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Previous Article | Next Article 
Molecular and Cellular Biology, March 2004, p. 2513-2525, Vol. 24, No. 6
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.6.2513-2525.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Program in Molecular Biology and Biotechnology, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599
Received 16 July 2003/ Returned for modification 13 August 2003/ Accepted 18 December 2003
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ABSTRACT |
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INTRODUCTION |
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Histone mRNAs are synthesized early in oogenesis, and synthesis is complete by the end of stage II of oogenesis. The histone mRNAs are also translated in stage II to provide a maternal store of histone proteins (39). In later stages of oogenesis there is little or no translation of the histone mRNA (39). When full-grown stage VI oocytes are treated with progesterone, they undergo maturation, initiating progression through meiosis, and also activate a translational regulation program necessary for the initial stages of embryogenesis. One of the mRNAs activated at oocyte maturation is histone mRNA (1).
The 3' end of histone mRNA is essential for efficient translation (9, 25). In Xenopus oocytes there are two stem-loop binding proteins (SLBPs) that can bind the 3' end of histone mRNAs: xSLBP1 and xSLBP2 (33). xSLBP1, which is the orthologue of the mammalian SLBP, is required for histone pre-mRNA processing (13), and it is also necessary for efficient translation of histone mRNA (25). In contrast, xSLBP2 is found only in oocytes and does not support translation of histone mRNA. xSLBP2 is associated with the maternally stored histone mRNA and is destroyed during oocyte maturation (33). The destruction of xSLBP2 may allow xSLBP1 to bind to the mRNA, activating its translation.
Histone mRNAs in Xenopus oocytes contain a short A tail, which is attached to the stem-loop (2). We report here that the oligo(A) tail plays an active role in translation repression. Histone mRNAs ending with an oligo(A) tail are not translated in the presence of xSLBP1 in Xenopus oocytes, although they are translated in reticulocyte lysates. In addition to xSLBP2, the oligo(A) tail also plays a role in repression of histone mRNA translation.
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MATERIALS AND METHOD |
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S1 nuclease mapping
To map the 3' end of the histone mRNA, we used two different probes. The S1 probe used to map the 3' end of the histone xH2a mRNA (Fig. 1B) was generated by PCR, using a previously cloned Xenopus H2a cDNA ending in 20 adenosines as the template (33). Standard RNAs ending in the histone stem-loop or in the stem-loop followed by 25 As were transcribed with T7 RNA polymerase from appropriate DNA templates. The 184-nucleotide (nt) PCR product contains 130 nt of histone mRNA and the 20-nt poly(A) tract followed by 34 nt that had no complementarity to the histone xH2a mRNA. This fragment was labeled at the 3' end with the Klenow fragment of DNA polymerase I (New England Biolabs) and [
-32P]dCTP. For the S1 analysis whose results are shown in Fig. 1C and 2A, the plasmid encoding the xH2a gene was digested with XmaI and labeled with the Klenow fragment of DNA polymerase I and [-32P]dCTP. Hybridization was done at 56°C, and S1 digestion was performed as previously described (10, 13) except that the digestion was carried out on ice with 4 U of S1 nuclease to preserve the oligo(A-T) hybrids (35). The protected fragments were resolved on a 10% polyacrylamide-7 M urea gel and detected by autoradiography.
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-32P]dCTP. Total RNA was extracted (using TRIzol reagent) (Invitrogen) from oocyte lysates, and 1 oocyte equivalent of total RNA was hybridized to 5 ng of probe. Hybridization, S1 digestion, and detection were performed as previously described (25).
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-32P]dCTP. Hybridization was done at 56°C. S1 digestion was performed at 4°C as described above. Analysis of xSLBP proteins The stem-loop probe and the conditions used for the mobility shift assay were as previously described (25). Purification of the anti-xSLBP antibodies and the conditions used for the Western blotting analysis were as previously described (25, 33). Unlabeled and labeled RNAs ending in the stem-loop or the stem-loop followed by oligo(A) for the mobility shift experiments were transcribed (using T7 RNA polymerase) from appropriate templates.
In vitro transcription and translation Transcription of capped and uncapped luciferase constructs (as well as of xSLBP1, xSLBP2, MS2, and MS2-hSLBP mRNAs) was performed as previously described (25). The synthesis of different SLBPs in vitro and the analysis of translation of the luciferase reporter mRNAs were done exactly as previously described, as were the protocols for microinjections, Western blotting, and luciferase assays (25).
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RESULT |
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TopAbstractIntroductionMaterials and MethodResultDiscussionReference |
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Oocyte histone mRNA has an A tail of 6 to 10 nt To quantitatively analyze the 3' end of Xenopus histone mRNAs, we developed an S1 nuclease protection assay that can measure the length of the A tail on the 3' end of histone mRNA (Fig. 1A). We prepared a 3'-labeled probe ending in the stem-loop followed by 20 As and 34 additional nucleotides. The probe was labeled at a site 130 nt from the end of the stem-loop, allowing us to precisely resolve even very short oligo(A) tails. For control mRNAs, we synthesized in vitro an mRNA ending at the stem-loop and one ending at the stem-loop with a 25-A extension. The probe containing 20 Ts will protect a 130-nt fragment from the wild-type histone mRNA and a 150-nt fragment from the mRNA ending in 20 As. A major challenge in the S1 mapping experiments is maintaining the integrity of the oligo(A-T) hybrids at the 3' end of the mRNA during the experiment. To enhance annealing of the oligo(A-T) sequence, we used the minimal amount of enzyme necessary to degrade the free probe and performed the S1 digestions on ice (35). For each set of S1 nuclease assays we included the two control mRNAs. This approach allowed us to control for the loss of the A-T tail by S1 digestion of the relatively unstable oligo(A-T) hybrid, which occurred to different extents among the sets of samples.
We used this assay to determine the 3' end of the Xenopus histone H2a mRNA during oogenesis. The control mRNA ending at the stem-loop protected a 130-nt fragment as expected, while the control mRNA ending in 25 As protected a major fragment of 150 nt (protecting the 20 Ts in the probe; Fig. 1B, lanes 3 and 4, respectively). The polyadenylated control mRNA displays a distribution of slightly smaller fragments as well as a small amount of the 130-nt protected fragment (Fig. 1B, lane 4). The 130-nt fragment could result either from "breathing" of the A-T hybrid, which allows the S1 nuclease to cleave the probe in the A stretch (resulting in subsequent complete removal of the A-T hybrid region), or from a small amount of the control mRNA that was not adenylated. We assayed total RNA from stage III and stage VI oocytes and matured oocytes. The oocyte histone mRNAs protected fragments longer than 130 nt. The most abundant fragments were 5 to 8 nt longer than those seen with the mature histone mRNA, with protected fragments up to 10 to 11 nt in length detected. Only a small proportion of the protected fragments were 130 nt, consistent with the great majority (if not all) of the mRNA ending in an oligo(A) tail (Fig. 1B, lanes 5 and 6). In contrast, RNA from in vitro-matured oocytes protected a major fragment of 130 nt, similar to the results seen with the standard synthetic histone mRNA (Fig. 1B, lane 7). These results are in general agreement with the results of Ballantine and Woodland (2).
We determined the state of the 3' end of the Xenopus histone mRNAs during oogenesis and early development. In stage I oocytes, most of the histone mRNAs do not contain A tails (Fig. 1C, lane 1). The mRNA from stage II oocytes was partially adenylated. In stages III and VI of oogenesis, the great majority of the histone mRNAs contained a short A tail (Fig. 1C, lanes 3 to 6). Since RNAs from the same number of oocytes were analyzed for each lane, this assay allows us to determine the relative amounts of histone mRNAs in different stages of oogenesis. Note that as is true for most other maternal mRNAs (7) and as previously reported for histone mRNAs (38), most of the histone mRNA accumulates very early in oogenesis and the steady-state level of histone mRNA remains constant after stage III of oogenesis.
Different stage II oocyte preparations gave different proportions of adenylated histone mRNAs (the distinction between oocyte stages is somewhat subjective during the sorting of the oocytes), but all preparations of stage I oocytes contained less histone mRNA with little or no adenylated mRNA. We conclude that in very early oogenesis (stages I and II), most of the histone mRNAs do not contain A tails and that by stage III the great majority (if not all) of the histone mRNAs contain A tails.
To determine whether the A tails were lost during oocyte maturation, we treated stage VI oocytes with progesterone and compared the histone mRNA levels in stage VI oocytes to those of oocytes which had matured after progesterone treatment (Fig. 1C, lanes 6 and 7). We also analyzed the histone mRNA from eggs in a parallel assay (Fig. 1C, lane 8). The histone mRNA from oocytes matured in vitro with progesterone (as well as the histone mRNA from eggs) was not adenylated. Therefore, treatment of the oocytes with progesterone recapitulated the loss of the oligo(A) tails from histone mRNA.
xSLBP2, which inhibits histone mRNA translation, is also degraded during oocyte maturation (33). To determine the relative timings of these two changes, we analyzed the amounts of xSLBP2 by Western blotting and the proportion of histone mRNAs containing the oligo(A) tails by S1 nuclease assay at 1-h intervals after progesterone treatment. The removal of the oligo(A) tails and the destruction of xSLBP2 occurred in parallel (Fig. 1D). This assay has limited temporal resolution, due to the lack of precise synchrony with which oocytes respond to progesterone and to the absence of phenotypic markers for the progression of maturation. Although the removal of the oligo(A) tails and the destruction of xSLBP2 could not be temporally separated, both had occurred extensively prior to the appearance of the white spot on the animal pole that is indicative of germinal vesicle breakdown and completion of oocyte maturation.
Deadenylation of histone mRNA and degradation of xSLBP2 at maturation can occur in the absence of the nucleus Messages that lack a cytoplasmic polyadenylation element (CPE) undergo default deadenylation at maturation, and their translational activity is inhibited (8, 31). An mRNA deadenylase called DAN has been purified from Xenopus oocytes, and there is a human orthologue called PARN. Removal of the nucleus or injection of antibodies against DAN (PARN) inhibits default deadenylation (14). To test whether removal of the histone mRNA oligo(A) tract at maturation requires the nucleus (like default deadenylation of non-CPE-containing mRNAs), enucleated oocytes were treated with progesterone to induce maturation. When enucleated oocytes were treated with progesterone, the histone mRNAs lost their oligo(A) tract to the same extent as whole oocytes treated with progesterone (Fig. 2A; compare lanes 3 and 5).
We also tested whether enucleated oocytes treated with progesterone degraded xSLBP2. Treatment of stage VI oocytes with progesterone resulted in a loss of xSLBP2 as analyzed by Western blotting (Fig. 2B, lanes 5 and 6). Identical results were obtained when the oocytes were enucleated prior to treatment with progesterone (Fig. 2B, lanes 7 and 8). We conclude that removal of the histone mRNA oligo(A) tail and the degradation of xSLBP2 can be mediated solely by cytoplasmic factors and is not dependent on germinal vesicle breakdown.
The presence of the oligo(A) tail does not affect binding of either xSLBP One function of the oligo(A) tail could be to help determine which xSLBP is associated with the histone mRNA. A second possibility is that another (or multiple) factor(s) could stably associate with the oligo(A) tail. Note that these tails are not long enough to be bound by the poly(A) binding protein (PABP), so it is unlikely that PABP binds to the oocyte histone mRNA via the poly(A) tract (24). To determine whether the two xSLBPs show different relative affinities for the histone stem-loop and the stem-loop with an oligo(A) tail, we performed mobility shift assays. We used extracts from stage I oocytes, which primarily form complexes containing xSLBP2 (33), and extracts from stage VI oocytes, which form complexes containing both xSLBP1 and xSLBP2, with xSLBP1 complexes being the more abundant species. We incubated each extract with either a radiolabeled stem-loop probe or with the stem-loop with 8 As (SLA8) attached. We then tested the ability of stem-loop and SLA8 unlabeled RNAs to compete for the binding of each xSLBP. Note that these assays are done using oocyte extracts, so any modifications of the xSLBPs that could alter their affinity for the stem-loop (which might occur at different stages of oogenesis) are likely to be retained. The two SLBPs bound the two probes with similar affinities, and the stem-loop and SLA8 unlabeled RNAs were equally effective in competing for the binding to either the SL or the SLA8 probe (Fig. 3A). Similar results were previously reported for the wild-type probe when the proteins were expressed in reticulocyte lysates (13).
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The SLBPs do not rapidly exchange in vitro A striking finding in the previous work of Wang et al. was that xSLBP1 was bound to histone mRNA early in oogenesis whereas xSLBP2 was bound to the histone mRNA late in oogenesis (33). When the amounts of active xSLBPs are determined by mobility shift assays, however, xSLBP2 is the major SLBP early in oogenesis and xSLBP1 is the major SLBP late in oogenesis (33). Since it is possible that one function of the poly(A) tail is to alter the exchangeability of the SLBPs, we tested whether the xSLBPs would interchange in vitro on either the wild-type or the oligo(A) stem-loop probe.
We synthesized the xSLBPs in vitro using rabbit reticulocyte lysates and labeled the proteins with [35S]methionine to allow us to estimate the amount of protein (there is no mammalian SLBP in reticulocyte lysates; see reference 34). We incubated the probe with an excess of one of the SLBPs under conditions in which the entire radiolabeled probe formed a complex with an SLBP after incubation for 5 min at 4°C. An approximately 10-fold excess of the second SLBP was then added to the extract and incubated for an additional 60 min on ice. As controls the proteins were mixed together prior to addition of the probe.
When we formed a complex with xSLBP1 on either the SLA8 (Fig. 3B, lane 1) or the stem-loop (Fig. 3B, lane 7) probe, all of the probe formed a single complex. When we mixed an excess of xSLBP2 with xSLBP1 prior to addition to the probe, about 90% of the complexes formed contained xSLBP2 with both probes (Fig. 3B, lanes 5 and 11). However, when the same amount of excess xSLBP2 was added after formation of the complex with xSLBP1, there was a less than 10% exchange of the proteins in 60 min at 4°C (Fig. 3B, lanes 2 and 8). When the experiment was reversed (forming the complexes on xSLBP2 first), the addition of excess xSLBP1 resulted in partial exchange during the 60-min incubation. Partial exchange was observed on both probes (Fig. 3B, lanes 4 and 10). When the proteins were premixed in the same amounts used in the exchange experiments, the xSLBP1 complex was the only complex detected with either probe (Fig. 3B, lanes 6 and 12). These results demonstrate that the proteins do not exchange rapidly, in agreement with previous results that the RNA does not rapidly dissociate from SLBP (37). The presence of the oligo(A) sequence did not affect the exchange of the SLBPs in vitro.
Effect of the oligo(A) tract on translation in vitro We have previously shown that xSLBP1 actively supports translation of a luciferase reporter mRNA ending in a stem-loop (both in reticulocyte lysates and in stage VI oocytes) whereas xSLBP2 does not (25). An MS2-SLBP fusion protein will also stimulate translation of luciferase reporter mRNAs that end in the histone stem-loop or the MS2 binding site (25). To determine whether the short A tail had any effect on the translation of the reporter mRNA ending in the stem-loop, we constructed a luciferase mRNA that ended in a stem-loop followed by 8 As (Luc-SLA8). As controls we constructed reporter mRNAs that ended in 8 Cs (Luc-SLC8) or in a random 8-nt sequence (Luc-SLN8). We also used mRNAs ending in the MS2 binding site (Luc-MS2) or in the MS2 site followed by 8 As (Luc-MS2A8). The constructs are shown in Fig. 4A.
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We compared the effect of the presence of xSLBP1 and xSLBP2 on translation of the Luc-SL, Luc-SLA8, and Luc-TL mRNAs in vitro. When xSLBP1 was synthesized in the lysate prior to the addition of the luciferase mRNAs, there was a six- to eightfold increase in the translation efficiency of both the Luc-SL and Luc-SLA8 mRNAs (Fig. 4B, lanes 2 and 3, and Fig. 4C) compared to the results seen with translation of Luc-TL mRNA (Fig. 4B, lane 1, and Fig. 4C), as determined both by luciferase assay and by autoradiography of the luciferase protein. Addition of a lysate containing xSLBP2 did not affect translation of any of the reporter mRNAs (Fig. 4B, lanes 4 to 6, and Fig. 4C). We conclude that the oligo(A) tail does not affect the ability of xSLBP1 to activate translation of the Luc-SLA8 mRNA in the reticulocyte lysate.
The oligo(A) tail reduces activation of translation of the Luc-SLA8 mRNA in Xenopus oocytes To assay translation of the reporter mRNAs in vivo (Fig. 5A) (25), we first injected a synthetic mRNA encoding an SLBP protein into stage VI oocytes. At 16 h later we injected equal amounts of a capped synthetic firefly luciferase mRNA (Luc-test) together with an internal standard capped Renilla luciferase mRNA ending in the histone stem-loop (R-Luc-SL). At different times after injection of the reporter mRNAs we harvested the oocytes and measured the firefly and Renilla luciferase activity levels. Using this assay we previously showed that xSLBP1 (but not xSLBP2) activates translation of the Luc-SL mRNA in vivo; the in vitro and in vivo assays gave essentially identical results (25).
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We measured (by Western blotting) the amount of xSLBP1 and xSLBP2 present in the oocytes injected with the synthetic SLBP mRNAs. The oocytes injected with the xSLBP2 or xSLBP1 mRNA expressed a large excess of the xSLBP both 16 and 24 h after injection of the reporter mRNAs (Fig. 5C) compared with the oocytes injected with buffer (Fig. 5C, lane 1, lanes 9 to 14, bottom panels).
We repeated this experiment with an independent set of oocytes. As additional controls we also used reporter mRNAs ending in a stem-loop followed by 8 Cs (Luc-SLC8) or by a random nucleotide sequence (Luc-SLN8; Fig. 4A). The oocytes were harvested 24 h after injecting these reporter mRNAs. In these oocytes, expression of exogenous xSLBP1 results in an approximately sixfold increase in translation of the Luc-SL reporter mRNA compared to the results seen with the Luc-TL mRNA. In contrast, translation of the Luc-SLA8 mRNA was stimulated only twofold in the oocytes expressing xSLBP1 (Fig. 6A). However, the Luc-SLC8 and Luc-SLN8 RNAs were translated with only slightly reduced efficiency compared with the Luc-SL RNA. Expression of xSLBP2 in the oocytes prior to injection of the reporter mRNAs again reduced translation of the Luc-SL mRNA compared to the results seen with injection of buffer, and all five reporter mRNAs were translated to the same extent (Fig. 6A). In these oocytes injected with buffer there was a twofold activation of the Luc-SL (compared to the results seen with Luc-TL mRNA reporter) due to the presence of endogenous xSLBP1 in the oocyte. There was no activation of the Luc-SLA8 mRNA; this RNA was translated to the same extent as the Luc-TL mRNA (Fig. 6A), although Luc-SLC8 and Luc-SLN8 were both translated at rates similar to those seen with the Luc-SL RNA. The levels of the exogenous proteins were the same in all extracts regardless of which reporter mRNAs had been injected (Fig. 6B). We also tested the ability of all five of these capped RNA preparations to translate in the rabbit reticulocyte lysate, and they were all equally active (not shown, but see Fig. 8).
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The probe used for the S1 protection assay in Fig. 7A would not detect any change that might occur on the oligo(A) tail of the Luc-SLA8 mRNA, since it extended only to the end of the stem-loop. To verify the integrity of the 3' end of the reporter mRNAs 16 h after injection, we designed a short probe that contained the 3' end of the Luc-SLA8 mRNA (Fig. 7B). To facilitate resolution of the small changes in length, this probe protected only 40 nt prior to the end of the stem-loop. We analyzed the RNA from oocytes that overexpressed xSLBP1 and that were injected with Luc-SL and Luc-SLA8 mRNAs. At 16 h after injection of the reporter mRNAs, the 3' end of the Luc-SLA8 mRNA still contained the eight adenosines (Fig. 7B, lane 2) whereas the Luc-SL mRNA did not have any adenosines added to the 3' end (Fig. 7B, lane 1). These protected fragments were absent from oocytes that did not have reporter mRNA injected (Fig. 7B, lane 3). Since the same probe was used in lanes 1 to 3, this experiment also confirms that the amounts of the Luc-SL and Luc-SLA8 mRNA were similar in the injected oocytes.
Translation repression in stage VI Xenopus oocytes requires the stem-loop and the oligo(A) tail Previously we showed that an MS2-SLBP fusion protein can activate translation of a reporter mRNA that ends in an MS2 binding site as well as of an RNA that ends in the stem-loop (25). We compared the ability of the MS2-SLBP fusion protein to activate translation of reporter mRNAs that end either in the MS2 site or the MS2 site followed by 8As (Luc-MS2A8) with its ability to activate translation of the Luc-SL and Luc-SLA8 mRNAs. We synthesized five capped reporter mRNAs (encoding firefly luciferase) with different 3' ends and mixed them with the internal control mRNA (encoding Renilla luciferase) ending in the stem-loop. All of these mRNAs were translated to the same extent in the reticulocyte lysate in the absence of SLBP (Fig. 8A).
These same RNA preparations were injected into Xenopus oocytes expressing either human SLBP, MS2 protein, or the MS2-SLBP fusion protein (Fig. 8B). Both the human SLBP and the MS2-SLBP fusion protein were overexpressed relative to the endogenous SLBPs, as measured by the mobility shift assay (Fig. 8C). The human SLBP activated translation of the Luc-SL mRNA but not that of the Luc-SLA8 mRNA or that of the mRNAs ending in the MS2 binding site. We tested whether placing an oligo(A) tail on the reporter mRNA ending in the MS2 site would affect activation of translation by the MS2-SLBP fusion protein. The MS2-SLBP fusion protein activated the levels of translation of the Luc-MS2 and the Luc-SL mRNAs to the same extent. However, the MS2-SLBP protein gave different results with the mRNAs that ended in 8 As. Like the hSLBP, the MS2-SLBP did not activate translation of the Luc-SLA8 mRNA. However, the MS2-SLBP did activate translation of the Luc-MS2A8 mRNA significantly more than it supported translation of the Luc-SLA8 mRNA. This result suggests that the inactivation of the Luc-SLA8 mRNA requires that the adenosines be attached to the histone stem-loop and suggests that there is a direct interaction between the stem-loop, an SLBP, and the eight adenosines and any associated factor(s) required for translational repression.
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DISCUSSION |
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Translational regulation of histone mRNA A major component of the regulation of translation of histone mRNA during Xenopus oogenesis is the presence of xSLBP1 and xSLBP2, two different proteins that bind the 3' end of these mRNAs (33). xSLBP1 stimulates translation in vivo and in vitro of reporter mRNAs that end with the 3' end of histone mRNA (25) and likely is required for efficient translation of histone mRNAs in vivo. xSLBP2, which is oocyte specific, does not support translation of histone mRNA and probably is involved in translational repression of histone mRNA.
Oocyte histone mRNAs differ from other histone mRNAs in that there is an oligo(A) tail added to their 3' end. This tail is too short to bind a molecule of PABP (24). The oligo(A) tail is added to the histone mRNA early in oogenesis and is associated with the translationally inactive histone mRNA. In vitro both xSLBP1 and xSLBP2 bind to the oligoadenylated histone mRNAs with similar affinities, and these proteins do not readily exchange. However, in vivo it is likely that other factors bind to the oligoadenylated 3' end and it is possible that these factors interact with xSLBP2. Other than xSLBP2, the protein components of the stored histone mRNA are not known. When stage VI oocytes were matured in vitro, we did not observe degradation of exogenously expressed xSLBP2 whether or not it was bound to histone mRNP or detect removal of the oligo(A) tail from injected in vitro-synthesized histone mRNAs (unpublished results). These results suggest that the xSLBP2/histone mRNA complex must be stored in a specific compartment or particle in the oocyte, which allows degradation of xSLBP2 and removal of the oligo(A) tail after oocyte activation. Exogenous xSLBP2 and/or histone mRNA may not be sequestered properly in the oocyte for degradation and deadenylation to occur at maturation.
A second possible function of the oligo(A) tail is to stabilize the histone mRNA for the long-term storage during oogenesis. We have recently characterized a mammalian 3' exonuclease that specifically binds the 3' end of histone mRNA (6), and this exonuclease would not bind to the oligoadenylated form of histone mRNA.
Strikingly, the oligoadenylated histone mRNA is not efficiently translated in Xenopus oocytes (even in the presence of a large excess of xSLBP1). This is not a result of a lower affinity of the xSLBP1 for the oligoadenylated histone mRNA, since xSLBP1 binds well to the oligoadenylated stem-loop and is equally active in stimulating translation of wild-type histone mRNA and oligoadenylated histone mRNAs in a reticulocyte lysate. The minimal xSLBP1 protein required for translation activation (a deletion of the first 68 amino acids and of the C-terminal domain; see reference 25) was also inactive in translation of the oligoadenylated histone mRNAs (data not shown). This result strongly suggests that there are oocyte-specific factors that interact with the oligoadenylated histone mRNA, inhibiting its translation by preventing xSLBP1 from interacting with its target in the initiation machinery. Activation of histone mRNA translation when oocytes are treated with progesterone requires both the removal of the xSLBP2 from the stored histone mRNA and the removal of the oligo(A) tail. Both of these events occur in enucleated oocytes that have been treated with progesterone. It is likely that a result of progesterone treatment is activation of the pathway leading to both xSLBP2 removal from the histone mRNA and destruction of xSLBP2 (as well as removal of the oligo(A) tail from the histone mRNA). It is reasonable to suspect that these events are mediated by the same signal.
Translation regulation of other mRNAs Extensive progress has been made recently in understanding the mechanism of translational regulation of the c-mos mRNA required for initiation of oocyte maturation (26, 28) as well as the regulation of translation of cyclin B mRNA (3, 29). Translation of these polyadenylated mRNAs is regulated by alterations in the length of the poly(A) tail and by proteins that interact with the untranslated regions (4, 20). c-mos mRNA is one of the earliest mRNAs whose translation is altered in response to progesterone treatment (26). Activation of c-mos translation requires the Eg2 kinase which is activated by treatment with progesterone (19, 20). c-mos translation activation also requires the CPE located in the 3' untranslated region of c-mos mRNA. CPEB, the protein that binds the CPE, is phosphorylated upon progesterone stimulation. Before oocyte maturation CPEB is linked to eIF-4E, the cap-binding protein, via the maskin protein. Maskin prevents the assembly of the translation preinitiation complex by impeding the interaction between eIF-4E and eIF-4G. Phosphorylation of CPEB in response to progesterone stimulation causes the release of CPEB-bound maskin from eIF-4E and promotes the recruitment of CPSF to c-mos mRNA (27). CPSF then recruits a poly(A) polymerase, possibly a novel poly(A) polymerase (32), that lengthens the poly(A) tail. These changes in translation of polyadenylated mRNAs important for cell-cycle regulation are occurring at the same time as the changes in the histone mRNA.
A second consequence of treatment of oocytes with progesterone is the shortening of the poly(A) tail on some of the oocyte mRNAs, resulting in translation arrest (8). The shortening of the poly(A) tail requires the presence of PARN, a 3' exonuclease specific for poly(A) tails (14). It has been reported that shortening of the poly(A) tails on oocyte mRNAs requires the breakdown of the germinal vesicle, since it does not occur in enucleated oocytes treated with progesterone (30). The results shown in Fig. 2 suggest that the mechanism for the removal of the oligo(A) tail from histone mRNA may be distinct from the removal of the poly(A) tails from other mRNAs. Whether the same signal transduction pathway triggers these events after progesterone treatment is not known. It is noteworthy that these two deadenylation reactions have opposite effects: translation of maternal adenylated mRNAs is inhibited, while histone mRNA translation is activated. Whether the signaling pathway that results in activation of histone mRNA translation requires the Eg2 kinase (the critical regulator of translation activation of polyadenylated mRNAs) is not known.
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ACKNOWLEDGMENTS |
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We thank Zbig Dominski and Eric Wagner for critical review of the manuscript.
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FOOTNOTES |
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