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Splicing of EXU mRNA

The Drosophila exuperantia gene encodes overlapping sex-specific, germline-dependent mRNAs. In this work, the structural differences between these sex-specific EXU mRNAs were determined by sequence analysis of 9 ovary and 10 testis cDNAs. The transformer 2 gene functions in sex determination of female somatic cells through its role in regulating female-specific splicing of Doublesex. tra-2 is required in male germ cells for efficient male-specific processing of EXU RNA; in the absence of tra-2, X/Y males produce a new mRNA that is processed at its 3' end so that it contains sequences normally specific to the female 3' untranslated region. Although the processing event that requires tra-2 occurs in an untranslated region of the EXU transcript, the isolation and characterization of a male-specific exu allele that deletes male 3' untranslated sequence indicates that this processing is biologically significant (Hazelrigg, 1994).

Interactions of Exu with mRNA

Localization of mRNAs, a crucial step in the early development of some animals, has been shown to be directed by cis-acting elements that presumably interact with localization factors. Exu binds to BLE1, an RNA localization element from the Drosophila Bicoid mRNA. Using mutations in BLE1, a correlation has been found between in vitro Exuperantia-like (Exl) binding (Exl has not been cloned) and an early previtellogenic phase of in vivo localization directed by BLE1, implicating Exl in that localization event. The same phase of localization is disrupted in exuperantia mutants, suggesting that exl and exuperantia proteins interact (Macdonald, 1995).

A cis-acting signal in the 3' UTR of the Drosophila Bicoid mRNA directs both the transport of the mRNA from the nurse cells to the oocyte and its anterior localization within the oocyte. The signal mediates redundant RNA recognition events, A and B, that initiate largely overlapping programs of mRNA localization during oogenesis. Recognition event A requires a region encompassing stem-loops IV/V of the predicted secondary structure, and can be eliminated by a single nucleotide mutation. Localization initiated through event B begins slightly later in oogenesis, and requires sequences that have not been narrowly defined. Using forms of the 3' UTR lacking this RNA recognition redundancy, the roles of the swallow, staufen, and exuperantia genes, which are all required for normal bicoid mRNA localization, were reexamined. The results reveal that exuperantia first becomes essential for localization at a time when well-defined microtubule tracks between the nurse cells and oocyte disappear. Thus, exuperantia may specifically facilitate a form of nurse cell-to-oocyte mRNA transport not dependent on the microtubule tracks (Macdonald, 1997).

Exu is involved in OSK mRNA localization

Whole mount in situ hybridization was performed on ovaries from wild-type and exu null females to determine whether exu is required for any aspect of OSK mRNA localization. In ovaries from wild-type females, OSK mRNA is often concentrated in apical patches in the nurse cells of stage 9 and 10 egg chambers. In contrast, OSK mRNA was dispersed in the nurse cells of exu egg chambers of the same stages. Thus, exu is required for OSK mRNA to be correctly localized within nurse cells. A role for Exu in OSK mRNA localization is consistent with several other findings: (1) Exu accumulates at both the anterior and posterior poles of the oocyte; (2) OSK mRNA transiently accumulates at the anterior pole along with BCD mRNA before its transport to the posterior; (3) one of the effects of exu mutants is to disrupt the localization of OSK mRNA to apical patches within nurse cells. This defect is identical to the nurse cell localization defect described for BCD mRNA in exu mutants and suggests that this step in the localization pathway may be common to both transcripts (Wilhelm, 2000 and references therein).

One of the reasons that exu mutants have not been examined previously for defects in OSK mRNA localization is that only a small percentage of embryos from exu mothers display posterior patterning defects. The examination of exu mutants reveals that the amount of OSK mRNA localized to the posterior pole is decreased in these mutants, suggesting that exu plays a role in localizing OSK mRNA within oocytes. However, since this defect is only partially penetrant, Exu-dependent posterior localization within the oocyte may be redundant with other localization mechanisms. In addition, the posterior patterning defects associated with the decrease in OSK mRNA localization in exu mutants during stages 9 and 10 of oogenesis may be rescued by localization of OSK mRNA during cytoplasmic streaming later in oogenesis. In support of this idea it has been shown that injected, fluorescently labeled OSK mRNA can be localized to the posterior at the time when cytoplasmic streaming occurs. Such localization most likely occurs by random motion during cytoplasmic streaming and specific anchoring of OSK mRNAs that come in contact with the posterior pole. These multiple mechanisms of localizing OSK mRNA account for the fact that exu mutants do not display pronounced defects in abdominal patterning (Wilhelm, 2000 and references therein).

The biochemical studies linking Ypsilon schachtel (Yps) to Exu and OSK mRNA suggest that this RNA-binding protein plays a role in posterior mRNA localization. This assertion is further supported by immunofluorescence studies showing that Yps and OSK mRNA have strikingly similar localization patterns throughout oogenesis: both accumulate in the early oocyte, transiently localize to the oocyte anterior during stages 8 and 9, and then assume their final positions at the posterior pole during stages 9 and 10. What role might Yps play in the localization complex? Yps belongs to the cold shock domain family of RNA-binding proteins that have been implicated in regulating translation and mRNA secondary structure. A notable example is FRGY2, which is complexed with mRNAs in the Xenopus oocyte and is thought to be important for translational silencing. Yps may serve a similar role, since OSK mRNA is translationally repressed until it reaches the posterior pole. Interestingly, Yps must also serve a function without Exu, since yps is expressed broadly, whereas exu expression is limited to the germ line. It is possible that Yps is a component of the mRNA localization machinery outside the germ line, since other components of the oocyte mRNA localization machinery, such as Staufen, are also used for mRNA localization in somatic tissues. Determining the precise involvement in transport and/or translational regulation of Yps in the oocyte and other tissues will be resolved in the future by mutational studies (Wilhelm, 2000 and references therein).

Ypsilon Schachtel, a Drosophila Y-box protein, acts antagonistically to Orb in the oskar mRNA localization and translation pathway

Several lines of evidence indicate that OSK mRNA, which encodes a primary organizer of the germ plasm, is a target of Ypsilon schachtel (Yps) activity. (1) OSK mRNA coimmunoprecipitates with both Yps and Exu proteins from ovary extracts. (2) OSK mRNA colocalizes with Yps and Exu throughout oogenesis. (3) There is a robust genetic interaction between yps and orb: keeping in mind orb's known regulation of OSK mRNA translation and localization, the yps null allele rescues orb-associated defects in OSK mRNA localization and translation (Mansfield, 2002).

In intermediate allelic combinations of orb, OSK mRNA fails to localize to the posterior pole of the oocyte, and Osk protein is not translated. The localization and translation of OSK mRNA is subject to a complex autoregulatory loop, whereby OSK mRNA must first be localized to the posterior pole of the oocyte to be translated, and subsequently Osk protein is required to maintain the localization of its own mRNA. Because the localization and translation processes are so entwined, it can be difficult to establish which process a regulatory factor affects. In the case of Orb, however, evidence suggests that its primary function may be translational regulation of OSK. In Xenopus, CPEB, which is virtually identical to Orb in its RNA-binding domain, regulates translation of stored maternal mRNAs by binding a U-rich region of 3'UTRs (the cytoplasmic polyadenylation element) and promoting cytoplasmic polyadenylation. The role of OSK's poly(A) tail in translation is controversial. Results from in vitro systems developed to study translation in Drosophila ovaries suggest that the length of OSK's poly(A) tail is not critical for regulating its translation. However, in vivo studies of OSK mRNA suggest that poly(A) tail length does affect its translation. These latter results indicate that polyadenylation of the OSK transcript is dependent on the function of orb, as is accumulation of Osk protein, suggesting that Orb serves a similar function to that of CPEB. In addition, Orb binds specifically to the OSK 3'UTR. Given this evidence, it appears that Orb may function as a translational enhancer of Osk, although a direct role in OSK mRNA localization cannot be ruled out (Mansfield, 2002 and references therein).

The orb genotypes that are rescued in double mutant combinations with ypsJM2 all include the orbmel mutation, a hypomorphic allele that produces some functional Orb protein. In contrast, females homozygous for a null allele (orbF343) or a strong allele (orbF303) show no rescue by the ypsJM2 mutation. These results indicate that rescue by ypsJM2 requires the presence of some functional Orb protein, and that Yps may normally act antagonistically to Orb. In the presence of Yps, the low amount of functional Orb protein present in orbmel mutants is not capable of promoting normal OSK mRNA localization and translation, whereas in the absence of Yps, the reduced Orb protein is sufficient (Mansfield, 2002).

The data indicate that yps is unlikely to regulate the expression or localization of Orb protein itself. (1) The distribution and levels of Orb produced by hypomorphic orb alleles are not altered in a ypsJM2 background. In addition, genetic analysis of yps, orb double mutants, shows that ypsJM2 specifically rescues defects in OSK mRNA localization and translation, but not orb-associated defects in dorsoventral chorion patterning or grk mRNA localization. Taken together, these results indicate a specific effect of yps on orb's function in localizing and/or translating OSK mRNA (Mansfield, 2002).

Previous work has shown that, in the minority of orbmel egg chambers in which Osk protein is detectable, Orb protein can be detected at the posterior pole as well. This correlation has been interpreted as evidence of a requirement for Orb for the on-site expression of Osk. When ovaries are doubly mutant for yps and orb, this correlation disappears. While Orb can rarely be detected at the posterior pole of the oocyte in yps;orb mutants, Osk protein is frequently present even in the absence of detectable Orb. However, loss of Yps cannot eliminate the requirement for Orb in Osk expression. It is possible that, in the absence of Yps, a very low concentration of Orb, which is undetectable by immunocytochemistry, is sufficient to localize or enhance the translation of OSK mRNA at the posterior pole. Alternatively, in the absence of Yps, the function of Orb might be accomplished at regions other than the posterior, since in yps;orb double mutants Orb protein is present throughout the oocyte (Mansfield, 2002).

Although OSK translation is significantly rescued in yps;orb ovaries, the amount of Osk present at the posterior appears reduced compared to wild type. In addition, Osk is not reliably detected in yps;orb egg chambers until stage 10. In wild-type ovaries, however, Osk can be detected in stage-9 oocytes, and sometimes as early as stage 8. It is thought that the temporal delay in detecting Osk is due simply to a reduction in Osk expression in yps;orb egg chambers during all stages of oogenesis, such that accumulation of the protein to detectable levels does not occur until stage 10. It is also hypothesized that, due to this reduction in the accumulation of Osk protein in yps;orb ovaries, OSK mRNA localization is not efficiently maintained. In late stage 9, 66% of yps;orb oocytes displayed localized OSK mRNA, while in stage 10 the percentage falls to 45%. This number closely parallels the percentage of yps;orb stage 10 oocytes with detectable Osk protein (43%) and the number of eggs (40%) that hatched from mutant mothers (Mansfield, 2002).

Biochemically an association between Yps and Orb has been detected. Orb protein coimmunoprecipitates with Yps. This association is mediated by RNA, since their coimmunoprecipitation is RNAse-sensitive. Similarly, Orb coimmunoprecipitates with Exu, in an RNA-dependent manner. Exu and Yps also coimmunoprecipitate, but independently of RNA, and bind each other in vitro, indicating that their interaction is probably direct (Wilhelm, 2000). Despite their direct association, Yps is localized normally in exu null ovaries, and Exu protein is localized normally in yps null ovaries. Thus Yps and Exu appear to be recruited independently to this ovarian complex. Do the associations detected by immunoprecipitation reflect biologically significant interactions that occur in vivo? Several other lines of evidence suggest that these proteins interact in vivo, and that OSK mRNA is part of this complex: (1) all three proteins, and OSK mRNA, colocalize throughout oogenesis; (2) OSK mRNA associates with both Exu and Yps (Wilhelm, 2000), and Orb binds directly to OSK mRNA; (3) the genetic results presented in this work are strong evidence for a biologically significant interaction of Yps and Orb in Drosophila ovaries (Mansfield, 2002).

Doubly mutant ypsJM2orbmel/ypsJM2orbF303 ovaries display a novel phenotype, not observed in ypsJM2 or orbmel/orbF303 females: a small proportion (5%) of mid- and late-stage egg chambers are bipolar. Strong allelic combinations of orb also generate a high proportion of egg chambers with the oocyte mispositioned, but these egg chambers arrest oogenesis before budding from the germarium, or shortly thereafter. The low frequency of late-stage bipolar egg chambers observed in ypsJM2orbmel/ypsJM2orbF303 females may result from partial rescue of egg chambers that would normally have arrested at very early stages in orbmel /orbF303 ovaries, with a phenotype similar to orbF303/orbF303 egg chambers. Alternatively, this may be a novel phenotype resulting from the additive loss of both yps and orb. In either case, this phenotype suggests an earlier, as yet uncharacterized function of yps. In support of this idea, yps is expressed in the germarium (Mansfield, 2002).

One model supported by the data is that Yps and Orb both bind to OSK mRNA, and have opposite effects on translation: Yps represses, and Orb activates translation. Immunoprecipitation experiments show that both proteins are present in an RNP complex and that their association is mediated by RNA, suggesting that both proteins simultaneously bind a common RNA target. Thistarget is likely to be OSK mRNA. OSK mRNA is a member of this RNP complex (Wilhelm, 2000). Orb is known to bind OSK mRNA, and the genetic results show that a yps loss-of-function mutation suppresses the defects in OSK mRNA localization and translation associated with reduced function orb alleles. Yps could prevent translation by preventing Orb from promoting cytoplasmic polyadenylation. At the posterior of the oocyte, where Orb and Yps both concentrate during mid-oogenesis, and where OSK mRNA is localized and translated, concentration differences between the two proteins could push the complex from being a negative to a positive regulator of translation. Additional factors at the posterior could also interact with either Orb or Yps to modify their functions, as might association with the actin cytoskeleton. This model accounts for why the yps mutation cannot eliminate the requirement for Orb, but can reduce the amount of Orb required for sufficient OSK translation. In the rescued genotypes, there may be enough Orb at the oocyte posterior to allow for on-site cytoplasmic polyadenylation of OSK mRNA, in the absence of negative regulation by Yps. It is also possible that, in the absence of Yps, Orb can stimulate polyadenylation of OSK mRNA before it becomes localized, although it remains subject to translational repression by other factors, such as Apontic and Bruno, until it reaches the posterior pole. Future studies will test this model by determining if Yps and Orb bind competitively to OSK mRNA, and if so, how their combined binding affects the translation of OSK mRNA, and its polyadenylation state. These studies should contribute not only to an understanding of localization-dependent mRNA translation in Drosophila, but also to a better understanding of the biological roles of the widespread family of Y-box proteins (Mansfield, 2002).

Protein interactions of Exuperantia

The complex between Exuperantia and the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps) contains OSK mRNA. This biochemical result has led to a reexamination of the role of Exu in the localization of OSK mRNA. exu-null mutants are defective in OSK mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and OSK mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. It is proposed that Exu is a core component of a large protein complex involved in localizing mRNAs, both within nurse cells and the developing oocyte (Wilhelm, 2000).

To identify the protein components of the Exu complex, GFP-Exu was immunoprecipitated from whole fly extracts prepared from a GFP-Exu-expressing fly line using an anti-GFP antibody. The GFP tag does not impair Exu protein function, since the gfp-exu transgene fully complements a null allele of exu. Seven polypeptides of 57, 74, 76, 78, 82, 88, and 147 kD coimmunoprecipitate specifically with GFP-Exu. A similar set of polypeptides coimmunoprecipitated with GFP-Exu from an extract made from hand dissected ovaries, indicating that this complex is present within the female germ line. This same set of polypeptides also coimmunoprecipitated with Exu from wild-type fly extracts using an antibody directed against a COOH-terminal peptide of Exu. Of the coimmunoprecipitated proteins, the 57-, 74-, 76-, 78-, and 82-kD proteins are present in amounts comparable to those of GFP-Exu, whereas the 88- and 147-kD proteins were clearly substoichiometric. When the extract is extensively treated with RNase A, only the 57-kD protein remains associated with GFP-Exu. Taken together with gradient analysis, these results suggest that Exu and p57 are components of a 7-S RNase-resistant core complex; the other polypeptides (p74, p76, p78, p82, p88, and p147) all require the presence of RNA in order to associate with Exu (Wilhelm, 2000).

To confirm that the 57-kD polypeptide is a bona fide Exu-associated protein, a different purification strategy was used to isolate Exu complexes. Using flies that express Exu with an NH2-terminal GFP tag and a COOH-terminal His6 tag, extracts were subjected to a two-step purification consisting of binding to an Ni-NTA column, elution with imidazole, and then immunoprecipitation with the anti-GFP antibody. The 57-kD protein consistently copurifies stoichiometrically with GFP-Exu-His6 through this two-step affinity purification, confirming that it is a true Exu-associated polypeptide. The other polypeptides in the 74-82 kD range were identified in some of these preparations, but their presence and amount is highly variable, possibly due to RNA degradation during the procedure or instability of the complex during imidazole elution from the column (Wilhelm, 2000).

To identify the 57-kD Exu-associated protein, three tryptic peptides from the purified protein were microsequenced. The sequence from two of the three peptides match a previously identified protein, the product of the yps gene. Yps is a member of the cold shock family of RNA-binding domain proteins and was identified as part of a degenerate PCR screen to identify cold shock domain containing genes from Drosophila melanogaster (Thieringer, 1997). However, the third peptide only matched the Yps sequence in a reading frame other than the published open reading frame. To rule out the possibility that yps expression is subject to ribosomal frameshifting or RNA editing, six independent yps ESTs (Berkeley Drosophila Genome Project) were obtained and sequenced. The original yps sequence was shown to contain several sequencing errors and the correct open reading frame contains all three microsequenced peptides. The cold shock domain of Yps shows extensive sequence identity to other cold shock domain proteins. This domain has been shown in several studies to bind RNA, although its ability to recognize specific substrates remains uncertain. Beyond the cold shock domain, Yps exhibits no significant homology to any other protein except YB-1, a cold shock domain protein from Drosophila silvesteris. Since the YB-1 protein is 70% identical to Yps across the entire length of the protein, it is likely to be a true ortholog of Yps. No function was assigned to either YB-1 or Yps in these prior studies (Wilhelm, 2000).

To further characterize Yps, affinity purified antibodies were prepared against bacterially expressed Yps (amino acids 1-160). These antibodies recognize the 57-kD Yps protein in crude extracts by immunoblot. Using the Yps antibody, Yps was found to comigrate with Exu in sucrose gradients and is distributed broadly in the 20-60 S size range. To rule out the possibility that Exu and Yps are components of distinct complexes of similar size, GFP-Exu was immunoprecipitated from individual gradient fractions and immunoblotted with the Yps antibody. This experiment shows that Exu and Yps coimmunoprecipitate together across the gradient, and argues strongly that Exu and Yps are part of the same complex (Wilhelm, 2000).

To provide further evidence for an Exu-Yps complex, GFP-Exu extracts were immunoprecipitated with anti-Yps polyclonal antibody. Immunoblots show that GFP-Exu specifically coimmunoprecipitates with Yps. However, immunoblots show only a weak GFP-Exu band in the Yps immunoprecipitate. The inefficient coimmunoprecipitation of GFP-Exu with Yps is probably due to the fact that the anti-Yps antibody may displace Exu from the complex, since it was raised against the Exu-binding region of Yps. The Yps immunoprecipitates also contain the same six proteins (p74, p76, p78, p82, p88, and p147) that strongly coimmunoprecipitate with GFP-Exu and are present in similar stoichiometries. The coimmunoprecipitation of these six proteins with Yps is diminished by RNase treatment, as is observed in Exu immunoprecipitation experiments. The ability of Yps and Exu antibodies to coimmunoprecipitate the same set of polypeptides argues that these proteins are bona fide components of the Exu-Yps complex (Wilhelm, 2000).

The coimmunoprecipitation of Exu and Yps after RNase A treatment suggests, but does not prove, that Exu and Yps bind directly to each other. To test this idea, the Exu-Yps interaction was examined in an in vitro translation reaction. Myc-tagged Yps was in vitro translated in the presence of [35S]methionine and then added to an unlabeled in vitro translation of HA-tagged Exu. Before mixing, each translation reaction was treated with RNase A to eliminate any residual RNA from the translation reaction. When HA-Exu was immunoprecipitated from the combined mixture with the anti-HA antibody, the 35S-labeled myc-Yps protein was coimmunoprecipitated. The amount of myc-Yps that coimmunoprecipitated with HA-Exu was approximately half of the amount of myc-Yps that was immunoprecipitated directly with the anti-myc antibody, showing that Yps is predominantly bound to Exu under these experimental conditions. These results demonstrate that the additional RNA and protein components of the native Exu complex are not required for Exu and Yps to associate stably with each other (Wilhelm, 2000).

To determine which region of Yps is important for binding to Exu, deletions of myc-Yps were assayed for their ability to bind HA-Exu in vitro. The NH2-terminal region (1-160 amino acids) of Yps, which contains the cold shock domain, binds to Exu at the same efficiency as the full-length protein. However, the minimal cold shock domain (56-151 amino acids) does not bind to Exu in this assay, suggesting that the sequences flanking the cold shock domain are likely to contribute to the Exu-binding site. The proline-rich COOH terminus of Yps presumably is not sufficient for binding. However, this could not be assessed experimentally, since this region did not stably express either in vitro or in bacteria (Wilhelm, 2000).

Exuperantia forms a complex with Me31B

A DEAD-box protein, Me31B, forms a cytoplasmic RNP complex with oocyte-localizing RNAs. During early oogenesis, loss of Me31B causes premature translation of oocyte-localizing RNAs within nurse cells, without affecting their transport to the oocyte. In early egg chambers that lack Me31B, at least two mRNAs in particles, OSK and Bicaudal-D mRNAs, are prematurely translated in nurse cells, though the transport of these RNAs to the oocyte is Me31B independent. These results suggest that Me31B mediates translational silencing of RNAs during their transport to the oocyte. These data provide evidence that RNA transport and translational control are linked through the assembly of RNP complex (Nakamura, 2001).

A visual screen was conducted with an ovarian GFP-cDNA library, in which fusion genes are expressed in germline cells during oogenesis. Transgenic flies were generated with this library and proteins were identified that distribute in a granular pattern during oogenesis. Screening ~3000 independent lines, one was isolated in which GFP signals were detected as cytoplasmic particles during oogenesis. The particles were dispersed in the cytoplasm of both nurse cells and oocytes but never detected within nuclei. The particles were frequently observed passing through ring canals, suggesting that the particles are assembled in nurse cell cytoplasm and transported to the oocyte (Nakamura, 2001).

The cDNA from this line was identified as me31B (De Valoir, 1991). In the cDNA fusion, almost the entire coding region of me31B, which lacks only the first four codons, was fused in frame with that of gfp. Me31B, a DEAD-box protein and therefore a putative ATP-dependent RNA helicase, was isolated as a gene expressed extensively during oogenesis (De Valoir, 1991). Me31B is a part of an evolutionally conserved DEAD-box protein group, which includes human RCK/p54 (71% identical), Xenopus Xp54 (73%), Caenorhabditis elegans C07H6.5 (76%), Schizosaccharomyces pombe Ste13 (68%) and Saccharomyces cerevisiae Dhh1 (68%). Furthermore, Me31B is phylogenetically close to two evolutionally conserved proteins, eIF4A and Dbp5/Rat8p but far from Vasa, which functions in germline development (Nakamura, 2001).

To examine distribution of the endogenous Me31B, antibodies were generated that specifically recognized Me31B. The distribution pattern of endogenous Me31B is identical to that of GFP-Me31B. No detectable signal in somatic follicle cells is observed at any stage of oogenesis. Me31B is first detected at a low level in germarium region 2B, where the signal is concentrated in the pro-oocytes. The signal remains concentrated in the oocyte until mid-oogenesis. In early egg chambers, a low level Me31B signal is detected in nurse cell cytoplasm. In both nurse cells and oocytes, the signal appears to be granular. Me31B signals in nurse cell cytoplasm become more evident from stage 5-6, when Me31B expression is drastically increased. In addition, Me31B is frequently enriched around nurse cell nuclei. Later, Me31B accumulates at the posterior pole of stage 10 oocytes. However, this posterior accumulation is transient, as revealed by uniform distribution of the signal in cleavage embryos. By cellular blastoderm stage, Me31B becomes undetectable in the entire embryonic region. No zygotic expression of Me31B was detected during embryogenesis (Nakamura, 2001).

Because Me31B is probably an RNA-binding protein that is transported to the oocyte, it was asked whether Me31B forms a complex with oocyte-localizing RNAs. Colocalization of OSK mRNA with Me31B was examined. OSK mRNA starts to accumulate in oocytes from germarium region 2B, with the concentration of OSK increasing over time. Posterior accumulation of OSK mRNA in the oocyte begins from stage 8 onwards. By fluorescent in situ hybridization, OSK mRNA exhibits particulate signals in the cytoplasm of both nurse cells and oocytes, and is frequently concentrated around nurse cell nuclei. This distribution pattern of OSK mRNA is essentially identical to that of Me31B, with colocalization present until OSK mRNA localizes to the posterior pole of stage 10 oocytes (Nakamura, 2001).

Colocalization of Me31B with other RNAs was also examined. Ovaries were doublestained for Me31B and BicD mRNA. In early egg chambers, BicD mRNA also produces particulate signals, and appears to localize in Me31B-containing particles. This colocalization becomes apparent from stage 5-6, when BicD mRNA expression is elevated. The oocyte-localizing RNAs examined [BCD, NOS, ORB, Polar granule component (PGC) and Germ cell-less (GCL)] all produce particulate signals in the cytoplasm of both nurse cells and oocytes, and colocalize with Me31B. In contrast, Vasa mRNA, which is not specifically transported to the oocyte, does not appear to be colocalized with Me31B. These results indicate that Me31B forms cytoplasmic particles that contain oocyte-localizing RNAs (Nakamura, 2001).

Whether Me31B and Exu colocalize was examined; distribution of GFP-Exu fusion protein is very similar to that of Me31B (Wang, 1994). GFP-Exu is concentrated to the oocytes from as early as stage 1, and transiently accumulates at the posterior pole of stage 10 oocytes (Wang, 1994). From stage 5-6 onwards, particulate GFP-Exu signals in the cytoplasm become apparent (Wang, 1994). To compare the distribution between Exu and Me31B directly, ovaries expressing GFP-Exu were immunostained with an anti-Me31B antibody. Almost all GFP-Exu particles contain Me31B. Colocalization between Exu and Me31B throughout oogenesis was further confirmed by immunostaining of ovaries expressing GFP-Me31B with an anti-Exu antiserum (Nakamura, 2001).

Colocalization of Me31B with Ypsilon Schachtel (Yps), which has been shown to be an Exu-binding protein in oogenesis (Wilhelm, 2000), was examined. In nurse cells and oocytes, Yps signals are observed in GFP-Me31B-positive cytoplasmic particles. These results indicate that Me31B, Exu and Yps are all components of a cytoplasmic RNP complex in nurse cells and oocytes (Nakamura, 2001).

Exu has been reported to be highly concentrated in a specialized RNA rich regions in the cytoplasm of nurse cells and oocytes, called the sponge bodies (Wilsch-Bräuninger, 1997). Sponge bodies have been proposed to be involved in the localization of RNAs in the oocytes. Whether Me31B is also concentrated in the sponge bodies was examined using immunoelectron microscopy. Me31B signals are detected in distinctive cytoplasmic regions, consisting of string-like materials embedded in an electron dense mass. The regions are in close proximity to mitochondria but not surrounded by membrane. All of these features completely satisfied the reported criteria of the sponge body. Therefore, it is concluded that Me31B is highly enriched in the sponge bodies, and that the sponge body is the region where cytoplasmic RNPs for oocyte localization are concentrated (Nakamura, 2001).

To characterize Me31B-containing particles in detail, the particles were immunoprecipitated with an anti-Me31B antibody from extracts of wild-type ovaries. The resulting immunoprecipitates were separated by SDS-PAGE and probed with antibodies against Me31B, Yps and Exu. Whether Me31B is coimmunoprecipitated with Yps was also examined. Both Yps and Exu coimmunoprecipitate with Me31B, and Me31B coimmunoprecipitates with Yps (Nakamura, 2001).

Exu has been shown to binds directly to Yps; these proteins coimmunoprecipitate several unknown proteins in an RNase-sensitive manner (Wilhelm, 2000). The effect of RNase treatment on the nature of the Me31B-Exu/Yps complex was examined. In the presence of RNase, Me31B-Yps and Me31B-Exu, coimmunoprecipitation is diminished. These results indicate that Me31B forms a complex with Yps and Exu via RNA (Nakamura, 2001).

To investigate the function of Me31B during oogenesis, me31B mutations were identified. A P element insertion, k06607, is located ~100 bp upstream of the 5' end of me31B. By mobilizing the k06607 insertion, three partial deletions of me31B (me31BDelta1, me31B Delta2 and me31B Delta3) were isolated. All the deletions start from the original P element insertion point, and their other breakpoints are within the me31B-coding region (Nakamura, 2001).

The three me31BDelta lines are all recessive lethal. They did not complement each other, and are also lethal in trans to a deficiency, Df(2L)J2, which deletes the entire me31B locus. Homozygous me31BDelta individuals die during the second- or third-instar larval stages without expressing any discernible morphological defects. The lethality associated with the me31BDelta chromosome is rescued by introducing a genomic DNA that contains the entire me31B locus and ~4.5 kb of the upstream region. It is concluded that the three me31BDelta mutations affect only me31B function, and that me31B is a vital gene (Nakamura, 2001).

To analyze the me31B function in oogenesis, the FLP-DFS mitotic recombination system was used to produce me31B germline clones. None of the egg chambers homozygous for me31B mutations complete oogenesis. Most me31B egg chambers degenerate during mid-oogenesis. In these egg chambers, cell membranes of germline cells are collapsed, and ring canals and nurse cell nuclei are concentrated to form large aggregates within egg chambers. Nurse cell nuclei in these egg chambers are then fragmented into small pieces. Occasionally, egg chambers are found that progressed as far as stage 10. In these egg chambers, oocytes do not grow normally, and the nurse cell nuclei are displaced posteriorly relative to the anterior border of columnar follicle cells, which normally surround the oocyte at stage 10 (Nakamura, 2001).

The complicated and redundant phenotypes observed in me31B- egg chambers in mid-oogenesis are unlikely to be the primary effect of loss of me31B function. Earlier phenotypes of me31B- egg chambers were examined using a FLP/FRT system to generate homozygous germline clones that are marked by the loss of Vas-GFP fusion protein. Based on Hoechst and phalloidin staining, me31B- egg chambers are morphologically normal until stage 4-5. From stage 6 onwards, oocytes in me31B- egg chambers fail to grow normally. At this stage, these egg chambers begin to degenerate. In early me31B- egg chambers, Exu signal is concentrated to the oocytes. Distributions of OSK and BicD mRNAs in me31B- egg chambers were examined. Both OSK and BicD mRNAs also accumulate in the oocytes of me31B- egg chambers until the chambers degenerate. Particulate signals for these RNAs are detectable in nurse cell cytoplasm in these egg chambers. These results indicate that Exu, OSK and BicD mRNAs can be transported to the oocyte even in the absence of Me31B. It is concluded that in early egg chambers, Me31B is dispensable for the transport of the molecules that form a complex with Me31B (Nakamura, 2001).

Whether loss of Me31B affects translation of OSK and BicD mRNAs was examined. Ovaries were immunostained with an anti-Osk antiserum. Although OSK mRNA is expressed during almost all stages of oogenesis, its translation is repressed to keep Osk protein level very low during early oogenesis. In me31B- egg chambers, Osk signal is significantly increased compared with that in the neighboring me31B+ egg chambers (Nakamura, 2001).

A similar increase of BicD signal in me31B- egg chambers is more evident. In wild-type egg chambers, BicD protein, like BicD mRNA, is highly concentrated in the oocytes starting from germarium region 2B. In the egg chambers lacking me31B, increased BicD signal is detected in nurse cell cytoplasm. These results suggest that loss of Me31B in germline cells causes derepression of OSK and BicD mRNA translation during their transport to the oocyte (Nakamura, 2001).

It remains unanswered where Me31B is assembled to form an RNP complex. It has been shown that shuttling hnRNP proteins remain associated with several localized RNAs, even in the cytoplasm, suggesting that cytoplasmic RNPs could be nucleated within the nucleus. Me31B is frequently enriched around the nuclear envelope in the nurse cell cytoplasm but is never detected within nuclei. Thus, the idea is favored that Me31B is assembled into RNP complexes containing oocyte-localizing RNAs in the perinuclear region of the nurse cell cytoplasm. It is notable that in yeast and mammalian cells, Dbp5p/Rat8p, a DEAD-box protein that is phylogenetically closed to Me31B, is also highly enriched in the perinuclear region, and is required for mRNA export from the nucleus. Although the Drosophila counterpart of Dbp5 has not yet been characterized, similar distribution of the two related proteins suggests an important link between nuclear RNA export and assembly of cytoplasmic RNP complexes. The perinuclear region would be the site where nuclear RNP complexes are reorganized into a cytoplasmic form, in which additional proteins involved in RNA transport and translational silencing are incorporated (Nakamura, 2001).

Egg chambers that lack me31B degenerate during mid-oogenesis. Since Me31B colocalizes with many oocyte-localizing RNAs other than OSK and BicD mRNAs, Me31B is likely to silence translation of these RNAs. Therefore, me31B- phenotypes in mid-oogenesis could be caused simply by translational derepression of many mRNAs in the particles. Similar pleiotropic effects, including germ cell degeneration during mid-oogenesis and invading of nurse cell nuclei into the oocyte, has been observed in mutations for vas, which encodes a germline-specific DEAD-box protein implicated in translational activation of several maternal mRNAs. It is proposed that translational control plays a fundamental and widespread role to advance oogenesis. Nevertheless, it is still an unanswered question whether Me31B has another function in oogenesis. Isolation of weak mutations of me31B may reveal additional functions, if any, of Me31B (Nakamura, 2001).

In Xenopus oocytes, Xp54, a protein homologous to Me31B, has been identified as a major integral component of stored RNP particles, in which maternal mRNAs are masked from translation, degradation and polyadenylation/deadenylation. Although the function of Xp54 in the RNP particles remains elusive, RNA masking is thought to involve the structural rearrangement of an RNP complex into a condensed form. Therefore, Xp54 may function to unwind the secondary structures of mRNAs to facilitate binding of RNA-packaging proteins such as FRGY2. Interestingly, Yps, a component of Me31B-containing particles, shares a conserved sequence motif with FRGY2. Although the function of Yps in the particles remains to be analyzed, these observations suggest that similar proteins mediate the translational silencing during oogenesis between two diverse organisms (Nakamura, 2001).

Me31B is highly concentrated in distinctive cytoplasmic regions, the sponge bodies. A similar electron-dense structure known as the mitochondrial cloud has been found in Xenopus oocytes. Numerous maternal mRNAs that are involved in the axis determination and germ plasm assembly, co-migrate with this structure to localize the vegetal cortex in growing oocytes. Some of the RNA components in the mitochondrial cloud are translationally silenced during oogenesis and translated only after the onset of embryogenesis. These data strongly suggest that the sponge body and the mitochondrial cloud are related cytoplasmic structures in terms of morphology and function, although it has not been determined whether they contain related protein components. Further analysis of composition and the role of the maternal RNP complexes in Drosophila and Xenopus will uncover the molecular mechanism of how localization and translation of maternal mRNAs is spatio-temporally regulated (Nakamura, 2001).

exuperantia: Biological Overview | Developmental Biology | Effects of Mutation | References

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