Musashi regulates the temporal order of mRNA translation during Xenopus oocyte maturation
2006; Springer Nature; Volume: 25; Issue: 12 Linguagem: Inglês
10.1038/sj.emboj.7601159
ISSN1460-2075
AutoresAmanda Charlesworth, Ania Wilczynska, Prajitha Thampi, Linda L. Cox, Angus M. MacNicol,
Tópico(s)Plant Molecular Biology Research
ResumoArticle8 June 2006free access Musashi regulates the temporal order of mRNA translation during Xenopus oocyte maturation Amanda Charlesworth Amanda Charlesworth Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Anna Wilczynska Anna Wilczynska Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Prajitha Thampi Prajitha Thampi Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Linda L Cox Linda L Cox Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Angus M MacNicol Corresponding Author Angus M MacNicol Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA The Arkansas Cancer Research Center, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Amanda Charlesworth Amanda Charlesworth Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Anna Wilczynska Anna Wilczynska Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Prajitha Thampi Prajitha Thampi Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Linda L Cox Linda L Cox Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Angus M MacNicol Corresponding Author Angus M MacNicol Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA The Arkansas Cancer Research Center, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Author Information Amanda Charlesworth1, Anna Wilczynska1, Prajitha Thampi1, Linda L Cox1 and Angus M MacNicol 1,2 1Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA 2The Arkansas Cancer Research Center, University of Arkansas for Medical Sciences, Little Rock, AR, USA *Corresponding author. Department of Neurobiology and Developmental Sciences, The Arkansas Cancer Research Center, Slot 814, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205, USA. Tel.: +1 501 686 8164; Fax: +1 501 686 6517; E-mail: [email protected] The EMBO Journal (2006)25:2792-2801https://doi.org/10.1038/sj.emboj.7601159 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A strict temporal order of maternal mRNA translation is essential for meiotic cell cycle progression in oocytes of the frog Xenopus laevis. The molecular mechanisms controlling the ordered pattern of mRNA translational activation have not been elucidated. We report a novel role for the neural stem cell regulatory protein, Musashi, in controlling the translational activation of the mRNA encoding the Mos proto-oncogene during meiotic cell cycle progression. We demonstrate that Musashi interacts specifically with the polyadenylation response element in the 3′ untranslated region of the Mos mRNA and that this interaction is necessary for early Mos mRNA translational activation. A dominant inhibitory form of Musashi blocks maternal mRNA cytoplasmic polyadenylation and meiotic cell cycle progression. Our data suggest that Musashi is a target of the initiating progesterone signaling pathway and reveal that late cytoplasmic polyadenylation element-directed mRNA translation requires early, Musashi-dependent mRNA translation. These findings indicate that Musashi function is necessary to establish the temporal order of maternal mRNA translation during Xenopus meiotic cell cycle progression. Introduction Regulated mRNA translation is emerging as a key mechanism to control temporal and spatial gene expression during numerous cellular and developmental processes including sex determination, anterior–posterior specification of the embryonic body axis, neuronal synaptic plasticity, neural stem cell self-renewal and control of vertebrate oocyte maturation (Kuersten and Goodwin, 2003). Regulatory elements within the 5′ and 3′ untranslated regions (UTRs) of target mRNAs are recognized by sequence-specific RNA protein complexes to mediate translational control (Wilkie et al, 2003; Colegrove-Otero et al, 2005; de Moor et al, 2005). Distinct mRNA translational regulatory pathways may function interdependently to coordinate and effect cellular and developmental decisions (Kuersten and Goodwin, 2003). The molecular mechanisms that effect these precise temporal and/or spatial changes in mRNA translation in response to cellular signal transduction pathways have not been well characterized. Hormonally induced maturation in oocytes of the frog Xenopus laevis requires the temporally regulated translation of maternally derived mRNAs for meiotic cell cycle progression. The translational induction of the mRNA encoding the Mos proto-oncogene is necessary for meiotic cell cycle progression (Sagata et al, 1988, 1989; Furuno et al, 1994; Sheets et al, 1995; Dupre et al, 2002) and occurs soon (2–3 h) after exposure to the maturation stimulus, progesterone. In the absence of early Mos mRNA translation and subsequent activation of mitogen-activated protein (MAP) kinase signaling, oocytes exhibit delayed germinal vesicle breakdown (GVBD), meiosis I and fail to transition to meiosis II (Furuno et al, 1994; Gross et al, 2000; Dupre et al, 2002). By contrast, translational induction of the mRNA encoding the Wee1 protein tyrosine kinase occurs later in maturation, coincident with completion of GVBD (Kobayashi et al, 1991; Murakami and Vande Woude, 1998; Charlesworth et al, 2000). Inappropriate early translation of the Wee1 mRNA blocks activation of maturation promoting factor (MPF) and prevents meiotic cell cycle progression (Murakami and Vande Woude, 1998; Howard et al, 1999; Nakajo et al, 2000). It has been recently recognized that the differential timing of Xenopus maternal mRNA translation is controlled through distinct 3′ UTR regulatory elements that direct the cytoplasmic polyadenylation of the target mRNAs (Charlesworth et al, 2002). The late polyadenylation and translational activation of the Wee1 mRNA is enforced by 3′ UTR cytoplasmic polyadenylation element (CPE) sequences (Charlesworth et al, 2000). By contrast, the early polyadenylation and translation of the Mos mRNA is controlled by a 3′ UTR polyadenylation response element (PRE) in a CPE-independent manner (Charlesworth et al, 2002). The PRE has been shown to be responsive to MAP kinase signaling (Charlesworth et al, 2002), whereas the CPE is responsive to MPF signaling (Paris et al, 1991; de Moor and Richter, 1997; Howard et al, 1999; Charlesworth et al, 2002). As progesterone-stimulated MAP kinase activation precedes MPF activation, the differential responsiveness of the 3′ UTR regulatory elements to these signaling pathways would effect temporal discrimination of maternal mRNA selection (Howard et al, 1999; Charlesworth et al, 2002). In contrast to the MAP kinase-dependent feedback (Matten et al, 1996; Roy et al, 1996; Howard et al, 1999; Charlesworth et al, 2002), initial Mos mRNA translation is induced in a MAP kinase-independent manner (Gross et al, 2000) and is a prerequisite for progesterone-stimulated Xenopus p42 MAP kinase activation (Roy et al, 1996; Dupre et al, 2002). Progesterone stimulation leads to a rapid decrease in oocyte cAMP levels (reviewed in Smith, 1989) and PKA activity (Wang and Liu, 2004). Initiation of Mos mRNA translational activation has been shown to be downstream of progesterone-mediated PKA inhibition (Daar et al, 1993; Qian et al, 2001). The mediators of the progesterone ‘trigger’ signaling pathway leading to Mos mRNA translational activation have not been elucidated, but presumably regulate a trans-acting factor that binds to the PRE in the Mos 3′ UTR. The identity of the trans-acting factor controlling early PRE-dependent mRNA translational activation has not been determined. In this study, we demonstrate that Xenopus Musashi is a trans-acting PRE binding protein that is necessary for maternal mRNA translational activation in Xenopus oocytes. Musashi has been previously shown to be essential for asymmetric cell divisions of Drosophila neural progenitor cell populations and in mammalian neural stem cell self-renewal, where it appears to function as an mRNA translational repressor (Okano et al, 2002, 2005). Our findings reveal a novel role for Musashi in mediating mRNA translational activation during Xenopus oocyte maturation. Inhibition of Musashi function through expression of a dominant inhibitory form of Musashi specifically attenuated maternal mRNA translational activation and oocyte meiotic cell cycle progression in response to progesterone stimulation. We report that Musashi-mediated mRNA translational activation is a prerequisite for the subsequent translation of CPE-dependent mRNAs, indicating a critical role for Musashi in controlling the temporal order of maternal mRNA translational activation. Results The Mos PRE is a target of the progesterone ‘trigger’ signaling pathway To determine if the Mos PRE was a direct target of the MAP kinase-independent progesterone trigger pathway, oocytes were treated with the MAP kinase pathway inhibitor U0126. U0126 significantly attenuated the rate of oocyte maturation (a 4 h delay to GVBD50; Figure 1A) and abolished progesterone-stimulated MAP kinase activation, as previously reported (Gross et al, 2000). However, the initiation of endogenous Mos mRNA polyadenylation occurred with similar kinetics in the presence or absence of U0126, although the length of the poly[A] tail extension was reduced in U0126-treated oocytes. By contrast, CPE-directed polyadenylation of the endogenous cyclin B1 mRNA was significantly delayed in the U0126-treated oocytes, occurring coincident with GVBD. As the β-globin 3′ UTR does not undergo any significant polyadenylation (Charlesworth et al, 2002, 2004), the Mos PRE was inserted into the β-globin 3′ UTR to directly determine if the PRE was sufficient to direct MAP kinase-independent polyadenylation. Initiation of PRE-directed polyadenylation of the β-globin 3′ UTR occurred with similar kinetics in the presence or absence of U0126 (Figure 1A), although some differences in overall length of poly[A] tail extension can be seen between the two treatments. Injection of the PKA inhibitor (PKI) has been shown to induce progesterone-independent oocyte maturation (Huchon et al, 1981; Davidson, 1986). To determine if the PRE was downstream of PKA inhibition, we analyzed PRE-directed polyadenylation following injection of PKI. PKI induced PRE-directed polyadenylation of the β-globin 3′ UTR reporter mRNA, even in the presence of U0126 (Figure 1B). These findings indicate that the Mos PRE is downstream of PKA inhibition and is a target of the MAP kinase-independent progesterone trigger signaling pathway. Figure 1.The Mos PRE is responsive to a MAP kinase-independent trigger pathway. (A) Immature oocytes were injected with RNA encoding the GST coding region fused to the β-globin 3′ UTR containing the Mos PRE (β-globin/PRE). Oocytes were pretreated where indicated with U0126 and then stimulated with progesterone. Protein and RNA were extracted from the same pools of oocytes at the specified times. Progression of maturation is indicated by whether the oocytes had (+) or had not (−) undergone GVBD. Top panel is a Western blot for active (phosphorylated) MAP kinase (arrowhead). Lysates from DMSO- and UO126-treated oocytes were processed and analyzed in parallel. Lower panels show polyadenylation (brackets) of the endogenous Mos and cyclin B1 mRNAs and the injected synthetic β-globin/PRE reporter mRNA, as assayed by RNA ligation-coupled RT–PCR from the same cDNA preparation. Retardation of the PCR products is indicative of polyadenylation. (B) Immature oocytes were injected with the β-globin/PRE reporter. Oocytes were pretreated where indicated with U0126 and then stimulated by injection of recombinant PKI protein. Top panel is a Western blot for active (phosphorylated) MAP kinase (arrowhead). Lysates from DMSO-and UO126-treated oocytes were processed and analyzed in parallel. Lower panels show polyadenylation of the injected synthetic β-globin/PRE reporter (brackets). Download figure Download PowerPoint Xenopus Musashi is a Mos 3′ UTR interacting protein We next sought to identify potential RNA sequence-specific binding proteins that control PRE function. The yeast three-hybrid system (SenGupta et al, 1996, 1999; Zhang et al, 1999; Bernstein et al, 2002) was used to screen a X. laevis pACT2 unfertilized egg cDNA library (Clontech) for proteins that specifically interact with a CPE-disrupted Mos UTR (Mos M1 48; Figure 2A). The CPE sequence was disrupted to prevent possible recovery of the CPE binding protein (CPEB1) in the library screen. In total, 1.3 × 107 initial yeast transformants were screened (Supplementary Table I) and two distinct cDNAs (designated A8 and C36) were recovered and shown to reconstitute specific RNA interaction with the Mos 3′ UTR (Figure 2B, M1 48). The protein encoded by the A8 cDNA interacts specifically with the Mos 3′ UTR but does not interact with the PRE sequence and will be described elsewhere (Charlesworth and MacNicol, in preparation). Figure 2.Identification of Mos UTR-specific binding proteins by yeast three-hybrid analyses. (A) Diagram of the RNA hybrid in the pIIIA/MS2 vector that was used to screen the Xenopus oocyte library. The last 48 nt of the Mos 3′ UTR (M1 48, bold line) containing the PRE (open rectangle), the canonical polyadenylation hexanucleotide (gray hexagon) and a disrupted CPE (‘X’, UUUUAU to UUUggU) were placed 3′ of the MS2 sites. (B) Clones A8 and C36 specifically bind to the Mos M1 48 hybrid RNA. pACT2 plasmids encoding A8 and C36 clones and IRP (iron response protein) hybrid proteins were co-transformed with pIIIA/MS2 plasmids specifying MS2 (empty vector), IRE/MS2 (IRE: iron response element) and MS2/M1 48 (M1 48) hybrid RNAs. Dark gray shows activation of the LacZ reporter, indicating interaction between protein and RNA. (C) Schematic of the Xenopus Musashi protein. The black boxes represent the RNA recognition motifs (RRM) of Musashi. Amino-acid position of the motifs and the fragment of Musashi (C36) that was recovered from the screen are indicated. Download figure Download PowerPoint The pACT2 C36 cDNA was identical to amino acids 17–347 of nervous system-specific RNP protein 1b (Nrp-1b), more recently identified as the Xenopus homolog of the Drosophila Musashi gene (Good et al, 1993, 1998). Xenopus Musashi has two RNA recognition motifs in the N-terminal region (Figure 2C) that are highly conserved (87% amino-acid identity) with the mammalian Musashi1 protein. The Xenopus Musashi protein localizes to the cytoplasm of the oocyte (Good et al, 1993), consistent with a potential role in the regulation of cytoplasmic mRNA translation. Musashi binds specifically to the PRE in the Mos 3′ UTR The 24-nt Xenopus Mos PRE (Charlesworth et al, 2002) contained a match to the SELEX-derived murine Musashi RNA binding consensus sequence (G/AU1−3AGU) (Imai et al, 2001), and included a 3′ U residue essential for PRE function (Charlesworth et al, 2002) (Figure 3A, white nucleotides). Electrophoretic mobility shift assays (EMSAs) were performed to verify that Musashi interacts specifically with the Mos 3′ UTR. GST-tagged N-terminal Musashi (GST N-Msi), but not the GST moiety alone or unprogrammed reticulocyte lysate, formed specific complexes with the wild-type Mos UTR probe (Figure 3B, left panel). Musashi did not form a complex with the Mos UTR when the Musashi binding site was disrupted (AUAGU to AUccU; Figure 3B, right panel). Moreover, the Musashi-specific complexes formed with GST N-Msi could be supershifted with GST antisera, but not GFP antisera (Figure 3B, right panel). To delineate the Musashi target sequence within the PRE, we performed a competition experiment using 50-fold molar excess of unlabeled wild-type Mos UTR probe or unlabeled mutant Mos UTRs (Figure 3C). Whereas Musashi-specific binding to the wild-type Mos UTR probe was competed by unlabeled wild-type Mos UTR, Musashi-specific complex formation was not competed with a Mos UTR carrying a disruption in the Musashi binding site (Msi mut) or a Mos UTR lacking the 24-nt PRE (ΔPRE). Two additional RNAs encoding PRE mutations 5′ of the Musashi binding site effectively competed Musashi complex formation (Figure 3C). These findings indicate that Musashi does not bind to the 5′ region of the PRE, although our data do not exclude a role for the 5′ region of the PRE in facilitating Musashi function. Musashi-specific complex formation with the wild-type Mos UTR was not observed with the RNA binding-deficient form of the truncated Musashi protein (N-Msi-bm; Figure 3D), which encodes three amino-acid substitutions in the first RRM domain (Imai et al, 2001). Taken together, these results demonstrate that Xenopus Musashi interacts specifically with the consensus Musashi binding site within the Mos PRE sequence. As an additional demonstration of specific RNA binding, the full-length Musashi (Msi) and N-Msi proteins were able to interact with the endogenous Mos mRNA but not with the IPP2 mRNA that lacks a consensus Musashi binding site in the 3′ UTR (Figure 3E). No association of either Mos or IPP2 mRNAs was detected with the GST moiety alone. Figure 3.Musashi binds to the PRE in the Mos 3′ UTR. (A) Schematic showing regulatory element composition within the last 50 nt of the Mos 3′ UTR. The Musashi consensus binding site is indicated by white nucleotides. (B) Left panel: Wild-type (WT Mos) or Musashi binding site mutant (Msi mut) Mos UTR probes were analyzed for interaction with the GST-tagged, N-terminal domain of Xenopus Musashi (GST N-Msi) by RNA EMSA. Specific Musashi binding complexes were only detected with the wild-type Mos UTR probe. Right panel: Musashi-specific complex formation with the wild-type Mos UTR probe can be supershifted with antisera to the GST epitope tag and not with GFP antisera. (C) Musashi-specific complex formation with the wild-type Mos UTR probe can be competed with a 50-fold excess unlabeled wild-type, PRE mut4 and PRE mut5 Mos UTR RNA. No competition is observed with a Mos UTR lacking the entire PRE (ΔPRE) or a Musashi binding site mutant (Msi mut) Mos UTR. (D) Disruption of the first RRM in Musashi (GST N-Msi-bm) prevents Musashi-specific complex formation with the wild-type Mos UTR probe (right panel). Equivalent levels of each GST fusion protein were expressed in the reticulocyte lysates (left panel). UP, unprogrammed lysate. (E) The full-length (GST Msi) and N-terminal domain of Musashi (GST N-Msi) interact with the endogenous Mos mRNA. Left panel: GST Western blot showing recovery of exogenously expressed GST fusion proteins using glutathione Sepharose beads. Equivalent levels of GST Msi and GST N-Msi and higher levels of the GST moiety were recovered in the pulldown. Right panel: RT–PCR of Mos and IPP2 mRNA from RNA extracted from the indicated GST fusion proteins shown in the left panel. GST-Msi and GST-N-Msi interact with the Mos mRNA but do not interact with the IPP2 mRNA. The GST moiety alone fails to interact with either Mos or IPP2 mRNAs. RT–PCR from total oocyte RNA was used as a positive control to indicate the relative position of the expected PCR products in the pulldown lanes. Download figure Download PowerPoint Musashi interaction with the Mos 3′ UTR is necessary for early translational activation To determine whether the Musashi consensus site was necessary for early, CPE-independent mRNA translational activation directed by the Mos PRE (Charlesworth et al, 2002, 2004), disruptive nucleotide substitutions were introduced into the Musashi consensus sequence (AUAGU to AUccU) and the polyadenylation and translation of reporter mRNAs analyzed. Insertion of the Mos PRE into the heterologous β-globin 3′ UTR confers progesterone-dependent polyadenylation and translational activation (two- to three-fold above β-globin alone; Figure 4A), whereas disruption of the Musashi binding site (PRE/msi mt) abolished this regulation (Figure 4A). These findings indicate that the integrity of the Musashi consensus site is critical for the translational activation directed by the Mos PRE. A role for the Musashi protein in mRNA translational activation in Xenopus oocytes was independently confirmed through the use of the MS2 tethered assay (Gray et al, 2000; Minshall et al, 2001; Collier et al, 2005). As can be seen in Figure 4B, fusion of Musashi to the MS2 RNA binding domain directed a two-fold stimulation of a firefly luciferase reporter mRNA containing 3′ UTR MS2 binding sites. No induction of the luciferase reporter mRNA was observed with the MS2 RNA binding domain alone, nor with a luciferase reporter mRNA lacking MS2 binding sites (ΔMS2; Figure 4B). In these experiments, the MS2-Dazl fusion protein serves as a positive control for translational induction (Collier et al, 2005). The firefly luciferase reporter mRNAs used in the tethered assay lack a consensus polyadenylation hexanucleotide, suggesting that tethering of Musashi can direct translation independently of polyadenylation. To test this directly, the polyadenylation hexanucleotide in the GST β-globin/PRE reporter mRNA was disrupted (AAUAAA to AAgAAA; Fox et al, 1989) to generate GST β-globin/PRE/hex mt. While eliminating progesterone-dependent polyadenylation, disruption of the polyadenylation hexanucleotide reduced, but did not eliminate, PRE-directed mRNA translation (approximately 1.5-fold above β-globin alone; Figure 4C). These findings suggest that the Musashi binding site in the Mos PRE directs both polyadenylation and translation in response to progesterone, but that polyadenylation is not obligatory for PRE-directed mRNA translation. Figure 4.Musashi directs mRNA translational activation. (A) Upper panel: Schematic representation of the reporter mRNAs analyzed. The GST open reading frame (box) was fused to the β-globin 3′ UTR, or β-globin 3′ UTRs containing either the wild-type (PRE) or Musashi binding site mutant (PRE/msi mt) Mos PRE sequences. Middle panel: Polyadenylation of the indicated reporter mRNAs was assessed by RNA ligation-coupled PCR from time-matched immature (I) or progesterone-stimulated (P) oocytes taken at GVBD50 from oocytes without white spots. Retarded migration of PCR products above the dotted line is indicative of polyadenylation. Lower panel: Western blot showing GST accumulation from time-matched immature or progesterone-stimulated oocytes taken at GVBD100. (B) Tethered assay demonstrating the ability of MS2-Msi and MS2-Dazl fusion proteins to induce firefly luciferase expression. Open and solid bars represent luciferase reporters lacking or containing MS2 binding sites in the 3′ UTR, respectively. Luciferase activity was determined in triplicate and normalized to expression of the coinjected Renilla luciferase mRNA. Error bars represent standard deviation of the mean. The experiment was repeated three times with similar results. (C) Oocytes were injected with GST reporter mRNAs fused to the β-globin 3′ UTR, the β-globin 3′ UTR containing the Mos PRE (PRE) or the β-globin 3′ UTR containing the Mos PRE (PRE) and a disrupted polyadenylation hexanucleotide (hex mt). The polyadenylation and translation of GST reporter mRNAs were analyzed as described in (A). Download figure Download PowerPoint A truncated form of Musashi blocks Xenopus oocyte meiotic cell cycle progression We reasoned that expression of a truncated form of Musashi encompassing only the RNA binding domain (N-Msi, aa 1–199) may function in a dominant inhibitory manner to block early Mos mRNA polyadenylation and translational activation in response to progesterone stimulation. Immature oocytes were injected with mRNA encoding either N-Msi or the RNA binding-deficient N-Msi-bm (Figure 3D). Compared to the N-Msi-bm-expressing oocytes, expression of N-Msi dramatically attenuated progesterone-stimulated oocyte maturation (Figure 5B, left panel). To determine the molecular basis for the block to oocyte maturation exerted by N-Msi, oocytes were analyzed for endogenous Mos and cyclin B1 mRNA polyadenylation. In contrast to the polyadenylation of the Mos mRNA in N-Msi-bm-expressing oocytes, Mos mRNA polyadenylation was attenuated in oocytes expressing N-Msi at all time points analyzed (Figure 5B, upper right panel). The polyadenylation of the cyclin B1 mRNA is a late event in progesterone-stimulated maturation and occurs coincident with oocyte GVBD (Sheets et al, 1994; Ballantyne et al, 1997; de Moor and Richter, 1997; Charlesworth et al, 2004). In N-Msi-bm-expressing oocytes, cyclin B1 mRNA polyadenylation occurred coincident with GVBD as expected, whereas expression of N-Msi protein prevented progesterone-stimulated GVBD and ablated cyclin B1 polyadenylation, even at late time points (Figure 5B, lower right panel). Two possible explanations could account for the inhibitory effect of N-Msi on cyclin B1 polyadenylation. First, N-Msi may exert an inhibitory effect by directly binding to the cyclin B1 3′ UTR and blocking CPE-dependent cytoplasmic polyadenylation. Alternatively, the effect of N-Msi may be indirect and occur through inhibition of Musashi-dependent mRNA translation events required for GVBD and/or the activation of CPE-dependent mRNA polyadenylation. To distinguish between these possibilities, oocytes were microinjected with cyclin B1 protein to induce activation of MPF independently of progesterone stimulation (Freeman et al, 1991) and reverse the relative order of MAP kinase and MPF activation (Figure 5A; Charlesworth et al, 2002). N-Msi did not block cyclin B protein-induced maturation and the kinetics of maturation were indistinguishable between N-Msi- and N-Msi-bm-expressing oocytes (Figure 5C, left panel). Cyclin B protein induced the polyadenylation of the endogenous cyclin B1 mRNA in both N-Msi- and N-Msi-bm-expressing oocytes (Figure 5C, right panel). These results indicate that CPE-dependent polyadenylation of the cyclin B1 mRNA can occur in oocytes expressing dominant-negative Musashi if MPF is activated independently of progesterone-triggered early signaling effectors. By contrast, cyclin B-induced Mos mRNA polyadenylation was abrogated in N-Msi-, but not N-Msi-bm-, expressing oocytes (Figure 5C, right panel) demonstrating the specificity of N-Msi-mediated inhibition on PRE-regulated mRNAs irrespective of initiating signal. Mos mRNA deadenylation was observed in the N-Msi-expressing oocytes after GVBD. We conclude that the dominant-negative Musashi specifically blocks early progesterone effector pathways (including Mos mRNA translational activation) and the inhibition of CPE-dependent mRNA polyadenylation is an indirect consequence of a block to upstream Musashi-regulated mRNA translation events. Consistent with a block to Mos mRNA polyadenylation, progesterone-stimulated Mos protein accumulation was abrogated in N-Msi (but not N-Msi-bm)-expressing oocytes (Figure 5D). N-Msi, but not N-Msi-bm, also prevented progesterone-dependent activation of MPF (Figure 5E). In these experiments, the N-Msi and N-Msi-bm mutant proteins were expressed to comparable levels (Figure 5F). Although we cannot exclude additional effects of the truncated Musashi protein (N-Msi) on processes upstream of mRNA polyadenylation and translational activation, the inability of an RNA binding-deficient form of the truncated protein (N-Msi-bm) to block progesterone-stimulated cytoplasmic polyadenylation or cell cycle progression indicates that effect of the dominant inhibitory Musashi is primarily due to interaction with mRNA targets. Taken together, our data provide compelling evidence that Musashi regulates maternal mRNA translation and MPF activation during progesterone-stimulated meiotic cell cycle progression. Figure 5.Dominant inhibitory Musashi blocks oocyte maturation and specifically prevents Mos polyadenylation and translation. (A) Schematic of experimental design. RNA encoding GST-tagged N-Msi or N-Msi-bm was injected into immature oocytes, which were left for 36 h to express the protein. Oocytes were then stimulated to mature either by addition of progesterone or injection of cyclin B protein. (B) Progesterone-stimulated maturation was scored by the appearance of a white spot at the animal pole and indicated by whether the oocytes had (+) or had not (−) undergone GVBD. The polyadenylation of endogenous Mos (upper panels) and cyclin B1 (lower panels) mRNAs was assessed by RNA ligation-coupled RT–PCR (brackets). In this experiment, progesterone-stimulated N-Msi-bm-expressing oocytes reached GVBD50 at 14 h. (C) Oocytes were injected with cyclin B1 protein and the effect on maturation and mRNA polyadenylation was assessed as in (B). (D) The expression of Mos protein in immature (I) or progesterone-stimulated (24 h) oocytes from exp
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