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Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation

2000; Springer Nature; Volume: 19; Issue: 6 Linguagem: Inglês

10.1093/emboj/19.6.1366

1460-2075

David Micklem,

Silk-based biomaterials and applications

Article15 March 2000free access Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation David R. Micklem David R. Micklem Present address: Department of Biochemistry, Beckman Center, Stanford University, Stanford, CA, 94305 USA Search for more papers by this author Jan Adams Jan Adams Wellcome/CRC Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author Stefan Grünert Stefan Grünert Present address: Institut für Molekular Pathologie, Dr. Bohrgasse 7, 1030 Wien, Austria Search for more papers by this author Daniel St Johnston Corresponding Author Daniel St Johnston Wellcome/CRC Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author David R. Micklem David R. Micklem Present address: Department of Biochemistry, Beckman Center, Stanford University, Stanford, CA, 94305 USA Search for more papers by this author Jan Adams Jan Adams Wellcome/CRC Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author Stefan Grünert Stefan Grünert Present address: Institut für Molekular Pathologie, Dr. Bohrgasse 7, 1030 Wien, Austria Search for more papers by this author Daniel St Johnston Corresponding Author Daniel St Johnston Wellcome/CRC Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author Author Information David R. Micklem2, Jan Adams1, Stefan Grünert3 and Daniel St Johnston 1 1Wellcome/CRC Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK 2Present address: Department of Biochemistry, Beckman Center, Stanford University, Stanford, CA, 94305 USA 3Present address: Institut für Molekular Pathologie, Dr. Bohrgasse 7, 1030 Wien, Austria ‡D.R.Micklem and J.Adams contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:1366-1377https://doi.org/10.1093/emboj/19.6.1366 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Drosophila Staufen protein is required for the localization of oskar mRNA to the posterior of the oocyte, the anterior anchoring of bicoid mRNA and the basal localization of prospero mRNA in dividing neuroblasts. The only regions of Staufen that have been conserved throughout animal evolution are five double-stranded (ds)RNA-binding domains (dsRBDs) and a short region within an insertion that splits dsRBD2 into two halves. dsRBDs 1, 3 and 4 bind dsRNA in vitro, but dsRBDs 2 and 5 do not, although dsRBD2 does bind dsRNA when the insertion is removed. Full-length Staufen protein lacking this insertion is able to associate with oskar mRNA and activate its translation, but fails to localize the RNA to the posterior. In contrast, Staufen lacking dsRBD5 localizes oskar mRNA normally, but does not activate its translation. Thus, dsRBD2 is required for the microtubule-dependent localization of osk mRNA, and dsRBD5 for the derepression of oskar mRNA translation, once localized. Since dsRBD5 has been shown to direct the actin-dependent localization of prospero mRNA, distinct domains of Staufen mediate microtubule- and actin-based mRNA transport. Introduction The establishment of cell polarity requires the targeting of specific proteins to the regions of a cell where they are required, and this is often achieved by localizing the mRNAs that encode them (St Johnston, 1995; Bashirullah et al., 1998). In many cases, mRNA localization is thought to be an active process that requires the cytoskeleton. For example, mating type switching in Saccharomyces cerevisiae is restricted to the mother cell by the myosin-dependent transport of ash1 mRNA into the emerging daughter cell, and the directed motility of cultured fibroblasts requires the actin-dependent localization of β-actin mRNA (Kislauskis et al., 1994; Bertrand et al., 1998). Other mRNAs are localized by microtubule-dependent mechanisms, such as Vg1 mRNA, which moves to the vegetal pole of the Xenopus oocyte, and bicoid (bcd) and oskar (osk) mRNAs, which localize to opposite poles of the Drosophila oocyte (Yisraeli et al., 1990; Pokrywka and Stephenson, 1991; Clark et al., 1994). The importance of microtubule-dependent mRNA localization has been most clearly demonstrated in the case of the latter two transcripts, since their positions define the anterior–posterior axis of the embryo. bcd mRNA localizes to the anterior of the egg, and is translated after fertilization to produce a morphogen gradient that patterns the head and thorax of the embryo; the localization of osk mRNA to the posterior of the oocyte directs the assembly of the pole plasm, which contains posterior and germline determinants (Ephrussi and Lehmann, 1992; Driever, 1993). The cis-acting signals that direct mRNA localization have been mapped in several transcripts, and in a few cases biochemical approaches have led to the identification of RNA-binding proteins that interact with these signals (Bashirullah et al., 1998). However, the best characterized example of an RNA-binding protein required for mRNA localization is Drosophila Staufen protein (Stau), which was identified in a genetic screen (Schüpbach and Wieschaus, 1986). Subsequent work has shown that Stau plays an essential role in the localization of three different mRNAs during development. It is required for (i) the localization of osk mRNA to the posterior of the oocyte (Ephrussi et al., 1991; Kim-Ha et al., 1991, 1995); (ii) the anchoring of bcd mRNA at the anterior of the egg (St Johnston et al., 1989); and (iii) the basal localization of prospero mRNA during the asymmetric divisions of embryonic neuroblasts (Li et al., 1997; Broadus et al., 1998; Matsuzaki et al., 1998; Schuldt et al., 1998; Shen et al., 1998). Although it has not been possible to test whether Stau binds specifically to these mRNAs, it contains five copies of a double-stranded (ds)RNA-binding domain (dsRBD), and the third of these has been shown to bind to dsRNA in vitro (St Johnston et al., 1992). When mutations that abolish the RNA-binding activity of dsRBD3 are incorporated into a full-length Stau transgene, this construct no longer rescues the localization of either osk or bcd mRNAs (Ramos et al., 2000). Thus, the dsRNA-binding activity of dsRBD3 is required for bcd and osk mRNA localization, strongly suggesting that Stau binds these RNAs directly. During stages 7–9 of Drosophila oogenesis, osk mRNA localizes transiently at the anterior of the oocyte, and then moves to the posterior pole at stage 9 in a microtubule-dependent manner (Ephrussi et al., 1991; Kim-Ha et al., 1991; Clark et al., 1994). In stau null mutants, however, osk mRNA fails to move to the posterior and remains at the anterior. Several lines of evidence indicate that Stau protein associates with osk mRNA to mediate its posterior localization. (i) Stau protein co-localizes with osk mRNA at the anterior of the oocyte and moves with the RNA to the posterior (St Johnston et al., 1991). (ii) Stau and osk RNA mislocalize to the same ectopic sites in mutants, such as gurken, which alter the polarity of the oocyte (González-Reyes et al., 1995; Roth et al., 1995). (iii) The posterior localization of Stau depends on osk mRNA (Ferrandon et al., 1994). In females carrying extra copies of an osk transgene, the increased quantity of osk mRNA produced induces a corresponding increase in the amount of Stau that localizes to the posterior pole. Thus, Stau is present in excess, and only the protein that is associated with osk RNA localizes to the posterior. Translation of unlocalized osk mRNA is repressed by the binding of Bruno protein to Bruno-response elements (BRE) in the 3′UTR (Kim-Ha et al., 1995; Gunkel et al., 1998). An osk transgene lacking the BRE (oskBRE−) is therefore translated prior to its localization, leading to the production of ectopic Osk, which causes a range of patterning defects in the resulting embryos. Low levels of ectopic Osk result in a loss of head and thoracic segments, while higher levels induce the formation of bicaudal embryos, with abdomens at both ends. Although the translation of oskBRE− mRNA no longer depends on its localization, it still requires Stau protein, since the bicaudal phenotypes are suppressed in a stau null mutant background. Thus, Stau plays a role in the translation of oskBRE− mRNA that is independent of its role in posterior localization, suggesting that it may also be involved in the translational regulation of wild-type osk mRNA. Finally, Stau has also been implicated in the anchoring of osk mRNA at the posterior. When a temperature-sensitive stau allele is kept under semi-restrictive conditions, Stau and osk mRNA localize to the posterior of the oocyte at stage 10, but are not maintained there in the embryo (St Johnston et al., 1991; Rongo et al., 1995). Although Stau is not involved in the initial anterior localization of bcd mRNA, it is required to anchor the mRNA during the final stages of oogenesis (St Johnston et al., 1989). bcd RNA is normally released from the cortex at some time between stage 12 of oogenesis and egg deposition, and remains tightly localized in a spherical region of cytoplasm at the anterior of the egg. In stau mutant eggs, however, the RNA forms a shallow anterior–posterior gradient, and the resulting embryos have head defects because there is insufficient Bicoid protein at the very anterior of the embryo. This function of Stau shows several parallels to its role in osk mRNA localization (Ferrandon et al., 1994). First, Stau protein co-localizes with bcd mRNA at the anterior of the egg, and this localization is bcd mRNA dependent. Second, when the bcd 3′UTR is injected into the egg, it recruits Stau into particles that localize to the poles of the mitotic spindles. Stau, therefore, mediates the microtubule-dependent localization of both bcd and osk mRNAs, but at two different stages of development. Furthermore, in each case the localization of Stau requires its interaction with the appropriate RNA, suggesting that Stau–RNA complexes are the substrate for localization. More recently, Stau has been shown to mediate the localization of prospero mRNA during the asymmetric divisions of embryonic neuroblasts (Li et al., 1997; Broadus et al., 1998; Fuerstenberg et al., 1998; Matsuzaki et al., 1998; Schuldt et al., 1998; Shen et al., 1998). In contrast to the localization of bcd and osk mRNAs, the localization of prospero mRNA–Stau complexes is disrupted by actin-destabilizing drugs, but not by microtubule-depolymerizing drugs (Broadus and Doe, 1997). The discovery that Stau can mediate both microtubule- and actin-dependent mRNA localization raises the question of how different Stau–mRNA complexes are coupled to distinct transport pathways. It has previously been shown that Miranda protein binds to the dsRBD5 of Stau to direct the basal localization of prospero mRNA (Fuerstenberg et al., 1998; Schuldt et al., 1998; Shen et al., 1998). However, it remains unclear how Stau links osk and bcd mRNAs to the microtubule-based transport machinery, or how the activation of osk mRNA translation at the posterior is achieved. In this paper, we address this question by analysing the domains of Stau to determine which regions of the protein are required for these functions. Results Conservation of Stau throughout the animal kingdom Since the functional domains of a protein can often be identified from their conservation during evolution, we cloned and sequenced homologues of stau from two other insect species, Drosophila virilis (Dvstau) and Musca domestica (Mdstau), which diverged from Drosophila melanogaster over 60 and 100 million years ago, respectively. In addition, Wickham et al. (1999) and Marión et al. (1999) have recently reported Stau homologues in Caenorhabditis elegans (Cestau), mouse (Mmstau) and human (Hsstau), and we also identified these on the basis of their homology to the insect genes and sequenced them in their entirety. The predicted amino acid sequences of the invertebrate homologues include five dsRBDs, corresponding to dsRBDs 1–5 of D.melanogaster Stau. In contrast, the human and mouse homologues include only four domains, which are most closely related to dsRBDs 2, 3, 4 and 5 of the invertebrate Stau (Figure 1D). Figure 1.A comparison of Staufen homologues from different species. (A) A diagram showing the positions of the conserved dsRBDs in D.melanogaster Staufen protein, and the percentage amino acid identity between these domains and the equivalent domains in other species. (B) A PlotSimilarity diagram of a ClustalW alignment of all six Staufen homologues. The positions of the dsRBDs are superimposed on the plot, and show that the similarity between homologues is almost entirely restricted to the dsRBDs. The arrow marks the only other region of similarity, which falls within the insertion in dsRBD2. Note that the degree of similarity shown for dsRBD1 appears lower than that for the other four dsRBDs because this domain is present in only 4/6 homologues. (C) Alignment of the conserved region in the dsRBD2 insertion. (D) An unrooted tree derived from a ClustalW alignment of dsRBDs from different proteins. Corresponding domains in the different Staufen homologues are more similar to each other than they are to any other dsRBDs, with the exception of CeStau dsRBD5, which is approximately similar to the other Stau dsRBD5s and to the third dsRBD of human TAR RNA-binding protein (Hstrbp) and Xenopus Xlrbpa (marked with asterisks). For simplicity, this diagram only includes dsRBDs described up to 1995. Download figure Download PowerPoint Despite the absence of a dsRBD1 equivalent in the mouse and human sequences, we believe that they represent vertebrate homologues of Stau because of the high degree of similarity to DmStau within the remaining four domains (Figure 1A). A ClustalW analysis of dsRBDs from many proteins reveals that apart from one minor exception for dsRBD5, a given Stau domain is most similar to the equivalent domain in each of the other Stau homologues (Figure 1D). This suggests that evolution is not acting simply to maintain similarity to a dsRBD consensus sequence, but rather that each domain has unique features that have been conserved during evolution. It is particularly notable that in all six homologues, dsRBD2 is split into two parts by up to 118 aa of non-dsRBD sequence. While dsRBDs have been identified in many proteins, the distinctive split in dsRBD2 has only been observed in these six Stau homologues. Analysis of an alignment of the Stau homologues reveals that the only portions of the protein to have been conserved during evolution are the five dsRBDs (Figure 1B). For example, the M.domestica and D.melanogaster proteins show an average of 67% amino acid identity within the dsRBDs, but <15% in the rest of the protein. dsRBD2 and dsRBD5 were originally described as 'half domains' showing similarity to the dsRBD consensus only over the C-terminal portion of the domain (St Johnston et al., 1992). However, the conservation extends over a region corresponding to the length of a whole domain, and these domains should therefore be considered as complete, albeit divergent, dsRBDs, in agreement with the results of Gibson and Thompson (1994). The only other obvious homology between these proteins is a short region within the insertion in the middle of dsRBD2 that is rich in proline and aromatic amino acids (Figure 1B and C). Since the regions of the protein essential for its activity are expected to be conserved during evolution, the dsRBDs and this proline-rich region are likely to mediate all of the functions of Stau, including its ability to bind both mRNA and the factors that localize Stau–mRNA complexes. To determine whether the dsRBDs are indeed the only part of Stau necessary for its function, we generated a transgene in which the large non-conserved N-terminal region of DmStau was deleted, and crossed this construct into a stau null mutant background (stauD3). Like the full-length protein expressed from the same vector (Staufull), StauΔN localizes normally to the posterior of the oocyte, and completely rescues the posterior localization and anchoring of osk mRNA (Figure 2A and B). Furthermore, the eggs laid by StauΔN females show a wild-type localization of bcd mRNA at the anterior pole (data not shown). This rescue of the maternal function of stau is also reflected in the phenotype of the embryos produced by StauΔN females. Whereas the embryos laid by stauD3 mutant females die with head defects and no abdomen, almost all of the progeny of StauΔN females hatch into larvae, and have normal heads and almost wild-type abdominal segmentation (Table I). Furthermore, a similar proportion of the adult offspring of StauΔN females had gametic ovaries (85%) compared with those of females carrying Staufull (88%), indicating that this construct leads to the production of the high levels of Osk activity that are necessary to specify the germline (Table I). Thus, the portion of Stau that includes the dsRBDs is able to mediate all of Stau's functions during oogenesis, including localization of both osk and bcd mRNAs, activation of osk translation and maintenance of pole plasm at the posterior. Figure 2.The dsRBDs are the only conserved regions of Stau required for osk mRNA localization. (A) The localization of Staufen protein (i and ii) and osk mRNA (iii and iv) in stage 9 and 10 oocytes from stauD3 T[Staufull]/stauD3 females. Full-length Staufen protein expressed from the transgene localizes normally to the posterior of the oocyte, and rescues the osk mRNA posterior localization defect of a staufen null mutation. (B) The localization of Stau protein (i and ii) and osk mRNA (iii and iv) in stage 9 and 10 oocytes from stauD3 T[StauΔN]/stauD3 females. Stau protein lacking the non-conserved N-terminal 282 aa also localizes normally and rescues osk mRNA localization and anchoring. (C) (i) In wild-type egg chambers, osk mRNA shows a transient localization to the anterior of the oocyte during stage 9. (ii) In stauD3 T[StauDmMd]/stauD3 egg chambers, osk mRNA fails to accumulate at the anterior margin at stage 9, and localizes instead to the centre of the oocyte. (iii) However, the mRNA shows a normal localization at the posterior pole by stage 10. (iv) In stauD3 egg chambers, all osk mRNA remains anchored at the anterior of the oocyte. (v) osk mRNA shows a transient localization to a point in the centre of the oocyte, when StauDmMd is expressed in the presence of wild-type Drosophila Stau protein. (D) A diagram showing the structure of the Stau proteins encoded by the Staufull, StauΔN, StauDmMd, StauΔloop2 and StauΔdsRBD5 transgenes. The boxes indicate the positions of the dsRBDs. The short leader peptide containing the myc epitope tag is labelled in blue, Drosophila sequences in green and M.domestica sequences in red or pink. Download figure Download PowerPoint Table 1. Rescue of the stau null phenotype by stau transgenes Genotype Average No. of abdominal denticle belts % adults with gametic ovaries % normal heads wild type 8 ± 0 100 100 stauD3 0.04 ± 0.002 n/a 0 stauD3 T[Staufull] 7.1 ± 0.21 88 100 stauD3 T[StauΔN] 6.9 ± 0.18 85 100 stauD3 T[StauDmMd] 7.4 ± 0.16 100 86 stauD3 T[StauΔloop2] 0.14 ± 0.06 n/a 74 stauD3 T[StauΔdsRBD5] 0.06 ± 0.03 n/a 68 As a more stringent test of whether the functional domains of Stau have been conserved during evolution, we generated transgenic lines in which the dsRBD-containing region of DmStau is replaced with the corresponding region from M.domestica (StauDmMd), and found that this transgene rescues all the phenotypes of a stau null mutation at least as well as the full-length Stau construct (Figure 2C; Table I). Since the only regions that are conserved between DmStau and MdStau are the five dsRBDs and the short sequence in the insertion in dsRBD2, it is likely that the most important functional domains of the protein reside in these regions. Although the localization of osk mRNA to the posterior of the oocyte during stage 9 appears normal in stauD3 females carrying StauDmMd, osk mRNA localizes through an atypical intermediate stage. In wild-type flies, osk mRNA shows a transient association with the anterior pole of the oocyte before moving to the posterior, whereas all of the mRNA remains at the anterior in stauD3 homozygous females (Figure 2C, i and iv). In StauDmMd ovaries, however, osk mRNA does not accumulate at the anterior of the oocyte, and instead forms a 'blob' in the middle of the oocyte, which disperses as localization proceeds (Figure 2C, ii and iii). Furthermore, this effect of StauDmMd is dominant: in the absence of endogenous D.melanogaster Stau this 'blob' is diffuse, but in a wild-type background it forms a much sharper, well defined spot (Figure 2C, v). The nature of the cytoplasmic blob is unclear, but it is intriguing that D.virilis osk mRNA is localized through a similar intermediate when introduced into D.melanogaster (Webster et al., 1994). dsRBD2 and dsRBD5 do not bind dsRNA The discovery that the dsRBDs of Stau are the only conserved regions of the protein that are required for its function raises the question of whether all of these domains bind to dsRNA, and we therefore examined the ability of the five dsRBDs to bind to dsRNA on Northwestern blots (Figure 3A). As previously reported, dsRBD3 binds strongly to dsRNA, whereas a control domain in which 5 aa that contact the RNA have been mutated does not (Ramos et al., 2000). dsRBDs 1 and 4 also bind dsRNA, irrespective of its sequence, although this binding is weaker than that observed with dsRBD3. In contrast, dsRBD2 and dsRBD5 do not bind to any of the dsRNAs tested in this assay. Figure 3.dsRBDs 2 and 5 do not bind to dsRNA in vitro. (A) A Coomassie-stained SDS–PAGE gel showing the expression of the Staufen dsRBDs fused to glutathione S-transferase (GST). Lane 1, GST alone; lane 2, GST–dsRBD1; lane 3, GST–full-length dsRBD2; lane 4, GST–dsRBD3; lane 5, GST–dsRBD4; lane 6, GST–dsRBD5; lane 7, GST–dsRBD2 in which the large insertion has been replaced by the short loop 2 from dsRBD3; lane 8, GST–dsRBD3 containing five amino acid substitutions in residues that contact dsRNA (Ramos et al., 2000). (B) A Northwestern blot of the same samples as in (A) probed with [32P]dsRNA. The right hand side of this blot has been exposed approximately four times longer than the left to reveal the weak dsRNA-binding activity of dsRBD4. (C) A comparison of the RNA-binding faces of dsRBDs 1, 3, 4 and 5, showing the amino acids in domain 3 that are required for dsRNA binding (yellow boxes), and the identity and conservation of the amino acids in equivalent positions in the other domains (yellow). The structures of dsRBDs 1, 4 and 5 have been modelled by 'threading' them onto the known structure of dsRBD3 (Bycroft et al., 1995). Blue, basic residues; red, acidic; yellow, non-polar; orange, polar and uncharged. The amino acids are numbered from the first conserved residue of the domain (Ramos et al., 2000). Download figure Download PowerPoint NMR and mutational analysis of Stau dsRBD3–dsRNA complexes have revealed that the dsRBD binds RNA through conserved amino acids that cluster on one face of the domain (Figure 3C) (Ramos et al., 2000). The corresponding positions in dsRBDs 1, 4 and 5 can be identified both by sequence alignment and by 'threading' their sequences onto the structure of dsRBD3. While dsRBDs 1 and 4 contain identical or similar conserved amino acids to dsRBD3, the amino acids on this face of dsRBD5 are much less well conserved, and are of a different type from those found in the other domains (Figure 3C). The lack of conservation of the dsRNA-contacting amino acids, coupled with the observed inability of dsRBD5 to bind dsRNA in vitro strongly suggest that this domain does not function as a dsRBD in vivo. However, the structural amino acids that comprise the hydrophobic core of the domain are highly conserved when compared with other dsRBDs, and amino acids on the other faces of dsRBD5 are also conserved across the different species. It is therefore likely that domain 5 folds into a typical dsRBD structure, but performs a distinct conserved function unrelated to dsRNA binding. A highly conserved feature of all six Stau homologues is the presence of the large loop interrupting dsRBD2, and this most probably accounts for the inability of this domain to bind dsRNA in vitro. The NMR structure of the dsRBD3–dsRNA complex reveals that the dsRNA-binding regions of the domain span one turn of a dsRNA helix, and that their relative positions within the whole domain are crucial for RNA binding (Ramos et al., 2000). The insertion in loop 2 separates the two halves of dsRBD2, and the RNA-binding amino acids are therefore unlikely to have the correct spacing to contact RNA. Although the presence of the insertion in loop 2 makes it impossible to predict the structure of dsRBD2, the sequence of the domain suggests that it could bind dsRNA if it adopted a conformation in which these two halves were juxtaposed. To test this hypothesis, we constructed a version of dsRBD2 in which the extended loop 2 is replaced by the corresponding 8 aa loop of dsRBD3. When examined in the Northwestern assay, this dsRBD2Δloop2 binds dsRNA almost as efficiently as dsRBD3 (Figure 3). Thus, dsRBD2 can bind dsRNA when the removal of the large insertion allows the correct folding of the domain, suggesting that this domain binds dsRNA in vivo in the context of full-length protein. The insertion in domain 2 is required for Stau–osk mRNA localization To determine what role, if any, the extended loop in dsRBD2 plays in Stau function, we constructed a transgene (StauΔloop2) in which the normal domain 2 is replaced by the truncated dsRBD2Δloop2 described above, and crossed this into a stauD3 mutant background. Although this transgene expresses high levels of a protein of the appropriate molecular weight (Figure 6B), it gives little or no rescue of the stau posterior phenotype; almost all osk mRNA and Stau protein fail to be transported to the posterior of the oocyte and remain trapped instead at the anterior margin (Figure 4A–E). However, a small amount of mRNA is occasionally seen at the posterior at stage 9. As a result of this defect in osk mRNA localization, none of the progeny of these flies hatch into larvae, and almost all develop less than one abdominal segment (Table I). Figure 4.The insertion in dsRBD2 is required for the posterior localization of osk mRNA. (A and B) Wild-type stage 9 and 10A egg chambers showing the normal localization of osk mRNA to the posterior of the oocyte. (C and D) stauD3 T[StauΔloop2]/stauD3 egg chambers, in which all osk mRNA remains anchored at the anterior of the oocyte. (E) A stauD3 T[StauΔloop2]/stauD3 stage 9 egg chamber, showing a small amount of osk mRNA at the posterior. (F) StauΔloop2 protein (yellow) co-localizes with osk mRNA to the anterior of the oocyte. (G) A cuticle preparation of a typical embryo from a stauD3;oskBRE− female, with a normal head and only one abdominal segment. (H and I) Typical embryos from StauΔloop2 stauD3;oskBRE− females, showing the loss of head structures and rescue of the abdomen (H), and the stronger symmetric bicaudal phenotype (I). Download figure Download PowerPoint Figure 5.dsRBD5 is required for osk mRNA translation. (A–D) Both StauΔdsRBD5 protein (A and B) and osk mRNA (C and D) localize normally to the posterior of stage 9 (A and C) and 10 oocytes (B and D). (E and F) Osk antibody stainings of wild-type (E) and stauD3 T[StauΔdsRBD5]/stauD3 oocytes (F). Download figure Download PowerPoint A trivial explanation for the inability of StauΔloop2 to localize osk mRNA is that the removal of the extended loop in dsRBD2 disrupts the folding of the protein and prevents it from binding to the RNA. While it is not possible to test the binding of full-length Stau to osk mRNA in vitro, three lines of evidence suggest that this is not the case. First, dsRBD2Δloop2 binds to RNA in vitro, whereas the wild-type domain does not, indicating that this deletion facilitates the folding of the domain into the RNA-binding configuration. Secondly, StauΔloop2 co-localizes with osk mRNA to the anterior of the oocyte (Figure 4F). The localization of Stau to the oocyte requires its association with osk mRNA, since the protein remains in the nurse cells in an osk mRNA null mutant (M.Weston and D.St Johnston, unpublished results). The normal co-localization of the mutant protein to the anterior of the oocyte with osk mRNA therefore indicates that it still binds to the RNA. The third argument to suggest that StauΔloop2 interacts with osk mRNA makes use of the oskBRE− transgene to uncouple osk mRNA translation from localization. In wild-type ovaries, the ectopic Osk protein produced from oskBRE− mRNA suppresses head development in about half of the embryos, and causes a duplication of abdominal segments at the anterior (bicaudal phenotype) in over a third (Table I). Even though the translation of this mRNA does not require its localization to the posterior, it is not efficiently translated in the absence of Stau protein: none of the embryos laid by stauD3;oskBRE− mothers develop the bicaudal phenotype, and almost all form only one abdominal segment at the posterior (Figure 4G).