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Dev Biol. Author manuscript; available in PMC 2010 March 16.
Published in final edited form as:
PMCID: PMC2839899

Glorund interactions in the regulation of gurken and oskar mRNAs


Precise temporal and spatial regulation of gene expression during Drosophila oogenesis is essential for patterning the anterior-posterior and dorsal-ventral body axes. Establishment of the anterior-posterior axis requires posterior localization and translational control of both oskar and nanos mRNAs. Establishment of the dorsal-ventral axis depends on the precise restriction of gurken mRNA and protein to the dorsal-anterior corner of the oocyte. We have previously shown that Glorund, the Drosophila hnRNP F/H homolog contributes to anterior-posterior axis patterning by regulating translation of nanos mRNA, through a direct interaction with its 3' untranslated region. To investigate the pleiotropy of the glorund mutant phenotype, which includes dorsal-ventral and nuclear morphology defects, we searched for proteins that interact with Glorund. Here we show that Glorund is part of a complex containing the hnRNP protein Hrp48 and the splicing factor Half-pint and plays a role both in mRNA localization and nurse cell chromosome organization, probably by regulating alternative splicing of ovarian tumor. We propose that Glorund is a component of multiple protein complexes and functions both as a translational repressor and splicing regulator for anterior-posterior and dorsal-ventral patterning.


Asymmetric mRNA localization is essential to establish and maintain polarity of the Drosophila oocyte. Protein asymmetries arising from localized mRNA translation also govern the patterning of the embryonic body axes and the segregation of the somatic and germline lineages. Localization of gurken (grk) mRNA to the posterior pole of the early oocyte results in local production of the Grk TGFα ligand, which signals to the Drosophila EGF receptor (EGF-R) on adjacent somatic follicle cells (González-Reyes et al., 1995; Roth et al., 1995). The follicle cells respond by inducing a reorientation of the oocyte microtubule cytoskeleton that promotes mRNA transport along the anterior-posterior axis of the oocyte (González-Reyes et al., 1995; Theurkauf et al., 1992). Consequently, grk mRNA is transported to the anterior margin of the oocyte and then to the future anterodorsal corner (MacDougall et al., 2003; Neuman-Silberberg and Schüpbach, 1993). Synthesis of Grk at this site results in the localized activation of EGF-R in the overlying follicle cells and the specification of dorsal fates, thereby defining the dorsal-ventral axis of the egg and, ultimately, the embryo (Nilson and Schüpbach, 1999; van Eeden and St Johnston, 1999). Concomitant with grk localization to the future dorsal anterior region of the oocyte, oskar (osk) mRNA accumulates at the posterior pole. Osk protein synthesized from localized osk mRNA nucleates the assembly of the germ plasm, which determines germ cell fate in the embryo. In addition, Osk-dependent assembly of germ plasm is essential for the posterior localization and translation of nanos (nos) mRNA, which is in turn required for abdomen formation in the embryo (Gavis and Lehmann, 1994; Wang et al., 1994).

Localization of grk and osk mRNAs is essential for their function, as mutations that abolish localization of either produce polarity defects. Furthermore, localization must be tightly coupled to translation, since precocious or ectopic translation of these mRNAs also produces deleterious defects in dorsal-ventral and anterior-posterior polarity. Genetic and biochemical studies have identified various proteins that participate in localization and translational regulation of grk and osk mRNAs. Among these, Squid (Sqd), Hrb27C/Hrp48 (referred to hereafter as Hrp48), and Ovarian tumor (Otu) are required both for anterodorsal localization and translational repression of grk mRNA. In mutants for these proteins, grk is mislocalized around the entire anterior cortex and this mislocalized grk is translated, producing dorsalized embryos (Goodrich et al., 2004; Norvell et al., 1999). Hrp48 and Sqd are both members of the heterogeneous ribonucleoprotein (hnRNP) A/B family and both bind to the grk 3'UTR. Hrp48 interacts with Sqd and Otu, suggesting that these three proteins are components of a grk RNP (Goodrich et al., 2004; Norvell et al., 1999). Intriguingly, Sqd, Hrp48, and Otu also participate in osk mRNA localization and/or translation (Huynh et al., 2004; Norvell et al., 2005; Tirronen et al., 1995; Yano et al., 2004) and Sqd and Hrp48 interact with osk mRNA in vitro (Huynh et al., 2004; Norvell et al., 2005; Yano et al., 2004).

Mutations in hfp also cause defects in both grk and osk localization (Van Buskirk and Schüpbach, 2002). hfp encodes the Drosophila homolog of the human RNA binding protein PUF60 and regulates alternative splicing of several ovarian transcripts including otu. Since the grk localization defect of hfp mutants can be rescued by expression of the Otu isoform (Otu-104) that is missing in hfp mutants, Hfp's primary contribution to grk regulation appears to be the generation of Otu-104 (Van Buskirk and Schüpbach, 2002). Mutation of hfp, as well as mutation of sqd, hrp48, and otu produces defects in nurse cell chromatin organization and, similarly to the grk localization defect, the chromatin defect of hfp mutants is rescued by expression of Otu-104 (Goodrich et al., 2004; Van Buskirk and Schüpbach, 2002). Together, these results suggest that Sqd, Hrp48, and Otu act together to regulate multiple mRNAs involved in different developmental processes during oogenesis and that Hfp plays a role in supplying Otu to this complex.

We have previously identified and characterized an hnRNP F/H family member, Glorund (Glo), that is required for translational repression of unlocalized nos mRNA in late oocytes. In addition to defects in nos regulation, a small proportion of glo mutant embryos show osk mRNA localization defects (Kalifa et al., 2006). Here we show that ovaries derived from glo mutant germline clones exhibit defects in dorsal-ventral polarity of the oocyte as well as defects in nurse cell chromosome organization. To better understand these different roles for Glo in oogenesis, we searched for proteins that interact with Glo. We provide evidence that Glo participates in a complex with Hrp48 and Hfp that functions in both grk mRNA localization and nurse cell chromosome dispersion by regulating otu.

Materials and Methods

Fly stocks

The following mutants and transgenic lines were used: glo162x and g-gloS (Kalifa et al., 2006), khc:lacZ (Clark et al., 1994), potu-104 (Sass et al., 1995). glo162x germline clones were induced by the dominant female sterile method (Chou et al., 1993) using the P{neoFRT}82B, P{ovoD1–18}3R chromosome. The yw67c23 and Oregon R strains (Lindsley and Zimm, 1992) were used as wild-type controls.

In situ hybridization and immunostaining

In situ hybridization with digoxigenin-labeled RNA probes for grk and osk was performed as described previously (Gavis and Lehmann, 1992). Anti-Grk and anti-Br-C immunostaining was performed according to Goodrich et al. (2004) using 1:10 monoclonal anti-Grk (1D12; Queenan et al., 1999) or 1:100 monoclonal anti-Br core (25E9.D7; Yakoby et al., 2008), followed by 1:1000 AlexaFluor 568 anti-mouse (Invitrogen/Molecular Probes). Anti-β-galactosidase immunostaining was performed according to Kalifa et al. (2006) using 1:1000 rabbit anti-βgal (ICN Cappel) and 1:1000 AlexaFluor 568 anti-rabbit antibodies. DNA and actin were visualized with DAPI and Oregon Green 488 phalloidin (Invitrogen/Molecular Probes), respectively.

Immunoprecipitation and immunoblot analysis

Ovaries of well fed females were dissected in PBS, washed twice with IP buffer [25 mM Hepes (Na+) pH 7.4, 150 mM NaCl, 2.5 mM MgCl2, 0.5 mM EDTA, 0.01% Triton X-100, 1× complete protease inhibitor cocktail (Roche), 10 µg/ml pepstatin], homogenized, and cleared by centrifugation at 13,000 rpm for 10 min at 4° C. The pellet was rehomogenized and centrifuged, and the cleared extracts were pooled. Aliquots of the extract were supplemented with either RNase [100 µg/ml RNase A and 100 units/ml RNase One (Promega)] or 1 unit/µl RNasin (Promega) and incubated with Dynabeads Protein G (Invitrogen) for 1 hr at 4° C. The pre-absorbed extract was then incubated overnight at 4° C with Dynabeads Protein G (Invitrogen) coated with one of the following antibodies: anti-Glo (monoclonals 5B7+1H2 or polyclonal mouse anti-Glo; Kalifa et al., 2006), rabbit anti-Hrp48 (Siebel et al., 1994), rabbit anti-βgal (Invitrogen/Molecular Probes), monoclonal anti-Sqd (8F3; Goodrich et al., 2004), monoclonal anti-Hfp (6G10; Van Buskirk and Schüpbach, 2002), monoclonal anti-Sxl (m104; Penn and Schedl, 2007), rabbit anti-GFP (Abcam). Beads were washed five times with IP buffer and bound protein was eluted by boiling in SDS-PAGE sample buffer. For co-immunoprecipitation of Sxl and Snf, nuclear extract (Deshpande et al., 1996) kindly provided by P. Graham was treated with RNase or RNasin as above and complexes were recovered using Protein A/G Plus-Agarose (Santa Cruz Biotechnology) pre-coated with anti-Sxl (m104) antibody.

Eluted proteins were resolved by SDS-PAGE and transferred to nitrocellulose membrane. Immunoblotting was carried out in 10 mM Tris-HCl pH 7.5/150 mM NaCl/5% nonfat dry milk with the following primary antibodies: 1:400 anti-Glo (5B7); 1:100 anti-Sqd (8F3); 1:20 anti-Hfp (6G10); 1:5000 anti-Hrp48; 1:2000 rabbit anti-Osk (Vanzo and Ephrussi, 2002); 1:10,000 monoclonal anti-Snf (4G3; gift of P. Schedl). Proteins were visualized by ECL (Roche).

GST pull-down assay

Recombinant full length GST-Hrp48 and GST-Hfp were expressed in E. coli from plasmids generously provided by T. Schüpbach and bound to glutathione-agarose resin (Sigma) according the manufacturer. Resin was equilibrated in IP buffer and incubated with purified MBP-Glo (Kalifa et al., 2006) for 1 hour at 4° C. After extensive washing with IP buffer, bound proteins were eluted with glutathione (Sigma) as specified by the manufacturer, resolved by SDS-PAGE, and immunoblotted with either anti-Glo or anti-GST (Santa Cruz Biotechnology) antibodies.


Multiple functions for glo during oogenesis

Since animals that are homozygous mutant for a glo null allele (glo162x) do not survive to adulthood (Kalifa et al., 2006), we investigated requirements for glo during oogenesis by generating homozygous glo162x germline clones in females heterozygous for glo162x using the dominant female-sterile method (Chou et al., 1993). Approximately 30% of eggs laid by females with glo162x germline clones have abnormal dorsal appendages, ranging from short, wide appendages to fused appendages that extend laterally around the anterior of the egg (Figs. 1B–D). Analysis of ovaries dissected from these females (referred to as glo162x ovaries) showed a higher frequency (55%) of dorsal appendage defects among late oocytes, suggesting that some of these oocytes never mature as eggs. In the wild-type ovary, the 15 germline derived nurse cells supply maternal mRNAs and other metabolites to the oocyte. As they complete their role, the nurse cells initiate apoptosis and rapidly transfer or "dump" their contents into the oocyte. In glo162x ovaries, we observed egg chambers that failed to undergo nurse cell dumping. Finally, DAPI staining of nuclei revealed a developmental defect in chromatin organization in nurse cells from glo162x ovaries. In wild-type ovaries, nurse cell chromosomes are initially polytene but disperse toward the middle stages of oogenesis. In nurse cells from glo162x ovaries, chromosomes fail to disperse during mid-oogenesis and maintain a polytene morphology (Figs. 1G, H; also see Figs. 5A, B). All of the observed phenotypes are rescued by a single copy of a genomic glo transgene (Kalifa et al., 2006), confirming that they result from loss of glo function.

Fig. 1
Regulation of grk by Glo
Fig. 5
Expression of Otu-104 rescues the polytene nurse cell chromosome defect of glo162x egg chambers

Mislocalization and ectopic translation of grk mRNA in glo mutant ovaries

The dorsal appendage defects exhibited by glo mutant eggs suggest a defect in specification of dorsal follicle cell fates and, consequently, that glo may be required for proper regulation of grk mRNA. We therefore investigated whether grk mRNA and protein localization are affected in glo162x oocytes. In wild-type mid-stage oocytes, grk mRNA is localized to the future dorsal-anterior corner of the oocyte overlying the oocyte nucleus (Fig. 1E). Grk protein is also restricted to this same region (Fig. 1G), where it induces dorsal follicle cell fates (Neuman-Silberberg and Schüpbach, 1993). Consistent with the dorsal appendage phenotype in glo162x egg chambers, grk mRNA appears mislocalized in a ring at the anterior of the oocyte in 30% of stage 8–9 egg chambers (n=73) as compared to wild-type oocytes at the same stage (Fig. 1F). Similarly, in approximately 25% of egg chambers, Grk protein is no longer restricted to the region adjacent to the oocyte nucleus, but is visible along the anterior margin of the oocyte (Fig. 1H). Thus, glo appears to be required for proper localization of grk mRNA to the dorsal-anterior region of the oocyte and the localized production of Grk protein.

The observed misexpression of Grk protein would be predicted to induce dorsal fates in a broader subset of anterior follicle cells, thereby producing broad or fused dorsal appendages that extend laterally. To determine whether follicle cell fates are indeed affected in glo162x egg chambers, we monitored the distribution of Broad-Complex (Br-C) protein. Br-C expression in dorsal follicle cells depends on Grk signaling and dictates dorsal appendage fate (Deng and Bownes, 1997). At stage 10, Br-C is expressed in anterior dorsolateral follicle cells where Grk levels are moderate, and repressed in the dorsal-most follicle cells where Grk levels are highest (Deng and Bownes, 1997; Fig. 1I). In glo mutant egg chambers, Br-C is detected in the dorsal-most follicle cells (Fig. 1J, K) and sometimes more posteriorly than in wild-type (Fig. 1J). This expanded Br distribution is consistent with a failure to restrict grk mRNA and its translation to the dorsal anterior corner of the oocyte. Furthermore, the resulting expansion of dorsolateral follicle cell fates is consistent with the observed glo mutant dorsal appendage defects.

Requirement for glo in osk localization

A small and variable fraction (5–10%) of embryos produced from glo162x germline clones exhibit defects in posterior localization as well as ectopic localization of both osk mRNA and protein (Kalifa et al., 2006). A variable fraction of stage 8–10 glo162x egg chambers lack osk mRNA at the posterior (Fig. 2A, B) and Osk protein levels are reduced in glo162x ovary extract, consistent with posterior localization defects observed in glo mutant embryos. We do not detect mislocalized osk mRNA or ectopic Osk protein during mid-oogenesis (data not shown), suggesting that the ectopic localization observed in glo162x embryos most likely occurs at late stages of oogenesis. Localization of both osk and grk is microtubule-dependent, and numerous mutations cause defects in localization of these and other mRNAs indirectly, through an effect on oocyte microtubule organization (reviewed in Steinhauer and Kalderon, 2006). To determine whether glo162x affects microtubule organization, we analyzed overall microtubule polarity in glo162x oocytes using a kinesin:β-galactosidase (kin:βgal) fusion protein that provides a marker for microtubule plus ends in the oocyte (Clark et al., 1994). During mid-oogenesis, kin:βgal is localized to the posterior in wild-type oocytes and this localization is unaffected in glo162x mutants (Fig. 2C, D). Thus, the defects in osk and grk localization in glo162x ovaries appear to occur independently of the microtubule cytoskeleton. Posterior transport of osk requires a splicing event that removes its first intron (Hachet and Ephrussi, 2004; Kim-Ha et al., 1993). Since mammalian hnRNP F and H have been implicated as general splicing factors as well as regulators of alternative splicing (Chen et al., 1999; Chou et al., 1999; Gamberi et al., 1997; Garneau et al., 2005), we tested whether the osk localization defect in glo162x ovaries results from a defect in osk splicing. By using RT-PCR with primers that flank the first intron, we do not detect a product corresponding to unspliced osk mRNA in glo162x ovaries (data not shown), indicating that glo is not required for osk first intron splicing.

Fig. 2
Defect in osk mRNA localization but not microtubule organization in glo mutant ovaries

RNA-independent association of Glo and Hrp48

The nuclear morphology, nurse cell dumping, and dorsal appendage defects observed in glo162x ovaries resemble those exhibited by various hrp48 mutant alleles. Moreover, different hrp48 alleles produce defects in localization and/or translation of grk and/or osk mRNAs (Goodrich et al., 2004; Huynh et al., 2004; Yano et al., 2004). These similarities suggest that Glo and Hrp48 might function in the same complex or by the same mechanism. To determine whether Glo physically associates with Hrp48, we assayed for co-immunoprecipitation of Glo and Hrp48 from ovary extracts. Glo and Hrp48 are co-immunoprecipitated by anti-Hrp48 antibody, but not by a control anti-βgal antibody (Fig. 3A). This interaction persists in extracts treated with RNase, under conditions that disrupt the known RNase sensitive complex of Sex-lethal (Sxl) and Sans-fille (Snf) proteins (Deshpande et al., 1996) (Fig. 3B). Thus, the association of Glo and Hrp48 in the same complex does not depend on the binding of these proteins to RNA.

Fig. 3
Glo interacts with Hrp48 but not with Sqd

Since Hrp48, Sqd, and Otu have been proposed to act in a complex to regulate grk (Goodrich et al., 2004), we tested whether Glo also interacts with Sqd and Otu. Glo is not detected in anti-Sqd immunoprecipitates, however, either in the absence or presence of RNA (Fig. 3C). Similarly, Sqd is not detected in anti-Glo immunoprecipitates (Fig. 3D). Because the monoclonal anti-Glo antibody used in this experiment (5B7) could block protein-protein interactions, we tested a second monoclonal anti-Glo antibody (1H2) as well as two polyclonal antibodies (Fig. 3D). In all cases, no interaction is detected between Glo and Sqd. Co-immunoprecipitation experiments also failed to detect any association between Glo and Otu (data not shown).

Glo interacts with the splicing factor Hfp

In a separate unbiased approach, we took advantage of transgenic animals expressing Glo-GFP to identify proteins that interact with Glo. The gfp-glo transgene rescues both the lethality and maternal effect phenotypes of glo162x, indicating that the fusion protein is functional. Glo-GFP was immunoprecipitated from ovary extracts and proteins in the immunoprecipitate were separated by SDS-PAGE. As a control, we performed parallel immunoprecipitations using ovaries from females carrying the mcp-gfp transgene (Forrest and Gavis, 2003). Proteins enriched in the Glo-GFP immunoprecipitate were analyzed by mass spectrometry. Among these, Hfp is one of the most highly represented, with 7 peptides distributed across 241 amino acids of the 637 amino acid protein.

Hfp was previously shown to be required for both the production of wild-type levels of grk transcript and for proper grk mRNA localization. In addition, hfp mutant egg chambers display defects in posterior localization of osk mRNA (Van Buskirk and Schüpbach, 2002). We confirmed directly that native Glo and Hfp can be co-immunoprecipitated from ovary extract, using antibodies to either protein, but not using a control anti-Sxl antibody (Figs. 4A, B). In each case, the interaction persists after RNase treatment of the extract, indicating that the association of Glo and Hfp is RNA-independent (Figs. 4A, B). To determine whether Glo, Hrp48, and Hfp could all be part of the same complex, we also tested whether Hfp and Hrp48 interact. Analysis of anti-Hrp48 immunoprecipitates showed that in addition to Glo (Fig. 3A), Hfp forms an RNA-independent association with Hrp48 (Fig. 4C). Like Glo, Hfp does not appear to interact with Otu (data not shown).

Fig. 4
Physical interaction of Glo and Hfp

Co-immunoprecipitation of Glo with Hrp48 and Hfp does not distinguish whether Glo interacts directly with these proteins or only indirectly, through interactions with additional components of the complex. To begin to distinguish between these possibilities, we performed in vitro binding assays using purified tagged recombinant Glo, Hrp48, and Hfp proteins. MBP-Glo binds to both GST-Hrp48 and GST-Hfp (Fig. 4D), consistent with a direct association of Glo with Hrp48 and Hpf in vivo.

Expression of Otu-104 rescues the glo mutant dorsoventral and nuclear morphology defects

Similarly to glo mutants, nurse cell nuclei in hfp mutant egg chambers show persistence of polytene chromatin and hfp mutant eggs are often shorter than wild-type, with dorsal appendages defects. To determine whether the glo mutant phenotype is due to an effect of glo on hfp, we assayed Hpf levels glo162x ovaries. Immunoblot analysis showed that Hfp protein levels are unaltered by glo162x (data not shown). Hfp has previously been shown to regulate the alternative splicing of otu pre-mRNA. otu can be alternative spliced into two protein isoforms, Otu-98 and Otu-104 (Steinhauer and Kalfayan, 1992) and Otu-104 levels are decreased in hfp mutant ovaries (Van Buskirk and Schüpbach, 2002). A tudor domain encoded within the differentially spliced exon of Otu-104 has been proposed to play a role in grk mRNA localization (Goodrich et al., 2004). Transgenic expression of Otu-104 rescues the nurse cell polytene chromosome and dorsal appendage defects of hfp mutant ovaries (Van Buskirk and Schüpbach, 2002), consistent with a requirement for Hfp in otu splicing. Expression of the potu-104 transgene (Sass et al., 1995) also partially rescues the hrp48 mutant nurse cell chromatin defect (Goodrich et al., 2004). Since mammalian homologs of Glo have been implicated in alternative splicing (Chen et al., 1999; Chou et al., 1999; Garneau et al., 2005), we hypothesized that Glo might also act through production of Otu-104. To test this idea, we expressed the Otu-104 isoform in glo162x females using potu-104. A single copy of potu-104 completely eliminates the dorsal appendage defects of late oocytes dissected from glo162x ovaries and reduces the nurse cell chromatin defect from 88% to 56% of egg chambers (Fig. 5, Table 1). PCR analysis to detect loss of the otu-104 splice form in previtellogenic glo mutant egg chambers, as previously performed for hfp mutants (Van Buskirk and Schüpbach, 2002), proved inconclusive. However, decreased production of otu-104 may be difficult to detect due to the variable contribution of glo mutant germline clones to ovarian tissue at previtellogenic stages, compounded by the variable penetrance of the glo mutant phenotype. Nonetheless, the physical interaction between Glo, Hfp, and Hrp48 and the phenotypic rescue of these mutants by Otu-104 suggest that Glo participates with Hfp and Hrp48 in regulating the alternative splicing of otu.

Table 1
Rescue of glo mutant dorsal appendage and polytene defects by Otu-104


HnRNPs contribute to numerous RNP complexes to regulate all aspects of mRNA metabolism, including splicing, nuclear export, intracellular localization, translational regulation, and stability (reviewed in Dreyfuss et al., 2002). We previously identified Glo, the Drosophila hnRNP F/H homolog, as an ovarian repressor of nos translation (Kalifa et al., 2006). Here we have investigated additional roles for Glo in Drosophila oogenesis through the identification of Glo interacting proteins. We provide biochemical evidence that Glo interacts directly with Hrp48 and Hfp, two proteins previously shown to regulate grk mRNA. Consistent with this interaction, ovaries lacking glo exhibit defects similar to those observed for hrp48 and hfp mutant ovaries, including abnormal dorsal appendages, a corresponding mis-regulation of grk mRNA localization and translation, and aberrant nurse cell chromosome organization. Together, these data suggest Glo, Hrp48, and Hfp act in a complex that regulates grk mRNA and nuclear morphology.

Hfp has been previously implicated in alternative splicing of otu to produce the Otu-104 isoform (Van Buskirk and Schüpbach, 2002). Moreover, the defects in nurse cell chromosome dispersion and grk mRNA localization exhibited by hfp, hrp48, and glo mutants are restored by transgenic expression specifically of the Otu-104 isoform (Goodrich et al., 2004; Van Buskirk and Schüpbach, 2002)(this work). Hrp48 regulates the tissue-specific alternative splicing of the P element third intron (Hammond et al., 1997; Siebel et al., 1994) and Ubx mRNA (Burnette et al., 1999) and we now show that Hrp48 interacts with Hfp. Although Glo has not previously been implicated in splicing, mammalian hnRNP F and H proteins have been shown to regulate alternative splicing of several mRNAs (Chen et al., 1999; Chou et al., 1999; Garneau et al., 2005). The RNA-independent association of Glo with two known Drosophila splicing proteins, Hfp and Hrp48, the shared mutant nurse cell chromosome dispersion and grk mislocalization defects, and the rescue of these defects by the potu-104 transgene, suggest that all three proteins participate in a splicing complex that contributes to two different processes, grk regulation and nurse cell chromosome dispersion, via alternative splicing of otu.

Hrp48 also participates in a complex with Sqd and Otu proteins to regulate grk mRNA localization and, independently, localization and/or translation of one or more as yet unidentified mRNAs required for nurse cell chromosome dispersion (Goodrich et al., 2004). Complexes containing Hrp48 and Sqd are thought to assemble on grk and potentially additional mRNAs in the nucleus and exit to the cytoplasm, where they are joined by Otu for assembly, transport, and/or anchoring of translationally repressed RNPs. How this complex regulates mRNA(s) required for nuclear chromosome dispersion remains to be uncovered. Hrp48 interacts with the osk 5' and 3'UTRs and colocalizes with osk mRNPs. These data together with results from genetic analysis implicate Hrp48 as a component of a translationally repressed osk transport particle (Huynh et al., 2004; Yano et al., 2004). In addition to hrp48 mutants, sqd and otu mutants exhibit osk localization defects (Norvell et al., 2005; Tirronen et al., 1995). Thus, it is possible that a Hrp48/Sqd/Otu complex regulates osk as well as grk. We do not detect either Sqd or Otu in co-immunoprecipitates with Glo or Hfp, indicating that the association of Hfp, Hrp48, and Glo represents a complex distinct from Hrp48/Sqd/Otu. Immunolocalization studies indicate that Hfp is a nuclear protein (Van Buskirk and Schüpbach, 2002), whereas Glo and Hrp48 are both nuclear and cytoplasmic (Goodrich et al., 2004; Huynh et al., 2004; Kalifa et al., 2006; Yano et al., 2004). We propose that Glo and Hrp48 participate together in a nuclear splicing complex but contribute independently to distinct complexes that regulate localization and/or translation of different target mRNAs in the cytoplasm. Intriguingly, one Hrp48-containing complex contributes to the formation of the other.


We thank P. Graham, A. Ephrussi, D. Rio, P. Schedl, T. Schüpbach, and N. Yakoby and S. Shvartsman for reagents and/or fly stocks, S. Chatterjee for technical assistance, G. Gray for fly media preparation, S. Kyin and the Princeton Mass Spectrometry Facility for protein analysis, and D. Lerit, R. Jain, and T. Schüpbach for comments on the manuscript. This work was supported by NIH grant GM061107 to E.R.G.


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