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Previous Article | Next Article 
Molecular and Cellular Biology, December 2006, p. 9508-9516, Vol. 26, No. 24
0270-7306/06/$08.00+0 doi:10.1128/MCB.01136-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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Institute for Cellular and Molecular Biology, Section of Molecular Cell and Developmental Biology, The University of Texas at Austin, Austin, Texas 78712-0159
Received 23 June 2006/ Returned for modification 24 July 2006/ Accepted 21 September 2006
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ABSTRACT |
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INTRODUCTION |
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These programs of posttranscriptional control of gene expression require RNA binding proteins that recognize regulatory elements within the mRNAs and mediate association with the localization or translational control machinery. Although it has proven difficult in most cases to demonstrate that a particular protein/RNA interaction contributes to regulation, multiple RNA binding proteins are required for correct expression of grk mRNA; these include Squid (Hrp40), Hrp48 (also known as Hrb27C), Bruno (Bru), Vasa, and Otu (8, 9, 13, 31, 41, 45, 49). Each of these proteins is also required for correct expression of osk (11, 14, 20, 30, 32, 40, 44, 47, 51), revealing substantial similarities in the control of grk and osk mRNAs.
Mutants defective for Sqd, Hrp48, and Otu have a common grk mRNA localization defect (9). Normally, grk mRNA is transiently localized to the anterior of the oocyte at stage 8 of oogenesis and then becomes restricted to the dorsal side of the anterior. In the mutants, grk mRNA persists along the anterior and fails to localize dorsally. Because localization of grk mRNA has been suggested to result from two vectorial movements—one toward the anterior and a second directed dorsally (19)—these genes could act specifically in the second movement. Sqd and Hrp48 have also been implicated in translational regulation and act to limit the translation of grk mRNA to the fraction of the mRNA that is properly localized at the dorsal side of the oocyte (9, 31). The mechanistic details of how these proteins contribute to localization and translational control remain to be determined, but it does appear that they function as part of a regulatory RNP complex, since Hrp48 interacts physically with both Sqd and Otu (9). Two components of the complex, Sqd and Hrp48, have been suggested to assemble with the mRNAs in the nucleus and associate with other factors in the cytoplasm (22-24, 31). It is likely that additional members of this complex have not yet been identified.
One candidate for another regulatory factor is Imp, the Drosophila homolog of a family of proteins that act in posttranscriptional regulation in a variety of animals (34, 50). One of the founding members of the family, ZBP-1, binds to a localization element in the chicken beta-actin mRNA (15) and appears to direct localization to the leading edge of embryonic fibroblasts (7). Another founding member, the Xenopus Vg1RBP/VERA protein, binds to signals directing localization of Vg1 and VegT mRNAs to the vegetal pole of the oocyte (4, 5, 10, 16). Mammalian homologs, the Imp proteins, have been suggested to act in mRNA localization (36), mRNA stability (6), and translational regulation (28). A recent report examined the RNA binding properties of Drosophila Imp protein, focusing specifically on the osk mRNA and its possible regulation by Imp (26). Although mutation of candidate Imp binding sites in the osk mRNA did block accumulation of Osk protein, loss of Imp activity did not cause a similar defect.
Here we also characterize the Drosophila Imp protein and show that it interacts with Sqd and Hrp48, two proteins that regulate expression of osk and grk mRNAs. Mutation of the Imp gene does not substantially alter grk or osk expression. Nevertheless, the Imp mutant partially suppresses a grk misexpression phenotype, arguing that it does contribute to grk regulation but may act redundantly and does not have an essential role. Consistent with this interpretation, overexpression of Imp interferes with localization of grk mRNA.
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MATERIALS AND METHODS |
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4-GAL4VP16] V37 driver.
Fly stocks l(1)G0072 (now called ImpG0072), Df(1)HCl33, P[mat4-GAL4VP16] V37 and Dp(1;Y)v+y+ were obtained from the Bloomington stock center. Secondary mutations on the w67c23 ImpG0072 chromosome were removed by extensive backcrossing to w1118 flies. The kinesin-LacZ reporter (3) was obtained from David Stein, the fs(1)K101 and sqd1 flies were from Trudi Schupbach, and the TauGFP flies were from Daniel St Johnston.
Plasmid rescue was performed to confirm that the P element of ImpG0072 is inserted into the Imp gene. The lethality of ImpG0072 was confirmed to be due to the P element by isolation and characterization of revertants: five excision lines were obtained using the P
23 transposase, and none of them shows the lethality observed in ImpG0072.
Homozygous ImpG0072 flies were obtained by the following cross scheme. Df(1)v-L2/Dp(1;Y)v+y+ males were crossed with ImpG0072/FM7c females. Progeny ImpG0072/Dp(1;Y)v+y+ males were crossed with ImpG0072/FM7c females to get ImpG0072 homozygous flies. Homozygous fs(1)K101 ImpG0072 flies were obtained by the same strategy, using an fs(1)K101 ImpG0072 chromosome obtained by recombination.
Generation of Imp antibody and purification of His-Imp. The coding region of the Imp gene was amplified by PCR and cloned into pET3b vector (Novagen). The Imp protein was expressed in Escherichia coli Codon-Plus (Stratagene) and partially purified. Polyclonal antibody against Imp was raised by Josman, LLC.
The Imp coding region was also cloned into PET15a vector (Novagen) to allow expression of Imp with an amino-terminal 6x His tag. The 6x His-Imp fusion protein was expressed in E. coli Codon-Plus RP (Stratagene) and purified using Probond resin (Invitrogen).
Immunodetection and in situ hybridization. Ovaries were dissected and stained as described previously (17, 18). Primary antibodies were used at the following dilutions: rabbit anti-Imp, 1:600; rat anti-Vasa, 1:500; rabbit anti-Oskar, 1:4,000; rat anti-Staufen, 1:100; rabbit anti-Stau, 1:1,000; mouse anti-Gurken [1D12 from the Developmental Studies Hybridoma Bank], 1:10; mouse anti-beta-galactosidase (40-1a; Developmental Studies Hybridoma Bank), 1:40. Secondary antibodies were labeled with Cy5 (Jackson Immunoresearch Laboratories) or Alexa Fluor 488 (Molecular Probes). Stained ovaries were mounted in Vectashield medium (Vector Labs) and imaged with a Leica TCS-SP confocal microscope.
Live imaging of egg chambers was performed as described previously (39) using flies expressing TauGFP maternally to mark microtubules (25).
To quantitate the loss of dorsal localization of Imp in sqd mutant oocytes, images acquired by confocal microscopy were analyzed for signal intensity using the ImageJ software (NIH). For each of four oocytes of each genotype, four nonoverlapping boxes were drawn at random within the dorsal cortical region adjacent to the nucleus or along the cortical region near the posterior pole. The signal intensity of each region was measured, to yield an average value, and the ratios of the dorsal and posterior values were determined. For the wild-type oocytes, the ratios were 1.54, 1.79, 1.83, and 2.00. For the sqd mutant oocytes, the ratios were 1.07, 1.14, 1.14, and 1.24.
In situ hybridization was performed as described previously (42). Linearized plasmids containing the osk 3' untranslated region (UTR) (pY107 cut by BamHI), the bcd 3' UTR (p908 cut by MluI), and the grk 3' UTR (p848 cut by BglII) were used as templates for synthesis of antisense RNA probes. The probes were labeled with digoxigenin-conjugated nucleotides (Roche Diagnostic GmbH).
Western blot analysis.
Protein samples were electrophoresed in a 10% sodium dodecyl sulfate-polyacrylamide gel and electroblotted to a polyvinylidene difluoride membrane. Proteins were detected by chemiluminescence (Western Light; Tropix). Primary antibodies were affinity-purified rabbit polyclonal anti-Imp at 1:3,000, mouse monoclonal anti--tubulin at 1:20,000 (gift from Tim Stearns), mouse monoclonal anti-Sqd at 1:100 (gift from Trudi Schubach), and rabbit polyclonal anti-Hrp48 at 1:20,000 (gift from Don Rio).
Immunoprecipitation. Ovaries of w1118 flies were hand-dissected in phosphate-buffered saline buffer, washed with lysis buffer (50 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% NP-40, 2 mM Pefabloc, 5 mM benzamidine. 2 µg/ml pepstatin, and 2 µg/ml leupeptin) three times, homogenized, and cleared by centrifugation at 13,000 rpm for 25 min at 4°C. Aliquots of the extract (300 µl; equivalent to 50 ovary pairs) were incubated with primary antibody at 4°C for 1 h. Subsequently, 20 µl of protein A/G PLUS agarose beads (Santa Cruz Biotechnology) preequilibrated with lysis buffer were added to the extract and incubated at 4°C for 30 min. Agarose beads were spun down and washed three times with lysis buffer. Next, they were incubated with lysis buffer with or without 50 ng/ml RNase A/T1 (Ambion) for 15 min at 4°C. Finally, beads were recovered by centrifugation and washed with lysis buffer three times. Sodium dodecyl sulfate loading buffer (2x, 50 µl) was added to the beads and boiled at 100°C for 5 min. Samples were assayed by Western blotting.
Filter binding assay. Probes were generated by in vitro transcription in the presence of [32P]UTP and gel purified. Details of the plasmids used to prepare the osk and grk RNAs described in the legend to Fig. 4 are available on request. Twenty microliters of reaction mix containing labeled probe (<0.1 nM in final concentration) and various amounts of purified Imp protein in filter binding buffer (10 mM Tris-Cl [pH 8.0], 25 mM NaCl, 0.2 mM EDTA, 0.1 mg/ml tRNA, 5 mg/ml heparin, 1 mM dithiothreitol) were incubated on ice for 1 h. Filter binding buffer (80 µl) was added to each reaction mixture, and the samples were filtered though nitrocellulose membrane filters (Millipore) preequilibrated with filter binding buffer at 4°C for at least 1 h. The membrane filters were washed three times with 1 ml filter binding buffer and assayed for radioactivity by scintillation spectrometry. Dissociation constants (Kd) were calculated using Kaleidagraph (Synergy Software).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Imp binds to grk mRNA. The RNA binding activity of purified Imp was monitored using a quantitative nitrocellulose filter binding assay. RNA probes for the assay were prepared from different parts of the grk and osk mRNAs (Fig. 4C) as well as from bcd mRNA 3' UTR, which serves as a negative control (there is no indication of any role for Imp in bcd expression). Imp binds with highest affinity to the grk mRNA 5' UTR (Kd is 134 nM) and the 5' part of the coding sequence (Kd of 192 nM) (Fig. 4A and D). Other parts of the grk mRNA, including the 3' coding region and 3' UTR, bind with much lower affinity (Kd of 1.6 and 4.3 µM, respectively). Weak binding is also observed for the osk 3' UTR (various segments bind with a Kd of 0.77 to 3.4 µM) (Fig. 4B and D). The negative control RNA is largely unbound at similar Imp protein concentrations. The high-affinity binding of Imp to grk mRNA, taken together with the colocalization of the mRNA and protein in the oocyte, strongly suggests that Imp binds grk mRNA in vivo. It also appears possible that Imp binds directly to osk mRNA in vivo. However, the lower affinity of the in vitro interaction with osk mRNA raises the possibility that binding occurs in the context of an RNP complex in which multiple proteins contact the mRNA and a high affinity is achieved through multiple contacts, much as has been suggested for the binding of a localization complex to the bcd mRNA (1). Obvious candidates for additional complex components are the Imp-associated proteins Sqd and Hrp48, both of which are concentrated with Imp and osk mRNA at the posterior pole of the oocyte (11, 30, 51).
Imp mutants have no overt ovarian phenotype but suppress the dorsalization of fs(1)K10. For genetic analysis of Imp function, we used a P element insertion mutant, ImpG0072, in which the transposon is inserted into the Imp gene. ImpG0072 is semilethal, with rare escapers surviving as adults for up to several days. Two forms of Imp protein are detected in wild-type ovaries by Western blot analysis (Fig. 1). The most abundant form is about 70 kDa, consistent with the reported structure of the Imp protein. Both protein forms are present at greatly reduced levels in ImpG0072/Df(1)H133 females. The ImpG0072 chromosome was extensively backcrossed to the wild type to remove secondary mutations. Reversion of the mutation by excision of the P element restored full viability and expression of Imp protein.
Homozygous ImpG0072 females, although unhealthy and prone to getting stuck in the growth medium, can produce eggs before they die. The eggs appear phenotypically normal and, if fertilized by wild-type sperm, form viable and fertile adults. Not surprisingly then, the Imp mutants have no substantial defects in distribution or activity of localized mRNAs that contribute to embryonic body patterning (data not shown). Nevertheless, Imp could play a redundant role, perhaps in regulation of grk or osk, given its concentration at the sites where these mRNAs are localized and its association with Hrp48 and Sqd.
As a more sensitive assay for a role in the dorsoventral pathway, we sought to determine whether the Imp mutation could modify an existing dorsoventral patterning defect. Mutation of fs(1)k10, which encodes a transcriptional factor, results in mislocalization of grk mRNA and protein along the anterior margin of the oocyte during midoogenesis, instead of restriction to the anterodorsal corner. Consequently, eggs laid by mutant fs(1)K10 mothers are strongly dorsalized and display expansion and fusion of the two dorsal appendages that normally lie near the dorsal midline (27, 35, 38, 48) (Fig. 5D). When fs(1)k10 females are also homozygous for the ImpG0072 mutation, the dorsalization phenotype is partially suppressed (Fig. 5C and D). In keeping with the absence of a detectable ImpG0072 mutant phenotype, the fs(1)K10 eggshell phenotype is only fully suppressed in very rare cases. More commonly, the embryos from the Imp fs(1)K10 mothers display some degree of reduced dorsalization, as scored by the effects on the dorsal appendages. No substantial change in the grk mRNA distribution of the fs(1)K10 mutant accompanies the partial suppression of the eggshell phenotype (data not shown). This is not surprising, given the normal appearance of grk mRNA distribution in Imp mutants. However, because the eggshell phenotype provides a very sensitive measure of grk patterning activity, it can presumably reveal defects not discernible by the in situ hybridization assay. We conclude that reduction of Imp activity has a very weak effect on dorsoventral patterning.
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In wild-type oocytes, grk mRNA is initially spread across the anterior of the oocyte and becomes tightly restricted to the dorsal anterior before stage 9. Because localization improves in the Imp overexpression oocytes as development of the oocyte advances, it is possible that the actions that restrict the mRNA dorsally are active but less effective or retarded. Alternatively, there may be mechanistically distinct phases in localization—one acting earlier and one later—with overexpression of Imp primarily or exclusively disrupting the early phase. Localization during the later phase would gradually restore the wild-type distribution of grk mRNA and protein.
Imp overexpression disrupts oocyte polarity and expression of osk. osk mRNA is tightly localized to the posterior pole of wild-type oocytes from stage 9 throughout the remainder of oogenesis, and Osk protein only accumulates after localization of the mRNA (14, 20, 32). In Imp overexpression ovaries, osk mRNA (as well as Stau protein, which typically marks the distribution of osk mRNA) (21) appears at two positions within individual oocytes: some of the osk mRNA is localized in a crescent at the posterior pole, the normal site of localization, and some of the osk mRNA appears in a discrete body in the ooplasm (Fig. 7D and G). These bodies, which are never seen in the wild type (Fig. 7C and E), are present in 79% of stage 9 egg chambers (n = 113) and 55% of stage 10 egg chambers (n = 99). Osk protein is present at both sites of osk mRNA concentration (Fig. 7H), demonstrating that translation of osk mRNA is not negatively affected by Imp overexpression. Moreover, the accumulation of Osk is no longer dependent on the posterior localization of the mRNA, revealing a loss of the regulation that normally prevents accumulation of Osk from unlocalized mRNA.
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DISCUSSION |
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The evidence that Imp, Sqd, and Hrp48 interact physically is complemented by striking similarities in grk and osk expression defects that arise from loss of sqd or Hrp48 activity or from overexpression of Imp. In each case, grk mRNA accumulates at the anterior of the oocyte, fails to become dorsally localized, and leads to misexpression of Grk protein. The defects of sqd and Hrp48 mutants in osk expression may result from both direct and indirect effects: a direct effect via binding to osk mRNA and an indirect effect owing to alterations in microtubule organization (11, 26, 30, 51). The same is true for Imp overexpression. In Imp overexpression oocytes, the posterior localization of Kin-ß-gal is disrupted, indicating some degree of microtubule defects. In addition, Imp, like Sqd and Hrp48, colocalizes with osk mRNA to the posterior pole of the oocyte.
The correlations between the consequences of excess Imp activity and loss of sqd or Hrp48 activity may be significant and suggest that Imp competes with these proteins at some level, either for binding to a common substrate or by exerting opposing effects on such a substrate. Alternatively, Imp could inactivate Sqd or Hrp48. Imp overexpression does not substantially alter the amount of Sqd or Hrp48 (data not shown), ruling out one form of inactivation. In addition, sqd and Hrp48 mutants display one phenotype, altered polytenization of nurse cell nuclei (9), which does not occur when Imp is overexpressed, arguing against any simple model in which Sqd and Hrp48 are inhibited by Imp.
Sqd and Hrp48 could compete with Imp at the level of RNA binding: excess Imp would displace Sqd or Hrp48 from shared or closely positioned binding sites on regulated mRNAs, yielding the same phenotype as if Sqd or Hrp48 were eliminated by mutation and thus not available for binding. This model seems unlikely, since Imp binds best to the 5' UTR and 5' coding regions of grk mRNA (regions implicated in grk mRNA localization) (43, 46), while Sqd and Hrp48 bind to the 3' UTR (9, 31) (osk mRNA is considered below). However, Imp does bind with lower affinity to the grk mRNA 3' UTR, and the assays with Sqd and Hrp48 have not tested for binding to the grk mRNA 5' UTR.
Competition could also occur for events that transpire after RNA binding, with bound Imp promoting one outcome for the mRNA and bound Sqd and Hrp48 promoting another. For example, localization of grk mRNA has been suggested to involve two vectorial movements within the oocyte, one directed anteriorly and one directed dorsally (19). In this model, Imp could promote the anterior movement, while Sqd and Hrp48 could contribute to the dorsal movement (a role in keeping with known phenotypes). Increasing the number of copies of one protein that become bound to the mRNA, even without a reduction in the binding of other proteins, could enhance association with the machinery that drives one vectorial movement and thus alter the balance between the two movements. This type of interpretation would explain the partial suppression of the fs(1)K10 ventralization phenotype by the Imp mutant. In the absence of K10, the competition would be skewed in favor of the Imp-promoted outcome. Removing Imp, even if it acts redundantly, could shift the competition back toward the balance normally achieved in wild-type ovaries. This model might appear to be at odds with the known distributions of Imp, Hrp48, and Sqd. Specifically, Imp is colocalized with grk mRNA even after the proposed second vectorial movement of localization, while Hrp48 and Sqd are never detectably colocalized with the mRNA. However, the proposed competition would not require displacement of Imp from the mRNA, and Hrp48 and Sqd might act very early in the localization process (perhaps beginning in the nucleus where the proteins are concentrated) to orchestrate events that only occur later. Thus, the positions of the proteins in the ovary only rule out the possibility that they are all persistently associated with one another but do not argue against the models described here.
The defect in grk mRNA localization caused by overexpression of Imp is accompanied by ectopic accumulation of Grk protein, whose distribution mirrors that of grk mRNA along the anterior of the oocyte. In wild-type ovaries, grk mRNA is transiently concentrated along the anterior of the oocyte at stages 7 and 8, but there is no corresponding anterior ring of Grk protein (37). Thus, the anterior accumulation of Grk when Imp is overexpressed reveals a defect in the control of grk mRNA translation, as well as localization. The premature translation could be an indirect consequence of derailing grk mRNA localization, or it could indicate a more direct effect of excess Imp on translation.
Does Imp act in regulation of osk mRNA? Our discussion of Imp has focused on regulation of grk mRNA, since this role is supported by multiple lines of evidence. Overexpression of Imp also dramatically alters osk expression, acting indirectly by altering microtubule organization and perhaps acting directly through binding to osk mRNA. The data implicating Imp in osk regulation, whether direct or indirect, are substantially less compelling than for regulation of grk. Most importantly, we have no loss-of-function evidence that implicates Imp in osk mRNA regulation or in control of microtubule organization. Second, the binding of Imp to the osk mRNA 3' UTR is relatively weak, with Kd values near or above 1 µM.
Munro et al. (26) specifically explored the possible regulation of osk by Imp. They identified sequences (Imp binding elements [IBEs]) in the osk mRNA as Imp binding sites. Inactivation of the IBEs eliminates accumulation of Osk protein. The osk mRNA initially localizes normally to the posterior of the oocyte but is later delocalized and dispersed in the ooplasm, apparently an indirect consequence of a failure to accumulate Osk protein, which is required for anchoring of osk mRNA (26). Because loss of Imp activity did not cause similar defects, they concluded that another factor (factor X) must bind the IBEs for osk mRNA translation. Factor X could act redundantly with Imp, or factor X alone could mediate the action of the IBEs. Munro et al. argue for the latter option and propose a regulatory interplay between Imp and factor X, in which they compete for binding. By that model, overexpression of Imp would be expected to have consequences similar to mutation of the IBEs. The consequences of Imp overexpression differ when comparing our work to that of Munro et al. They present evidence that Imp overexpression reduces the level of Osk at stage 10 and do not report on osk mRNA localization. We also find a reduction in the accumulation of Osk protein at the posterior pole, but this is accompanied by mislocalization of a fraction of Osk protein to a discrete body in the ooplasm, a feature not observed for the IBE mutants. This body also contains osk mRNA, a localization defect that is clearly different from the dispersal of osk mRNA caused by mutation of the IBEs. To consider the possibility that the difference between our results and those of Munro et al. may reflect different levels of Imp overexpression, we varied the dosage of the P[UAS-Imp] transgene: increasing from one to two copies greatly enhanced the shift of osk mRNA and protein to multiple discrete bodies in the ooplasm but did not eliminate Osk accumulation (data not shown). Thus, in our extensive analysis, the effects of Imp overexpression on osk mRNA localization and translation are markedly different from the IBE inactivation phenotype. We cannot explain why our results differ from those of Munro et al.; further characterization of their Imp overexpression mutant might provide insight.
Imp mutants do not have an osk misexpression phenotype, but the in vitro binding properties of Imp and the consequences of Imp overexpression suggest that Imp plays a redundant role, much as we have argued in the case of grk mRNA. It would not be surprising for Imp to act in regulation of osk, as well as grk, since Imp is associated with proteins known to regulate both mRNAs.
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ACKNOWLEDGMENTS |
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Members of the Macdonald lab, especially Mark Snee, Nan Yan, and Eric Arn, provided useful discussion and comments on the manuscript. We thank the Bloomington Stock Center, Trudi Schupbach, Daniel St Johnston, Don Rio, Tim Stearns, and Tom Hays for fly stocks, cDNAs, or antibodies. Some antibodies were obtained from the Developmental Studies Hybridoma Bank, which was developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, Iowa.
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FOOTNOTES |
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Published ahead of print on 9 October 2006.
Supplemental material for this article may be found at http://mcb.asm.org/.
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