Enforcing temporal control of maternal mRNA translation during oocyte cell-cycle progression
2009; Springer Nature; Volume: 29; Issue: 2 Linguagem: Inglês
10.1038/emboj.2009.337
ISSN1460-2075
AutoresK. Arumugam, Yiying Wang, Linda Hardy, Melanie C MacNicol, Angus M. MacNicol,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle3 December 2009free access Enforcing temporal control of maternal mRNA translation during oocyte cell-cycle progression Karthik Arumugam Karthik Arumugam Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USAPresent address: Department Genetics and Development, Institut de Genetique Humaine, 141, rue de la Cardonille, 34396 Montpellier Cedex 5, France Search for more papers by this author Yiying Wang Yiying Wang Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USAPresent address: Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, US FDA, 3900 NCTR Rd., Jefferson, AR 72079, USA Search for more papers by this author Linda L Hardy Linda L Hardy Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Melanie C MacNicol Melanie C MacNicol Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Center for Translational Neuroscience, 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 Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Winthrop P Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Karthik Arumugam Karthik Arumugam Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USAPresent address: Department Genetics and Development, Institut de Genetique Humaine, 141, rue de la Cardonille, 34396 Montpellier Cedex 5, France Search for more papers by this author Yiying Wang Yiying Wang Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USAPresent address: Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, US FDA, 3900 NCTR Rd., Jefferson, AR 72079, USA Search for more papers by this author Linda L Hardy Linda L Hardy Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Melanie C MacNicol Melanie C MacNicol Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Center for Translational Neuroscience, 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 Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA Winthrop P Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Author Information Karthik Arumugam1, Yiying Wang1, Linda L Hardy2, Melanie C MacNicol2,3 and Angus M MacNicol 1,2,4 1Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA 2Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, USA 3Center for Translational Neuroscience, University of Arkansas for Medical Sciences, Little Rock, AR, USA 4Winthrop P Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA *Corresponding author. Department of Neurobiology and Developmental Science, University of Arkansas for Medical Sciences, 4301 W Markham, Slot 814, Little Rock, AR 72205, USA. Tel.: +501 686 8164; Fax: +501 686-6517; E-mail: [email protected] The EMBO Journal (2010)29:387-397https://doi.org/10.1038/emboj.2009.337 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Meiotic cell-cycle progression in progesterone-stimulated Xenopus oocytes requires that the translation of pre-existing maternal mRNAs occur in a strict temporal order. Timing of translation is regulated through elements within the mRNA 3′ untranslated region (3′ UTR), which respond to cell cycle-dependant signalling. One element that has been previously implicated in the temporal control of mRNA translation is the cytoplasmic polyadenylation element (CPE). In this study, we show that the CPE does not direct early mRNA translation. Rather, early translation is directed through specific early factors, including the Musashi-binding element (MBE) and the MBE-binding protein, Musashi. Our findings indicate that although the cyclin B5 3′ UTR contains both CPEs and an MBE, the MBE is the critical regulator of early translation. The cyclin B2 3′ UTR contains CPEs, but lacks an MBE and is translationally activated late in maturation. Finally, utilizing antisense oligonucleotides to attenuate endogenous Musashi synthesis, we show that Musashi is critical for the initiation of early class mRNA translation and for the subsequent activation of CPE-dependant mRNA translation. Introduction Meiotic cell-cycle progression in Xenopus laevis oocytes requires a strict temporal order of translation of pre-existing mRNAs encoding cell cycle-control proteins (Freeman et al, 1991; Roy et al, 1991; Sheets et al, 1995; Murakami and Vande Woude, 1998; Ferby et al, 1999; Howard et al, 1999; Nakajo et al, 2000). Translation of mRNAs is classed as early (i.e. occurring before cell division cycle 2 (cdc2) activation and oocyte germinal vesicle (nucleus) breakdown (GVBD) or late (i.e. coincident with or after cdc2 activation and GVBD). The translation of the early class mRNA encoding the Mos proto-oncogene results in MAP kinase activation that contributes to cdc2 activation and is necessary for the transition from metaphase I to metaphase II (Sagata et al, 1988, 1989; Furuno et al, 1994; Sheets et al, 1995; Gross et al, 2000; Dupre et al, 2002) (Kosako et al, 1994; Gotoh et al, 1995; Huang and Ferrell, 1996a; Palmer et al, 1998). By contrast, the mRNA encoding cyclin B1 is a late class mRNA, and translational activation requires previous cdc2 activation (Ballantyne et al, 1997; de Moor and Richter, 1997; Howard et al, 1999; Barkoff et al, 2000). The timing of translational activation of early and late class mRNAs is enforced by regulatory elements in their 3′ untranslated regions (3′ UTRs), which are bound by element-specific RNA binding proteins. Two regulatory elements that direct the timing of oocyte mRNA translation are the cytoplasmic polyadenylation element (CPE) and the Musashi/polyadenylation-response element (forthwith referred to as a Musashi-binding element, MBE) (Fox et al, 1989; McGrew et al, 1989; Charlesworth et al, 2002, 2006). The CPE sequence has general consensus U5AU (Radford et al, 2008), although a number of variations have been documented (Charlesworth et al, 2004; Pique et al, 2008). The consensus MBE sequence is (G/A)U1−3AGU (Imai et al, 2001). The roles of CPE and MBE sequences in regulating the temporal order of mRNA translation in response to progesterone-induced signalling pathways have not been fully resolved. A recent study assigned roles for CPEs according to their responsiveness to signalling pathways, where cdc2-independent mRNA translation was defined as 'early' and cdc2-dependant mRNA translation was defined as 'late' (Pique et al, 2008). Using these temporal definitions, an analysis of signalling dependancy for translational activation of B-type cyclin mRNAs led to the suggestion that position and arrangement of CPEs within the 3′ UTR are the primary determinants of timing of translational activation (Pique et al, 2008). Specifically, CPE sequences positioned within 100 nucleotides of the polyadenylation hexanucleotide have been proposed to direct early translational activation of the cyclin B5 and cyclin B2 mRNAs, whereas a CPE which overlaps the polyadenylation hexanucleotide acts dominantly to direct late translation of the cyclin B1 and B4 mRNAs. These results were extrapolated to a generalized model for predicting the timing and extent of translation of CPE-containing mRNAs in Xenopus and potentially in other organisms (Pique et al, 2008; Richter, 2008). However, these conclusions are at odds with previous findings that indicate the early translational activation of multiple early class mRNAs may be independent of CPE sequences (Charlesworth et al, 2004, 2006). Analysis of translation of the mRNA encoding the Mos proto-oncogene occupies the center of discussion on temporal control of maternal mRNA recruitment. Early translational activation of the Mos mRNA is essential for progression through the oocyte meiotic cell cycle (Sagata et al, 1988; Sheets et al, 1995; Dupre et al, 2002) and for the subsequent translation of late class mRNAs (Ballantyne et al, 1997; de Moor and Richter, 1997). As the Mos mRNA 3′ UTR contains both a MBE and a CPE, initiation of Mos mRNA translation has been variously ascribed to both CPE-dependant and Musashi-dependant mechanisms. A role for CPE-directed control was proposed based on indirect evidence from experiments employing a dominant inhibitory form of the CPE-binding protein (Mendez et al, 2000) and inferred from computational predictions based on the CPE combinatorial code model (the Mos 3′ UTR contains a single CPE in an 'early' configuration, (Pique et al, 2008)). However, mutational disruption of the Mos CPE does not affect the timing of Mos mRNA translational activation. Rather, the MBE in the Mos 3′ UTR was shown to be critical for Mos mRNA translational activation (Charlesworth et al, 2002). Consistent with a Musashi-dependant regulatory mechanism, a dominant inhibitory form of Musashi prevents the translational induction of Mos and several additional early class mRNAs that contain both MBE and CPE sequences (Charlesworth et al, 2006). These findings suggest that although their 3′ UTRs appear to satisfy the requirements of the proposed early CPE combinatorial code, the early activation of these mRNAs is actually directed through the MBE. However, a role for Musashi in directing early class mRNA translational activation has come under question because the presence of a MBE is not per se an indicator of early mRNA translation as MBE sequences are also present in multiple late class mRNAs (Charlesworth et al, 2006). In this study we have re-evaluated the role and requirement for Musashi in controlling the timing of CPE-independent and CPE-dependant mRNA translation. Utilizing direct analyses of the timing of activation of endogenous B-type cyclin mRNAs, we have tested the prediction of the CPE combinatorial code model. We found that the CPE combinatorial code does not correctly predict the temporal translational activation of the cyclin B2 mRNA and although the cyclin B5 mRNA is translationally activated early as predicted by the CPE code, this activation is independent of the CPEs in the cyclin B5 3′ UTR. Further, we demonstrate that Musashi is necessary to mediate the initial progesterone 'trigger' pathway independently of CPEB and that early Musashi-dependant mRNA translation is required for subsequent CPE-dependant mRNA translation. We propose a model in which oocyte cell-cycle progression is controlled through early MBE-directed mRNA translation followed by CPE-directed translation. Results The CPE combinatorial code does not predict the timing of cyclin B2 mRNA translational activation A direct analysis of the timing of translational activation of mRNAs containing specific CPE 'code' sequences has not been carried out. We thus analyzed the timing of endogenous Mos, cyclin B5, cyclin B2 and cyclin B1 mRNA polyadenylation following progesterone stimulation (where extension of the 3′ UTR polyadenylate tail is indicative of translational activation (Rosenthal et al, 1983; Dworkin et al, 1985; Vassalli et al, 1989)). The Mos and cyclin B5 mRNAs were polyadenylated early after progesterone stimulation (before GVBD), as predicted by the CPE-combinatorial code model (Figure 1A). Despite Mos and cdc2-independent regulation (Pique et al, 2008 and Supplementary Figure S1), the cyclin B2 mRNA exhibited late class translational activation that was indistinguishable from the established late class cyclin B1 mRNA profile (Figure 1A). The late timing of the cyclin B2 mRNA reported here is, thus, contrary to the CPE code prediction, but is consistent with an earlier independent study (Sheets et al, 1994). Figure 1.The cytoplasmic polyadenylation element (CPE) combinatorial code does not correctly predict cyclin B2 mRNA polyadenylation. (A) Oocytes treated with or without progesterone for the indicated times were analyzed for initiation of polyadenylation of multiple endogenous mRNAs by RNA ligation-coupled PCR from the same cDNA preparation at each time point. Oocytes reached GVBD50 (see Materials and methods) at 5.5 h and were segregated into those that had not (−) or had (+) completed GVBD. An increase in the size of the mRNA population is indicative of polyadenylation (bracketed). An asterisk indicates when polyadenylation of each mRNA initiated as evidenced by a shift in mRNA population size above the basal size in immature oocytes (indicated by a dotted line). On the right of the panel, a schematic representation of each mRNA 3′ untranslated region (UTR) shows the position of consensus Musashi-binding sites (solid black square), consensus CPEs (white circle), non-consensus CPEs (diagonal bar circle) and consensus polyadenylation hexanucleotides (grey hexagon). (B) β-globin reporter 3′ UTRs were generated by inserting a consensus Musashi-binding site (solid black square) or a consensus CPE (white circle) 5′ of the polyadenylation hexanucleotide (grey hexagon). Separate pools of oocytes were injected with the indicated mRNA reporter constructs, incubated for approximately 16 h and then left untreated or stimulated with progesterone. When 50% of the progesterone-treated oocyte population reached GVBD, oocytes were segregated based on whether they had (+WS) or had not (−WS) completed GVBD and RNA isolated. RNA was also isolated from time matched immature oocyte samples. The polyadenylation status of the reporter mRNAs analyzed by RNA-ligation coupled PCR using appropriate forward primers. It should be noted that in the absence of inserted elements, the β-globin 3′ UTR does not undergo progesterone-dependant polyadenylation (Hyman and Wormington, 1988). The experiment was repeated three times with identical results. Download figure Download PowerPoint We noted that the cyclin B5 mRNA, like the early class Mos mRNA, contained an MBE in addition to an 'early' CPE arrangement. In the case of the Mos mRNA, the MBE, rather than the CPE, has been previously demonstrated to be the critical determinant of early translational activation (Charlesworth et al, 2002). When examined in the context of a reporter mRNA, an MBE directs early polyadenylation (before GVBD), whereas a single CPE (in a predicted 'early' configuration, Pique et al, 2008) directs late polyadenylation coincident with the completion of GVBD (Figure 1B). We thus hypothesized that the MBE directed early translational activation of the cyclin B5 mRNA. Early translational activation directed by cyclin B5 3′ UTR requires the MBE To test the role of the MBE in the early translation activation of cyclin B5 mRNA, we utilized reporter mRNAs fused to either the wild-type cyclin B5 mRNA 3′ UTR (cyclin B5 wt) or a mutant cyclin B5 3′ UTR (cyclin B5 mbm) containing a disruption in the MBE that attenuates Musashi binding (Charlesworth et al, 2006). It is important to note that the MBE mutation was engineered to leave the adjacent CPE intact within the cyclin B5 3′ UTR (Figure 2A and Supplementary Figure S2). Functional integrity of the CPE sequences in the MBE-disrupted cyclin B5 3′ UTR was demonstrated by showing that both the wild-type and MBE-disrupted cyclin B5 3′ UTR were able to compete for interaction with the CPE-binding protein, CPEB, for a labelled CPE-containing cyclin B1 probe (Figure 2B). As expected, a CPE-disrupted cyclin B5 3′ UTR was not able to compete for CPEB interaction. Incubation with the GST moiety alone (expressed at equivalent levels to GST–CPEB, Figure 2C) did not result in specific complex formation (Figure 2B). Figure 2.Early translational activation of the cyclin B5 3′ untranslated region (UTR) requires a Musashi-binding element (MBE). (A) Schematic showing the regulatory elements of cyclin B5 3′ UTR fused to a Firefly luciferase reporter (see legend to Figure 1 for symbol definitions). The β-globin 3′ UTR lacks progesterone-responsive elements and so serves as an unregulated control luciferase reporter mRNA. (B) RNA EMSA utilizing a biotin-labelled cyclin B1 3′ UTR and reticulocyte expressing either the GST moiety alone or GST fused to the C-terminal RNA binding domain of CPEB (ΔN-CPEB). A specific cyclin B1 RNA binding complex is formed with ΔN-CPEB but not GST (arrowhead). This complex can be effectively competed with unlabelled wild-type (wt) cyclin B5 3′ UTR and the Musashi-binding mutant form of the cyclin B5 3′ UTR (mbm). By contrast, no competition for ΔN-CPEB binding is seen with a cytoplasmic polyadenylation element (CPE)-disrupted cyclin B5 3′ UTR (CPE mut). (C) GST Western blot demonstrates equivalent levels amount of GST and GST-ΔN-CPEB protein expression in the reticulocyte lysates used in (B). (D) Wild-type (wt) or Musashi-binding mutant (mbm) cyclin B5 3′ UTRs were fused to a GST open reading frame and the resulting transcribed RNA injected into immature oocytes. The polyadenylation status of the reporter RNAs was assessed by RNA ligation PCR where an increase in the size of the mRNA population is indicative of polyadenylation (bracketed). Imm, immature oocytes. Whether oocytes had (+) or had not (−) completed GVBD is indicated above each time point. (E) Oocytes were co-injected with a Renilla luciferase mRNA and a Firefly luciferase RNA under the control of either the wild-type (wt) or polyadenylation hexanucleotide mutant cyclin B5 UTR and incubated for 16 h before progesterone treatment. When the oocytes reached GVBD50, oocytes were segregated based on whether they had or had not completed GVBD. For these experiments, three pools of five oocytes were harvested for each analysis from the oocytes, which had not yet completed GVBD (and so are considered to be in the early phases of maturation). In addition, three pools of five oocytes were harvested from time matched immature oocyte samples. The triplicate sets of Firefly:Renilla ratios of the cyclin B5 UTR constructs from progesterone-stimulated oocytes were normalized to the Firefly:Renilla ratios for the same 3′ UTR constructs in immature oocytes (ratio P:I). The data presented are the mean±SEM from three independent experiments. (F) Oocytes were co-injected with a Renilla luciferase mRNA and a Firefly luciferase RNA under the control of either the wild-type (wt) or Musashi binding mutant (mbm) cyclin B5 UTR. Luciferase analyses were performed as described in (E) and the data are presented from two independent experiments with the SEM shown for each individual experiment. We confirmed that the reporter mRNAs were expressed to similar levels (Supplementary Figure S3). Download figure Download PowerPoint When assessed directly in a polyadenylation assay, the wild-type cyclin B5 3′ UTR directed robust polyadenylation (Figure 2D), with significant polyadenylation observed before the completion of GVBD (Figure 2D, 6 h time point, GVBD— lane). By contrast, disruption of the MBE in the cyclin B5 3′ UTR dramatically attenuated progesterone-stimulated polyadenylation (Figure 2D). We confirmed that the progesterone-stimulated polyadenylation directed by the cyclin B5 3′ UTR results in mRNA translational activation. When fused to a Firefly luciferase-coding region, wild-type cyclin B5 3′ UTR exerted about a three-fold induction of translation in response to progesterone (Figure 2E). Disruption of the polyadenylation hexanucleotide in the cyclin B5 3′ UTR mitigated the majority of the progesterone-dependant induction of translation (Figure 2E). Taken together, these results indicate that the MBE directs progesterone-dependant polyadenylation of the cyclin B5 3′ UTR and consequent progesterone-stimulated translational induction. Consistent with this interpretation, a direct comparison of reporter mRNAs fused to wild-type or MBE-disrupted mutant cyclin B5 3′ UTRs indicated that the MBE mediates translational activation in response to progesterone stimulation (Figure 2F). We note that the cyclin B5 mbm reporter did undergo limited polyadenylation and translation in response to progesterone (Figure 2D and F). A modest general progesterone-dependant increase in basal translation of reporter mRNAs lacking either CPE or MBE sequences has been previously observed (Charlesworth et al, 2002), which likely contributes to the residual regulation observed in the cyclin B5 mbm reporter. In addition, the MBE mutation may not completely inhibit Musashi regulation of cyclin B5 3′ UTR (see Figure 3C). Figure 3.Antisense oligonucleotides targeting endogenous Musashi1 and Musashi2 mRNAs inhibit oocyte maturation. (A) Immature oocytes were injected with water, 200 ng control antisense oligonucleotide (Control AS) or a combination of 100 ng xMsi1 and 100 ng xMsi2 antisense oligonucleotides (xMsi1+xMsi2 AS) and incubated for 16 h. The oocytes were then stimulated with progesterone and GVBD scored over the course of the experiment. (B) Pools of 10 oocytes from each experimental condition in panel (A) were harvested when control AS-injected oocytes reached GVBD50. Time-matched immature and progesterone-treated samples were analyzed for MAP kinase and cdc2 activation. In these analyses, phosphorylated MAP kinase (pMAPK) indicates activation and loss of phosphorylated (and inhibited) cdc2 (pcdc2) indicates activation. Ringo and CPEB protein levels were also assessed in the same samples by western blotting, with tubulin serving as an internal control for protein loading. The data are representative of three separate experiments. (C) Oocytes were injected with water, 200 ng control antisense oligonucleotide (AS), or a combination of 100 ng xMsi1 and 100 ng xMsi2 AS and incubated for 16 h before progesterone stimulation. Pools of 10 oocytes were harvested for each condition when control AS-injected oocytes reached GVBD50. Similar to (A), the xMsi1/2 AS-injected oocytes failed to mature in response to progesterone stimulation. The polyadenylation of endogenous cyclin B5, Mos, TATA BP2, cyclin B2 and cyclin B1 mRNAs was determined using RNA ligation-coupled PCR from the same cDNA preparations at each time point. Progesterone-dependant polyadenylation of each mRNA is indicated (bracket). The experiment was repeated three times with similar results. Download figure Download PowerPoint Antisense knockdown of Musashi blocks the early translational activation of endogenous cyclin B5 mRNA To confirm that Musashi mediates the early translational activation of the endogenous cyclin B5 mRNA, we utilized antisense oligonucleotide attenuation of endogenous Musashi mRNA translation. There are two isoforms of Musashi in Xenopus: Musashi1 (xMsi1, also known as Nrp1A/B) and Musashi2 (xMsi2, also known as Xrp1) (Good et al, 1993; Sakakibara et al, 2001). The Musashi1 mRNA appears to be the predominant form and is expressed at levels at least five-fold higher than the Musashi2 mRNA (Good et al, 1993). Injection of antisense oligonucleotides targeting both xMsi1 and xMsi2 mRNAs (Msi1/2 AS) inhibited progesterone-stimulated maturation of oocytes (Figure 3A). Musashi has been previously shown to control early translation of the Mos mRNA (Charlesworth et al, 2006). Consistent with loss of Mos mRNA translation, the downstream signalling pathways of MAP kinase and cdc2 were not activated in Msi1/2 AS injected oocytes (Figure 3B). It is interesting to note that the use of either Musashi1 or Musashi2 antisense oligonucleotides alone delayed rather than inhibiting oocyte maturation (Supplementary Figure S4), thus suggesting that like mammalian Musashi, each isoform can partially compensate for the loss of the other (Sakakibara et al, 2002). No inhibition of oocyte maturation or cellular signalling was observed using an equivalent level of control antisense oligonucleotides that do not target Musashi1 or Musashi2 mRNAs (Figure 3A and B). To address the point of action of the antisense Msi1/2 oligonucleotides during progesterone stimulated oocyte maturation we examined additional early and late signalling events. In response to progesterone, Ringo mRNA translation occurs very rapidly, with endogenous Ringo protein accumulation detectable 30–60 min after stimulation (Gutierrez et al, 2006; Padmanabhan and Richter, 2006). We found that the accumulation of the endogenous Ringo protein was not affected in Msi1/2 antisense oligonucleotide injected oocytes (Figure 3B). We conclude that progesterone-stimulated Ringo mRNA translation is independent of Musashi function. Further, this finding indicates that Musashi antisense oligonucleotide injections did not adversely affect general mRNA translation in the oocyte. We also assessed the levels of CPEB in the antisense oligonucleotide injected oocytes. CPEB protein is expressed in immature oocytes, but undergoes cdc2-dependant phosphorylation at GVBD which targets a proportion of the protein population for proteosomal degradation (Reverte et al, 2001; Mendez et al, 2002). This CPEB degradation is linked to the activation of late class CPE-dependant mRNAs that contain a CPE overlapping the polyadenylation hexanucleotide (e.g. cyclin B1; Mendez et al, 2002). In contrast to water and control antisense oligonucleotide injected oocytes that demonstrated CPEB destruction at GVBD (Figure 3B), the injection of Msi1/2 antisense oligonucleotides prevented CPEB destruction and CPEB levels were equivalent irrespective of whether the oocytes were or were not stimulated with progesterone. Our findings suggest that the CPEB protein present in the Musashi antisense oligonucleotide injected oocytes is unable to mediate oocyte maturation or CPE-dependant mRNA translational activation when Musashi function is attenuated. Consistent with this hypothesis, endogenous polyadenylation of the late class cyclin B1 and B2 mRNAs was blocked in the Msi1/2 antisense injected oocytes (Figure 3C). Similarly, the polyadenylation of the MBE-regulated early class mRNAs Mos and cyclin B5 (Figure 2) was blocked as expected, as was the polyadenylation of the MBE-containing early class TATA BP2 mRNA which lacks a functional CPE (Charlesworth et al, 2006). The effective inhibition of endogenous cyclin B5 mRNA polyadenylation in Musashi antisense oligonucleotide injected oocytes suggests that the residual polyadenylation and translation in the cyclin B5 mbm reporter (Figure 2) may be a consequence of incomplete attenuation of Musashi function by the introduced 3′ UTR MBE mutation. To address the specificity of the Msi1/2 antisense inhibition of oocyte maturation, we attempted to rescue the inhibition of GVBD and cellular signalling events by injecting an mRNA encoding wild-type Xenopus Musashi fused to an N-terminal GST moiety (GST–Msi). As can be seen in Figure 4A, injection of GST–Msi1 efficiently rescued the ability of Msi1/2 antisense injected oocytes to undergo GVBD in response to progesterone. In addition to the extent of rescue, wild-type Musashi1 also rescued normal kinetics of maturation (Supplementary Figure S5). By contrast, an RNA-binding deficient form of Xenopus Musashi1 (GST–Msi bm; Charlesworth et al, 2006) was unable to effect a rescue of oocyte maturation (Figure 4A, Msi1/2+Msi bm) indicating that Musashi must be able to interact with target mRNAs to mediate cell-cycle progression to GVBD. Similarly, overexpression of a GST–CPEB protein was unable to rescue the maturation defect (Figure 4A). Likewise, only the wild-type GST–Musashi protein was able to generate high MAP kinase activation and downstream cdc2 activation in Msi1/2 injected oocytes (Figure 4B). The lower level of Msi1/2 antisense oligonucleotides used in this experiment was less effective at suppressing MAP kinase signalling (compared with Figure 3B). Nonetheless, the lower levels of Msi1/2 antisense oligonucleotides did still exert efficient inhibition of oocyte maturation (Figure 4A). These findings are in agreement with previous studies that have observed a threshold of progesterone-stimulated Mos accumulation and MAP kinase activation necessary for cell-cycle progression (Huang and Ferrell, 1996b; Chen and Cooper, 1997; Chen et al, 1997) and indicate that the Musashi1/2 antisense oligonucleotides function in a dose-dependant manner to suppress Mos mRNA translation and subsequent MAP kinase activation. The Musashi1/2 antisense oligonucleotides only reduced cellular Musashi1 protein levels by about 20% (±4.24% s.d.; n=2, Supplementary Figure S6), in progesterone stimulated oocytes. We have not been able to definitively ascertain the effect of the antisense oligonucleotides on the minor Musashi2 protein isoform because of the inadequacy of available antibodies. Our findings
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