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a Department of Biology and Center for Research on Reproduction
b Women's Health, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10-fold greater to the full-length Prm1 mRNA than to a 37-nucleotide sequence derived from the 3' UTR, and gel shift assays indicate that multiple MSY2 molecules bind to a single Prm1 mRNA. MSY2 binding to luciferase mRNA at ratios of protein to mRNA that are likely to exist in the oocyte also leads to a moderate inhibition of protein synthesis in vitro. Given the abundance of MSY2 in mouse oocytes (2% of total oocyte protein), these data suggest that MSY2 packages mRNAs in vivo with relatively little sequence specificity, which may lead to both stabilization and translation repression of maternal mRNAs.
developmental biology, gamete biology, gene regulation, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Y-box proteins are multifunctional. Many members can bind to the Y-box element 5'-CTGATTGG(C/T)(C/T)AA-3' (or reverse CCAAT box) present in the promoters of many viral and cellular genes, including many germ cell-specific genes such as the oocyte-specific hsp70 gene in Xenopus and the testis-specific Prm1 gene in mouse, and regulate the transcription of these genes [4, 10, 12]. Y-box proteins are also found in the messenger RNPs (mRNPs), which implicates these proteins in the translational regulation of mRNAs [8, 13–15]. The role of Y-box proteins in the translational control is well studied for the germ-line member FRGY2 in Xenopus. Using a method to select specific RNA sequences from a degenerate pool of short (25 nucleotides [nt]) RNAs (Selex method), recombinant FRGY2 can recognize specific RNA sequences; this sequence-binding specificity requires the presence of the cold-shock domain [16]. Binding of FRGY2 to mRNAs in vivo, however, is likely to be relatively independent of RNA sequence because 80% of the maternal mRNAs synthesized in the Xenopus oocyte are packaged in mRNP particles, of which FRGY2 is a major component. Both the ability of FRGY2 to inhibit translation in vitro and to participate in mRNP assembly in vivo requires the relatively nonspecific interaction of the C-terminal tail domain with mRNA [17].
In the female mouse, the expression of MSY2, which was cloned from an expression library screen with anti-FRGY2 antibodies [1], is restricted to the oocyte and constitutes 2% of total oocyte protein [18]. Oocyte mRNAs are very stable during the growth phase (with a half-life of 8–12 days [19]), and the abundance of MSY2 protein is consistent with its role as a global regulator of mRNA stability and translational control during this period of tremendous growth. In male germ cells, MSY2 is present in mRNPs, which package 75% of total paternal mRNAs in round spermatids [14]. These data obtained from both male and female germ cells suggest that MSY2 binds mRNAs in vivo in a relatively sequence-independent manner.
Recent reports, however, suggest that MSY2 displays a high sequence specificity for binding to RNA [20, 21]. MSY2 is a component of the protamine (Prm) mRNA-binding activity that is present in mouse testis extracts [20]. Prms are small arginine-rich proteins that are involved in chromatin condensation in the nucleus of spermatozoa [22]. Prm mRNAs are synthesized in round spermatids and stored in translationally repressed mRNPs until their translation in elongated spermatids 2–8 days later [23]. Two regions in the Prm1 3' untranslated region (UTR), the 3'-most 67 nt and the 5'-most 37 nt (Prm11–37(wt)), are involved in the translational delay, and MSY2 binds to the Prm1 5'-most 37 nt [20, 24]. RNA electrophoretic mobility shift assays using testis extract and a yeast three-hybrid assay with the Prm1 5'-most 37 nt variants as probes or bait, respectively, suggest that MSY2 binds RNA with a high degree of sequence specificity; MSY2 did not bind to a probe carrying a single nt change [20, 21].
In this study, we demeonstrated that although purified MSY2 recombinant protein binds to short RNA sequences derived from the Prm1 5'-most 37 nt with a limited sequence specificity, little if any sequence specificity is observed when full-length Prm1 mRNA is used. Moreover, several molecules of MSY2 bind to the full-length Prm1 mRNA with an affinity that is 10-fold greater than the binding of MSY2 to the short Prm1 mRNA sequences. Binding of MSY2 to mRNA at ratios of MSY2 to mRNA that approximate those in mouse oocytes leads to a modest inhibition of protein synthesis in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MSY2 recombinant protein bearing a T7 tag and a His tag at the carboxyl terminus was expressed in BL-21(DE3) cells (Novagen, Madison, WI) and purified as previously described [18]. For in vitro RNA binding and in vitro translation assays, the protein was dialyzed against 20 mM Hepes, pH 7.9, containing 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol (DTT), and 20% glycerol.
Production of MSY2 Antibody
To generate a polyclonal antibody against MSY2, purified recombinant MSY2 was used to inoculate New Zealand White rabbits, and the polyclonal antibody was affinity purified using MSY2 recombinant protein conjugated to agarose beads. The affinity-purified antibody, which was generated and purified by Covance (Richmond, CA), was diluted to 1 mg/ml and stored at -80°C until use.
RNA Probe Synthesis
The Prm1 full-length cDNA was amplified from testis cDNA (5' primer Prm1F, 5'-TACAAGCTTACAGCCCACAAAATTCCACCT-3'; 3' primer Prm1R, 5'-CGCGGATCCTTGTTCCTTAGCAGGCTCCT-3'), and the sequence was cloned into the HindIII and BamH1 sites of pSP64(polyA) (Promega, Madison, WI). Mutations were introduced into the 5'-most 37-nt region of Prm1 full-length cDNA as follows. Initial polymerase chain reactions (PCRs) with primers Prm1F/Prm11–37(C26A)R(5'-GCAGGAGTTTTTATGGACTT-3') and Prm1R/Prm11–37(C26A)F (5'-AAGTCCATAAAAACTCCTGC-3') yielded the 5' and 3' half of the Prm1(C26A) full-length cDNA. The single-stranded (ss) DNA of the 5' half of Prm1(C26A) was generated from the 5' half of Prm1(C26A) full-length cDNA using PCR with the primer Prm1F, and the ssDNA of the 3' half of Prm1(C26A) was generated from the 3' half of Prm1(C26A) full-length cDNA using PCR with primer Prm1R. The 5' and 3' half ssDNAs of Prm1(C26A) (PCRs) were mixed together and denatured at 94°C for 5 min and then annealed by gradual cooling to room temperature over a period of 3 h. Following annealing, 1 µl of 10 mM dNTPs and 1 µl of pfu polymerase (Stratagene, La Jolla, CA) were added, and the sample was incubated for 10 min at 72°C to fill in the 3' ends of Prm1(C26A) cDNA. The sample was then run on a 2% agarose gel, and the Prm1(C26A) full-length cDNA was purified with a gel purification kit according to the manufacturer's instructions (Qiagen, Valencia, CA). The Prm1(C26A) full-length cDNA was further amplified using primers Prm1F and Prm1R and cloned into the HindIII and BamH1 sites of pSP64(polyA). The Prm1(mut) full-length cDNA was generated with the same method, but primers Prm1(mut)R (5'-GAGTTTTGTTCAGGTATATATTCTGTGCATCTAGTAT-3') and Prm1(mut)F (5'-TATACCTGAACAAAACTCCTGCGTGAGAA-3') were used instead of Prm11–37(C26A)R and Prm11–37(C26A)F. All the PCRs using pfu polymerase were carried out as follows: initial denaturation for 5 min at 94°C, 35 cycles of 30 sec at 94°C, 45 sec at 55°C, and 45 sec at 72°C, followed by 10 min at 72°C. All the clones were sequenced to confirm the mutations introduced. For in vitro transcription reactions, the pSP64(polyA) plasmids were linearized with EcoRI.
To make the templates of the 5'-most 37-nt region of the 3' UTR of Prm1 cDNA for in vitro transcription, the SP6 promoter was introduced by PCR. Primers SP6prm11–37(wt)F (5'-GCTATTTAGGTGACACTATAGTAGATGCACAGAATAGCAAGTCCATCAAAACTCCTGC-3') and Prm11–37(wt)R (5'-GCAGGAGTTTTGATGGACTT-3') were used to amplify the prm1(wt) full-length cDNA, and primers SP6prm11–37(mut)F (5'-GCTATTTAGGTGACACTATAGTAGATGCACAGAATATATACC-3') and Prm11–37(mut)R (5'-GCAGGAGTTTTGTTCAGGTATA-3') were used to amplify prm1(mut) full-length cDNA. Both PCR products were ligated into pCRII vector using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). The template for the Prm11–37(wt) probe was amplified from the Prm11–37(wt) ligation reaction with the T7 primer (5'-GTAATACGACTCACTATAGGGC-3') and Prm11–37(wt)R. The template for the Prm11–37(C26A) probe was amplified from the Prm11–37(wt) ligation reaction with the T7 primer and Prm11–37(C26A)R. The template for the Prm11–37(mut) probe was amplified from the Prm11–37(mut) ligation reaction with the T7 primer and Prm11–37(mut)R. All the PCRs using pfu polymerase were carried out as decribed above. The PCR products were purified using the PCR purification kit (Qiagen).
The in vitro transcription reactions were carried out as described in the SP6 MAXIscript kit (Ambion, Austin, TX). The RNA probes were extracted once with phenol/chloroform and once with chloroform and then were precipitated by adding 0.1 volume of 5 M ammonium acetate and 1 volume of isopropanol. For the 37-nt probes, 10 µg glycogen was added to facilitate precipitation. The samples were chilled at -20°C for 30 min, and the precipitated material was collected by centrifugation at 14 000 x g for 20 min at 4°C. The pellets were washed with 75% ethanol twice and resuspended in RNase-free H2O.
RNA Electrophoretic Mobility Shift Assay
The gel shift assays were carried out in 20 µl containing 1x binding buffer (20 mM Hepes, pH 7.9, 2 mM MgCl2, 100 mM KCl, 1 mM DTT, and 24 µg/ml BSA); 0.15 ng of each 37-nt probe (2.5 x 105 cpm) and 0.62 ng of each Prm1 full-length RNA probe (435 nt, 2.9 x 105 cpm) were used in the reactions. The reaction mixtures were incubated at room temperature for 20 min and then run on a 5% nondenaturing 37:5:1 acrylamide gel (0.5x TBE: 45 mM Tris base, 50 mM boric acid, 1 mM EDTA). The gels were vacuum dried, exposed on a PhosphorImager cassette, and scanned with a Storm 860 system. Quantification was carried out with ImageQuant software.
Nitrocellulose Filter Binding Assay
The binding reactions were carried out as in the RNA electrophoretic mobility shift assays. Following incubation at room temperature for 20 min, the reactions were filtered for 45 min through 0.45-µm HA MF-nitrocellulose membrane filters (no. 9004-70-0; Millipore, Bedford, MA) initially made wet with the 1x binding buffer. The membranes were washed twice with 0.5 ml 1x binding buffer before being subjected to liquid scintillation counting. The experiments were repeated three times for each probe.
Luciferase mRNA Preparation
The luciferase (Luc) cDNA from pGL3-Control vector was cloned into the HindIII and XbaI sites of the pSP64(polyA) vector (Promega). The plasmid was linearized with EcoRI for in vitro transcription, and capped and polyadenylated Luc mRNA (1754 nt) was transcribed using the SP6 mMessage mMachine kit (Ambion). The transcription mixture was incubated at 37°C for 2 h followed by a 15-min incubation with 1 µl of RNase-free DNase I (2 units/µl) to digest the DNA template. The RNA was phenol/chloroform extracted and precipitated with 1 volume of isopropanol.
In Vitro Translation
The in vitro transcribed Luc mRNA was heated at 65°C for 5 min and then immediately cooled on ice. Different amounts of MSY2 recombinant protein were incubated with 0.25 µg Luc mRNA for 20 min in a volume of 8.5 µl containing 20 mM Hepes, pH 7.9, 2 mM MgCl2, 100 mM KCl, and 1 µl RNasin (40 U/µl; Promega) to allow the binding of MSY2 to the Luc mRNA. Following addition of the translation mixture containing 15 µl of a rabbit reticulocyte lysate, 0.5 µl of 1 mM amino acids mixture (-met; Promega), and 1 µl of [35S]met (10 mCi/ml; Amersham, Piscataway, NJ), the sample was incubated for 90 min at 30°C. Following incubation, 10 µl of each translation reaction was mixed with 10 µl of 2x SDS loading buffer (100 mM Tris-HCl, pH 6.8, 200 mM DTT, 4% SDS, 20% glycerol, 0.2% bromophenol blue), and the sample was boiled for 5 min prior to being subjected to electrophoresis in a 9% SDS-polyacrylamide gel. The gels were vacuum dried, exposed on a PhosphorImager cassette, and scanned with a Storm 860 system. The amount of Luc protein synthesized was quantified with ImageQuant software.
Northern Blotting
Following in vitro translation, the Luc mRNA was extracted with Trizol reagent (Gibco/Invitrogen) and loaded onto a 1% formaldehyde agarose gel. Northern blotting was carried out as previously described [18]. The blot was exposed on a PhosphorImager cassette and scanned with a Storm 860 system.
Immunoprecipitation
Following in vitro translation, 0.5 ml IP buffer (20 mM Hepes, pH 7.9, 100 mM KCl, 2 mM MgCl2, 0.1% Tween-20, 0.1% Empigen, 24 µg/ml BSA, 100 U/ml RNasin) was added to each reaction. The samples were cleared by adding 30 µl of protein A agarose beads (50% slurry; Gibco/Invitrogen) previously washed with 0.5 ml IP buffer and then incubated with rocking for 1 h at 4°C. The agarose beads were removed by centrifugation at 14 000 x g for 1 min at 4°C. To immunoprecipitate MSY2, 5 µl of the affinity-purified MSY2 antibody (1 mg/ml) was added to the supernatant, the sample was incubated with rocking at 4°C for 1 h, 30 µl of fresh protein A agarose beads (previously washed with 0.5 ml IP buffer) was added, and the sample was incubated at 4°C for an addition 1 h. Preimmune serum was used instead of MSY2 antibody as a control. The immunoprecipitates were collected by centrifugation at 14 000 x g for 1 min at 4°C and washed five times with 0.1 ml IP buffer. RNA was extracted from the immunoprecipitates using Trizol reagent, and Northern blotting was conducted as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MSY2 represents about 2% of total protein in the fully grown mouse oocyte [18] and is present in the mRNPs, which package about 75% of paternal mRNAs in round spermatids [14]. These findings suggest that MSY2 binds mRNAs in a non-sequence-specific manner. However, in a recent report, the authors proposed that MSY2 binds RNA with a high degree of sequence specificity, i.e., that MSY2 binds the consensus sequence UACCACAUCCACU (Y-box recognition site, YRS) [20, 21]. To resolve this potential discrepancy, we conducted both electrophoretic mobility shift and nitrocellulose filter binding assays using purified recombinant MSY2 protein. The probe used in these studies was the 5'-most 37 nt from the Prm1 3' UTR (Prm11–37(wt)), which contains the consensus site (UCCAUCA) (Fig. 1A). We also introduced mutations into this sequence (Fig. 1A). Prm11–37(C26A) contains a single mutation in the consensus site, and Prm11–37(mut) has eight mutations, with three in the consensus site; these mutations are the same ones used in the previous study [20].
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Electrophoretic mobility shift assays demonstrated that the recombinant MSY2 protein was functional and could bind to the probes (Fig. 1, B and C); the observed binding was specific because no electrophoretic shift of the probe was observed when glutathione S-transferase (GST) was added. Consistent with a previous report [20], little binding was observed to the mutated probes when small amounts of MSY2 were used. With increasing concentrations of MSY2, however, significant binding to the mutant probes was observed, although the degree of binding was less than that seen with the wild-type probe. Similar results were obtained from competition studies in which increasing amounts of nonradiolabeled probe were added to the binding reactions (data not shown). Thus, a limited degree of sequence-specific binding was observed.
The differences in MSY2 binding to the different probes observed in the electrophoretic mobility shift assays were not as apparent when nitrocellulose filter binding assays were used (Fig. 2). The apparent dissociation constants calculated from the binding curves were 2.1 x 10-7 M for the Prm11–37(wt), 3.3 x 10-7 M for Prm11–37(C26A), and 7.2 x 10-7 M for Prm11–37(mut).
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These results suggest that the binding of MSY2 to these short sequences displays some sequence specificity, with relatively low affinity binding in the range of 10-7 M.
MSY2 Binding to the Full-Length Prm1 RNA
The average length of mRNAs is 1500–2000 nt, and hence the sequence specificity of binding of MSY2 to long RNA sequences may differ substantially when compared with its ability to bind short RNA sequences. Accordingly, we prepared full-length Prm1 RNA probes (435 nt) with the aforementioned mutations introduced into the 37-nt region (Fig. 3A) and determined MSY2 binding by both electrophoretic mobility shift and nitrocellulose filter binding assays (Fig. 3, B and C).
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When small amounts of MSY2 were used (10 ng) for electrophoretic mobility shift assays, MSY2 could shift both the Prm1(wt) and Prm1(C26A) probes but not the Prm1(mut) probe, i.e., some sequence binding specificity was detected (Fig. 3B). Similar binding to all probes, however, was observed with increasing amounts of MSY2. Moreover, with increasing amounts of MSY2, multiple bands were observed (up to five), suggesting that multiple MSY2 molecules could bind to a single mRNA molecule. The apparent dissociation constants calculated from the filter binding assays were 5.5 x 10-8 M for the Prm1(wt), 6 x 10-8 M for Prm1(C26A), and 5.4 x 10-8 for Prm1(mut). This increase in binding affinity of 10-fold could reflect the binding of multiple MSY2 molecules to the mRNA, i.e., it represents a form of cooperation. The stronger signal in the upper bands of Prm1(mut) observed in the electrophoretic mobility shift assay might be due to differences in secondary structure in the Prm1(mut) mRNA (as indicated by slower electrophoretic mobility of the free Prm(mut) mRNA) that facilitated the binding of multiple MSY2 molecules to the same RNA molecule.
Results of these experiments indicated that MSY2 had a significantly higher affinity for full-length mRNAs and that multiple MSY2 molecules can bind to a single RNA molecule. Little if any sequence specificity of MSY2 binding to full-length mRNAs was observed at higher concentrations of MSY2.
Effect of MSY2 on In Vitro Translation of mRNA
MSY2 has been suggested to modulate translation of maternal mRNAs in mouse oocytes [18]. To ascertain whether MSY2 binding to mRNA could modulate the translatability of mRNAs, we examined the effect of MSY2 on the in vitro translation assays of Luc mRNA. We conducted these experiments over a range of MSY2:mRNA molar ratios based on the molar MSY2:mRNA ratio of about 70:1. This ratio is based on a total of 80 pg of mRNA in an oocyte, an average mRNA length of 1500 nt, and 0.5 ng of MSY2 protein per oocyte [18].
Increasing amounts of MSY2 protein resulted in a progressive decrease in the translatability of the Luc mRNA, such that when physiological ratios were obtained the inhibition was about 70% (Fig. 4, A and B). This inhibition was specific; no inhibition was observed when similar amounts of GST were added. The inhibition could not be attributed to the degradation of the input Luc mRNA because the amount of Luc mRNA present at the end of translation reactions, as determined by Northern analysis, was similar in the control (no MSY2 added) and in reactions in which MSY2 was added (Fig. 4C). Moreover, this inhibition was correlated with binding of MSY2 to the Luc mRNA, because Luc mRNA, as detected by Northern analysis, was coprecipitated by the affinity-purified MSY2 antibody (Fig. 4D); preimmune serum did not allow coprecipitation of Luc mRNA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In other studies, MSY2 binding has displayed high sequence specificity. For example, when using a yeast three-hybrid system that employs a LexA-RNA-binding bacteriophage MS2 coat protein and MSY2-Gal4 activation domain to drive the expression of the HIS3 gene, only the wild-type Prm1 37-nt sequence enabled the yeast to grow on histidine-free medium [20]. Neither the Prm1 probe containing a single mutation nor the Prm1 probe containing several mutations supported growth on the histidine-free medium. We also detected such sequence specificity in the binding of recombinant MSY2 protein in electrophoretic mobility shift assays, but only when low concentrations of MSY2 were employed. When higher concentrations of MSY2 were used, MSY2 binding was readily observed, albeit at reduced levels. In the yeast three-hybrid assay, the amount of MSY2 fusion protein expressed relative to the amount of bridging RNA probe was not determined. Moreover, the binding differences that we observed were not as prominent when binding was assayed by filtration, which likely reflects intrinsic differences between the electrophoretic mobility shift and filtration assays. In the electrophoretic mobility shift assay, there may be some loss of the MSY2-RNA complex because of dissociation of MSY2 during electrophoresis. In contrast, the filtration assay is very rapid compared with the electrophoretic mobility shift assay, and hence less loss of the complex is likely to occur. If the off rate is the major contributing factor to the apparent dissociation constant that we calculated, the electrophoretic mobility shift assay may be biased towards detecting smaller differences in binding affinity when compared with the filtration assay.
When full-length Prm1 mRNA was used in the binding assays, there was little if any difference in MSY2 binding to any of the mRNAs tested, i.e., wild-type and mutant forms, although the binding affinity was increased by 10-fold. The apparent dissociation constant (5 x 10-8 M), which was obtained with the short Prm1 mRNA, could even be lower for longer mRNAs. This increase in binding affinity may be due to the binding of multiple MSY2 molecules to the mRNA, as indicated by the ladder of bands observed in the electrophoretic mobility shift assay (see Fig. 3).
The consensus sequence UACCACAUCCACU to which MSY2 appears to bind preferentially under certain experimental conditions [21] is present in many mRNAs and frequently as multiple copies in an mRNA, e.g., ß-actin contains nine of these consensus sequences. Our finding that MSY2 preferentially binds to wild-type Prm1 mRNA at low MSY2 concentrations (Fig. 3B) suggests that the consensus sequence could serve to prime MSY2 binding, which is then followed by binding of additional MSY2 molecules, visible as the banding ladder that develops at higher MSY2 concentrations. Thus, although the presence of this consensus sequence may provide a modest specificity in MSY2 binding in vivo to mRNAs by serving as a nucleation or priming center, results of our studies and mass action considerations suggest a more global function of MSY2 in RNA binding in vivo. The concentration of MSY2 in the fully grown oocyte is 50 µM, whereas the concentration of total mRNA is 0.7 µM, assuming that the oocyte contains 0.5 ng of MSY2 and 80 pg of mRNA with an average length of 1500 nt. Based on an apparent dissociation constant of 5 x 10-8 M for the binding of MSY2 to full-length Prm1 mRNA, it is likely that most of the mRNA in the oocyte is complexed with MSY2.
We previously demonstrated that following permeabilization of oocytes with Triton X-100 75% of the MSY2 remains associated with an insoluble fraction; the conditions of permeabilization result in the total loss of soluble proteins [25]. The proposed global association of MSY2 and its association with this insoluble fraction could sequester mRNAs from the translation machinery. Reverse transcription PCR analysis of mRNAs present in the insoluble and soluble fractions following permeabilization suggests that the bulk of the mRNA is associated with the insoluble fraction (unpublished results). Such an association, mediated either directly or indirectly by MSY2, could be a major factor contributing to the marked stability of mRNAs during oocyte growth, because translation is linked with mRNA degradation [26, 27]. Likewise, the degradation of MSY2 that follows fertilization and is essentially complete by the late two-cell stage [18] could facilitate the global degradation of maternal mRNAs that occurs during this time.
The ability of MSY2, like the ability of FRGY2 [17], to inhibit translation in vitro could also contribute to mRNA stability during oocyte growth. MSY2 recombinant protein was capable of moderately repressing the translation of Luc mRNA in rabbit reticulocyte lysates at a calculated physiological MSY2:mRNA molar ratio of about 70:1; similar results were obtained with AKAP7 mRNA (data not shown). This modest repression contrasts with the total inhibition of translation of histone H1 mRNA by FRGY2 at a molar ratio of 30:1 [17]. Because 25% of the MSY2 in the mouse oocyte is present in the soluble fraction following Triton X-100 permeabilization, mRNAs associated with the soluble MSY2, e.g., actin and tubulin, would be capable of being translated, albeit at a reduced level. The role of MSY2 phosphorylation [18] (or other proteins that interact with MSY2 but are absent in the in vitro assays) in RNA binding and translation repression is not known and could be a contributing factor.
The accumulation of mRNAs that occurs during oocyte growth means that the mRNAs must be very stable because of the prolonged length of the growth phase and the absence of cell division. Generating oocytes that lack MSY2 function should afford insights into the proposed role of MSY2 in mRNA stability and translation during oocyte growth and development.
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FOOTNOTES |
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2 Correspondence: Richard Schultz, Department of Biology, University of Pennsylvania, 415 South University Ave., Philadelphia, PA 19104-6018. FAX: 215 898 8780; rschultz{at}mail.sas.upenn.edu
Received: 25 March 2002.
First decision: 24 April 2002.
Accepted: 26 April 2002.
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