-pathway gene. Analysis of a newly-induced kinesin light chain (klc) allele shows that kinesin motor activity is required for both efficient grk mRNA localization and oocyte centrosome integrity. It was also shown that initiation of the dorso-anterior localization of grk mRNA precedes centrosome localization, suggesting that MT self-organization contributes to breaking axial symmetry to generate a unique dorsoventral axis (Hayashi, 2014).

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