Article |
Address correspondence to Kimberly L. Mowry, Box G-J2, Brown University, Providence, RI 02912. Tel.: (401) 863-3636. Fax: (401) 863-1201. email: kimberly_mowry{at}brown.edu
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Abstract |
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Key Words: RNA-binding protein; Vg1; VegT; Xenopus; oocyte
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Introduction |
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During Xenopus oogenesis, maternal mRNAs localized along the animalvegetal axis can influence patterning later during embryogenesis (for review see Mowry and Cote, 1999). The vegetal hemisphere is a repository of developmental information; both mesodermal and endodermal determinants reside there (for review see Chan and Etkin, 2001). Indeed, one mRNA localized to the vegetal hemisphere is VegT, a T-box transcription factor (Lustig et al., 1996; Stennard et al., 1996; Zhang and King, 1996; Horb and Thomsen, 1997) required for endoderm and mesoderm specification (Zhang et al., 1998). Also localized to the vegetal hemisphere is Vg1 mRNA, a member of the TGF-ß growth factor superfamily (Weeks and Melton, 1987) that has been implicated in mesoderm and endoderm specification (Dale et al., 1993; Thomsen and Melton, 1993; Kessler and Melton, 1995; Joseph and Melton, 1998). Misexpression of either Vg1 or VegT in the animal hemisphere leads to induction of mesoderm in cells that would normally form ectoderm (Dale et al., 1993; Thomsen and Melton, 1993; Zhang and King, 1996), underscoring the importance of regulating the localization of these RNAs.
Vegetal localization of Vg1 and VegT RNAs is directed by localization elements (LEs) contained within their 3' untranslated regions (Mowry and Melton, 1992; Bubunenko et al., 2002; Kwon et al., 2002). Within the LEs, repeated sequence elements are critical for proper function (Deshler et al., 1997; Gautreau et al., 1997; Betley et al., 2002; Bubunenko et al., 2002; Kwon et al., 2002; Lewis et al., 2004). Two such elements, VM1 (YYUCU; Gautreau et al., 1997; Cote et al., 1999; Lewis et al., 2004) and E2 (A/U,YCAC; Deshler et al., 1997, 1998), function as binding sites for specific RNA-binding proteins. Clustering of VM1 and E2 sites within LEs appears to be critical for function (Betley et al., 2002; Bubunenko et al., 2002; Kwon et al., 2002; Lewis et al., 2004), perhaps by facilitating interactions between protein components of the localization machinery. Identified protein components include a set of RNA-binding proteins that interact directly with the Vg1 LE (Schwartz et al., 1992; Mowry, 1996; Deshler et al., 1997, 1998; Havin et al., 1998; Cote et al., 1999; Zhao et al., 2001; Kroll et al., 2002). Two of these RNA-binding proteins, hnRNP I (VgRBP60; Cote et al., 1999; Lewis et al., 2004) and Vg1RBP/vera (Deshler et al., 1997, 1998; Havin et al., 1998), bind to VM1 and E2 sites, respectively. Roles in vegetal localization for these proteins were revealed through mutational analyses in which base changes in VM1 or E2 sites both disrupted protein binding in vitro and abolished localization of the RNA in vivo (Deshler et al., 1997, 1998; Cote et al., 1999; Lewis et al., 2004). Both hnRNP I and Vg1RBP/vera colocalize with Vg1 RNA at the vegetal cortex (Cote et al., 1999; Zhang et al., 1999), as do two additional RNA-binding proteins implicated with key roles in vegetal RNA localization, Prrp (Zhao et al., 2001) and XStau (Yoon and Mowry, 2004). Although these proteins bind to and colocalize with Vg1 RNA, when and where they assemble onto Vg1 RNA in vivo is still not known.
Cytoplasmic RNA localization relies on interactions between cis-acting sequences and multiple trans-acting factors, and it has been hypothesized to occur in the context of an RNP complex (Mowry, 1996; Ross et al., 1997; Arn et al., 2003). Indeed, in some instances, large RNP granules have been visualized during RNA transport (Barbarese et al., 1995; Bertrand et al., 1998; Rook et al., 2000; Krichevsky and Kosik, 2001; Wilkie and Davis, 2001). Formation of a localization-specific RNP is arguably an early or initiating event in the localization pathway and, until recently, it has been assumed that assembly of the transport RNP occurs in the cytoplasm. More recent findings have hinted that the process could instead initiate in the nucleus (for review see Farina and Singer, 2002). For example, a growing number of trans-acting localization factors have been identified as hnRNP or otherwise predominantly nuclear proteins. This list includes mammalian hnRNP A2 (Hoek et al., 1998), Drosophila squid (Norvell et al., 1999), Xenopus hnRNP I (Cote et al., 1999), yeast Loc1p (Long et al., 2001), and vertebrate ZBP2/KSRP (Gu et al., 2002). In addition to their roles in cytoplasmic RNA localization, many of these proteins have functions in nuclear events in RNA biogenesis such as splicing and nuclear export (Patton et al., 1991; Matunis et al., 1992; Mayeda et al., 1994; Min et al., 1997; Bilodeau et al., 2001). Intriguingly, a potential link between splicing and mRNA localization has been uncovered through analyses of mRNA localization in Drosophila (for review see Palacios, 2002). In motile fibroblasts, ZBP1 is required for localization of ß-actin mRNA to the leading edge (Kislauskis et al., 1994; Ross et al., 1997), and colocalizes with both nuclear and cytoplasmic ß-actin transcripts (Oleynikov and Singer, 2003). Although these analyses suggest a link between nuclear factors and cytoplasmic localization, they do not provide insight into the biochemical interactions underlying potential roles for nuclear factors in RNA transport.
Here, we show that hnRNP I and Vg1RBP/vera bind to Vg1 and VegT RNAs in both the nucleus and the cytoplasm, providing biochemical evidence that RNA localization initiates in the nucleus rather than in the cytoplasm. Upon export from the nucleus, the core RNP complex is remodeled and additional factors, including Prrp and XStau, are recruited. These results define distinct nuclear and cytoplasmic steps in the localization pathway, and suggest that the binding of specific RNA-binding proteins in the nucleus can direct an RNA to its final destination in the cytoplasm.
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Results |
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hnRNP I and Vg1RBP/vera interact both in vitro and in vivo
Interaction between Vg1RBP/vera and hnRNP I may be important for vegetal RNA transport; both proteins are required for localization of Vg1 and VegT RNAs (Deshler et al., 1997, 1998; Havin et al., 1998; Cote et al., 1999; Bubunenko et al., 2002; Kwon et al., 2002), they interact with these RNAs in the nucleus (Fig. 2), and their binding sites are clustered in close proximity (Bubunenko et al., 2002; Lewis et al., 2004). To test whether Vg1RBP/vera and hnRNP I can interact with one another, we immunoprecipitated hnRNP I from S10 lysates and tested for the presence of Vg1RBP/vera by immunoblot (Fig. 3 A). Indeed, Vg1RBP/vera is associated with hnRNP I (Fig. 3 A, lane 4). To further test this interaction, FLAG-tagged Vg1RBP/vera was translated in vitro and incubated with oocyte S10 lysate. After immunoprecipitation with anti-FLAG antibodies, immunoblot analysis with anti-hnRNP I showed that Vg1RBP/vera complexes contain hnRNP I (Fig. 3 B, lane 6). To determine whether the Vg1RBP/verahnRNP I interaction relied on binding to the same target RNA, binding reactions were treated with RNase A before immunoprecipitation. The interaction was insensitive to RNase in vitro (Fig. 3 B, lane 7), suggesting the possibility of a direct interaction between the two proteins. To test this, in vitrotranslated Vg1RBP/vera and hnRNP I were mixed and were immunoprecipitated with anti-Vg1RBP/vera, and bound hnRNP I was detected by immunoblotting with anti-hnRNP I. Indeed, the two in vitrotranslated proteins can interact in the absence of any other Xenopus proteins or mRNA (Fig. 3 C, lane 4). These results indicate that hnRNP I and Vg1RBP/vera are capable of interacting with one another in vitro, perhaps directly.
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XStau associates with the vegetal RNA localization RNP in the cytoplasm
Recent results have implicated an additional RNA-binding protein, XStau, with a role in Vg1 RNA transport (Yoon and Mowry, 2004). To ask where XStau associates with the vegetal localization RNP, we probed interactions with Prrp (Fig. 5 B) and Vg1RBP/vera (Fig. 5 C). In cytoplasmic fractions prepared from oocytes expressing Prrp-myc, XStau was found in Prrp-containing complexes (Fig. 5 B, lane 4). The interaction between Prrp and XStau appears indirect, as the association was disrupted by RNase A treatment (Fig. 5 B, lane 5). To test whether XStau can associate with the core localization RNP in the nucleus, Vg1RBP/vera-FLAG was expressed in oocytes, and nuclear and cytoplasmic fractions were isolated. As shown in Fig. 5 C, Vg1RBP/vera-FLAG complexes contained XStau only in the cytoplasm (lane 8), and not in the nucleus (lane 5). A similar interaction was observed between XStau and hnRNP I (unpublished data). Together, these data suggest that Prrp, Vg1RBP/vera, hnRNP I, and XStau are present in a cytoplasmic RNP containing vegetally localized RNAs.
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Discussion |
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Previously, we suggested that the clustering of the binding sites for hnRNP I and Vg1RBP/vera may demarcate a consensus LE RNA and serve to facilitate interactions (Bubunenko et al., 2002; Lewis et al., 2004). Here, we show that these proteins are capable of interacting in vitro and in vivo (Fig. 3 and Fig. 4). However, the association in the nucleus and the cytoplasm is distinct; the interaction may be direct in the nucleus, but is dependent on RNA binding in the cytoplasm (Fig. 4). The possibility that hnRNP I and Vg1RBP/vera can interact directly in the nucleus may suggest that the two proteins associate with one another before binding the vegetally localized RNA. Alternatively, one of the proteins may bind to the vegetally localized RNA first and recruit the second protein to the RNA, thereby nucleating assembly of the nuclear localization RNP.
The dynamic nature of the Vg1RBP/verahnRNP I interaction between the nucleus and the cytoplasm (Fig. 4) suggests a remodeling of the localization RNP complex upon export to the cytoplasm (Fig. 6). Our observations that distinct sets of factors comprise the nuclear and cytoplasmic vegetal RNPs lend support to this idea as well. Remodeling of the localization RNP may be linked to the phosphorylation state of hnRNP I, as phosphorylation is required for nuclear export of the mammalian homologue (Xie et al., 2003). The phosphorylation state of the Xenopus protein similarly reflects its nucleocytoplasmic distribution (Xie et al., 2003) and could alter its interactions with other proteins. Although the role of hnRNP I phosphorylation in localization is still unknown, it will be important to determine if it influences the composition of Vg1 or VegT RNPs.
In contrast to Vg1RBP/vera and hnRNP I, both Prrp and XStau associate with the vegetal RNP only in the cytoplasm, indicating that these factors may function in the cytoplasm to direct vegetal RNA localization at later stages during the transport pathway. Neither a nuclear localization signal nor a nuclear export signal has been identified in Prrp; however, Zhao et al. (2001) noted a COOH-terminal domain of the protein that has a similar amino acid composition to the M9 nucleocytoplasmic shuttling domain of hnRNP A1 (Michael et al., 1995a; Siomi and Dreyfuss, 1995). Although our results do not provide evidence for Prrp function in the nucleus, a nuclear role for Prrp cannot be excluded. Although XStau may be present in both the nucleus and the cytoplasm (Fig. 5 C), the protein is most abundant in the cytoplasm, and we observed an interaction of XStau with the localization RNP proteins only in the cytoplasm (Fig. 5, B and C). Hence, our data indicate that both Prrp and XStau join the core localization RNP once it is exported to the cytoplasm (Fig. 6).
We have uncovered distinct nuclear and cytoplasmic steps in the vegetal RNA localization pathway through biochemical analysis of the Vg1 and VegT RNPs. Moreover, we suggest that vegetal RNP assembly in the nucleus may be required for the formation of a cytoplasmic localization-competent RNP, thus linking nuclear and cytoplasmic steps in the vegetal RNA transport pathway. Connections between steps in RNA biogenesis are increasingly appreciated (for review see Reed, 2003). For example, recent analyses on mRNA localization in Drosophila have revealed a requirement for components of the exon junction complex (Hachet and Ephrussi, 2001; Mohr et al., 2001). We have shown that Xenopus hnRNP I and Vg1RBP/vera first bind to vegetally localized RNA in the nucleus, but roles for these factors in other post-transcriptional functions in the oocytes remain to be determined. Nonetheless, it is tempting to speculate that such proteins could link cytoplasmic RNA localization to earlier nuclear events in RNA biogenesis such as transcription and splicing. The challenge now is to determine how the nuclear and cytoplasmic steps in the vegetal RNA localization pathway are interlinked to ensure proper regulation and coordination of RNA localization, from initiation in the nucleus to its final destination the cytoplasm.
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Materials and methods |
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Vg1RBP/vera-FLAG and hnRNPI-FLAG were in vitro translated in wheat germ extract (Promega) according to the manufacturer's instructions, and were used directly for immunoprecipitation. For antibody production, the NH2-terminal 136 amino acids of hnRNP I (including RRM1) were amplified from pBShnRNP I and cloned into pGEX4T1 (Amersham Biosciences) to generate pGST-N+RRM1. GST-N+RRM1 was expressed and purified by GST chromatography as in Coligan et al. (1995), followed by Mono QTM chromatography.
Immunoblot analysis
Immunoblotting was performed as described in Denegre et al. (1997) with primary antibodies as follows: anti-hnRNP I, anti-XStau (Yoon and Mowry, 2004), anti-Vg1RBP/vera (supplied by J. Yisraeli, Hebrew University, Jerusalem, Israel, and by A. Git and N. Standart, University of Cambridge; Zhang et al., 1999; Git and Standart, 2002), or anti-tubulin (Sigma-Aldrich). Secondary antibodies were peroxidase-conjugated goat antirabbit IgG or rabbit antimouse IgG (Sigma-Aldrich). All incubations were performed in S-Blotto (50 mM Tris, pH 7.5, 250 mM NaCl, 5% powdered milk, and 1% Tween 20) except for the final wash (S-Blotto minus milk) before detection.
Immunolocalization
Immunofluorescence was performed as in Denegre et al. (1997), except stage I/II and III/IV oocytes were fixed for 12 h, and stage V/VI were fixed overnight in MEMFA (0.1 mM MOPS, pH 7.4, 2 mM EGTA, 1 mM MgSO4, and 3.7% formaldehyde). The primary antibody was anti-hnRNP I at 1:250 for stage IIV and 1:100 for stage VVI, and the secondary antibody was Alexa® 568conjugated goat antirabbit (Molecular Probes, Inc.) at 1:100. After fixation, oocytes were mounted in 2:1 benzyl benzoate/benzyl alcohol and analyzed on a confocal microscope (LSM 410; Carl Zeiss MicroImaging, Inc.) using a 10x Plan-Apochromat objective with 0.45 NA (Carl Zeiss MicroImaging, Inc.) at RT. Images were obtained using Renaissance software v.2.17 (Microcosm) and prepared using Adobe Photoshop® 7.0.
Oocyte microinjection
Stage III/IV oocytes (as in Dumont, 1972) were obtained surgically from X. laevis females (Nasco), and were defoliculated by incubation in 2 mg/ml collagenase (Sigma-Aldrich). RNA for injection was transcribed from pSP64ThnRNPIFLAG, pSP64TVg1RBP/veraFLAG, or pCS3+MT-prrp using the mMESSAGE mMACHINE® kit (Ambion). After microinjection with 2 nl of in vitrotranscribed RNA at 500 nM, oocytes were cultured for 18 h as in Gautreau et al. (1997).
Preparation of lysates
Defolliculate oocytes were placed in YSS buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 0.01% Ipegal [Sigma-Aldrich], 1 U/ml RNasin [Promega], 0.1 µg/ml leupeptin, 0.1 µg/ml aprotonin, 0.1 µg/ml trypsin inhibitor, 0.4 mM Pefabloc®, 1.0 mM DTT, and 100 mM sucrose). Oocytes were lanced with a 26-gauge needle and the nucleus was squeezed from the oocyte, leaving the cytoplasm intact within the oocyte membrane. For preparation of S10 lysates, nuclei or enucleated oocytes (cytoplasm) were homogenized in YSS (1 nucleus/µl or 1 cytoplasm/µl) and were centrifuged for 10 min at 10,000 g. S10 supernatants were collected and used immediately for analysis. For RNase treatment of lysates, RNase A (Sigma-Aldrich) was added at 0.1 µg/µl and incubated for 20 min at 37°C; mock treatment was with RNasin (Promega) at 0.12 U/µl, incubated for 20 min at 37°C.
Immunoprecipitation and RT-PCR
For immunoprecipitations, 5-µl anti-hnRNP I, anti-Vg1RBP/vera (Zhang et al., 1999; Git and Standart, 2002), anti-myc (9E10; Sigma-Aldrich), or control (anti-VP67; Volodina et al., 2003) antibodies were incubated overnight at 4°C with 10 µl protein GSepharose beads (Amersham Biosciences) in 1 ml YSS. FLAG immunoprecipitations were performed using 10 µl anti-FLAG beads (Sigma-Aldrich). After four washes with YSS, 20 µl of nuclear or cytoplasmic S10 lysates or 4 µl of in vitro translation mixture was added to the beads in 1 ml YSS. When indicated, RNase treatment was performed as above. After overnight incubation at 4°C, beads were washed 4x with 1 ml YSS, resuspended in sample buffer, and separated by SDS-PAGE. For RT-PCR, samples were resuspended after immunoprecipitation in 100 µl YSS. Isolation of RNA and RT-PCR were performed as in LaBonne and Whitman (1994). Vg1 primers were 5'-CGATGACATCCACCCAACAC-3' and 5'-GAGGGTCACAGTCAGCAAGG-3', and the VegT primers were as in Zhang and King (1996). PCR products were analyzed by agarose gel electrophoresis along with an amplification control from total cDNA generated from nuclear or cytoplasmic lysates.
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Acknowledgments |
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T.L. Kress was a predoctoral trainee, supported in part by a grant (5-T32-GM07601) from the National Institutes of Health. This work was supported by a grant (R01 HD30699) from the National Institutes of Health to K.L. Mowry.
Submitted: 24 September 2003
Accepted: 19 March 2004
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