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Agathe Devaux, and
Nancy Standart*
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom
Received 12 November 2004/ Returned for modification 13 December 2004/ Accepted 29 June 2005
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of RNA localization and translational control have been best characterized for Drosophila melanogaster, where, for example, the establishment of the anterior-posterior axis relies on the hierarchical localization and coordinate translational control of mRNAs encoding a number of key determinants, including those for Nanos and Oskar (reviewed in references 7, 24, and 43).
Characterization of the mechanisms of translational control that are utilized by a variety of different organisms has revealed that, most frequently, regulation occurs via the action of trans-acting factors binding to specific motifs in the 3'-untranslated regions (UTRs) of the regulated mRNAs (6, 29, 62). For example, repression of the oskar mRNA is dependent upon the binding of Bruno to Bruno response elements in the 3'-UTR (27), while translational control of nanos is effected by the trans-acting factor Smaug, which interacts with a 90-nucleotide translation control element found in the nanos 3'-UTR (9, 18, 50).
The Xenopus laevis Vg1 mRNA encodes a member of the transforming growth factor ß family implicated in mesoderm formation and the establishment of left-right asymmetry in the developing embryo (22, 58). Vg1 mRNA is localized to the vegetal cortex of the Xenopus oocyte during oogenesis (36). Localization begins during stage II of oogenesis when the mRNA becomes restricted to a wedge-shaped region within the vegetal hemisphere. By stage IV of oogenesis, Vg1 mRNA is confined to the vegetal cortex and it remains here until the oocytes meiotically mature after stage VI. The mRNA is not translated until stage IV when localization is completed (10, 53). We previously described a 250-nucleotide element in the Vg1 3'-UTR, the Vg1 translation element (VTE), which represses the translation of a reporter mRNA to the same extent as the full-length Vg1 3'-UTR (42) and which is encompassed by the larger translational control element described by Wilhelm et al. (60). The VTE is located downstream of, and does not overlap with, the previously described Vg1 localization element (VLE) (39). Translational repression by the VTE was observed in both stage III and stage VI oocytes since the injected RNAs are not localized under the conditions utilized. Further analysis of this element revealed the presence of AU-rich sequence domains at the 5' and 3' ends of the VTE, and deletion studies showed that these AU-rich sequences are required for the translational control (42). AU-rich elements (AREs) had previously been demonstrated to destabilize RNAs (5), and they were also shown to support translational repression in Xenopus oocytes. For example, the 3'-UTRs of cytokine mRNAs, which contain destabilizing AREs, repress the translation of reporter RNAs injected into Xenopus oocytes (28). Subsequently, additional RNAs have been shown to be translationally regulated by AREs in their native context (reviewed in reference 13). However, the mechanisms of repression have not been elucidated.
The ELAV family of RNA binding proteins, named after their founding member in Drosophila (embryonic lethal and abnormal vision) (4), is known to interact with AREs. Vertebrates possess four homologues, the human forms being named HuR (HuA), HuB (Hel-N1), HuC, and HuD. HuR is ubiquitously expressed and HuB is expressed in neurons, testes, and ovaries, while HuC and HuD are strictly neuronal (1). In Xenopus, the equivalent homologues are called ElrA, ElrB, ElrC, and ElrD and show a similar pattern of expression (19). The ELAV proteins were initially shown to bind to AREs in the 3'-UTRs of unstable RNAs, such as those encoding cytokines, and to have a stabilizing effect upon these RNAs (15, 45). More recently, these proteins have been implicated as playing a part in translational control, both in a stimulatory (2, 23, 32, 35) and in a repressive (30) manner.
Another family of ARE binding proteins that has been shown to be involved in translational control is the TIA-1 family, comprising TIA-1 and TIAR. These closely related proteins have a role in the general arrest of translation that occurs upon exposure to environmental stress, promoting the formation of stress granules in which untranslated mRNAs are sequestered. This behavior requires the general RNA binding activity of TIA-1/TIAR (26). In addition, TIA-1 and TIAR are capable of acting as translational silencers of specific mRNAs, such as tumor necrosis factor alpha (46) and cyclooxygenase-2 (12), through interactions with the AREs present in the 3'-UTRs of these mRNAs.
Here we describe that the major protein interacting with the VTE is the Xenopus homologue of HuB, ElrB. ElrB is expressed in Xenopus oocytes in a temporal pattern consistent with a role in the translational repression of the Vg1 mRNA. We report that disruption of ElrB binding does indeed abolish repression by the VTE of a reporter gene, while multiple copies of the ElrB binding site can support translational repression. In addition, injection of an antibody against ElrB into stage III oocytes results in the appearance of Vg1 protein. ElrB, therefore, appears to play an important role in translational control during Xenopus oogenesis.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To synthesize the pLUC-5xEBS and pGEM-5xEBS constructs, complementary oligonucleotides encoding five copies of the ElrB binding site were utilized: 5'-CTTATTTGTTTTTAAATTATTTGTTTTTAAATTATTTGTTTTTAAATTATTTGTTTTTAAATTATTTGTTTTTAAAG-3' and 5'-GATCCTTTAAAAACAAATAATTTAAAAACAAATAAT-TTAAAAACAAATAATTTAAAAACAAATAATTTAAAAACAAATAAGAGCT-3'. Annealing of these oligonucleotides generated overhanging ends that allowed cloning into the SacI and BamHI sites of pLUC and pGEM1.
In vitro transcription. pLUC constructs were linearized with PvuII to give polyadenylated transcripts with an (A)50 tail, while pRLUC and pGEM constructs were linearized with XbaI. Transcriptions were performed as described previously (42).
Preparation of oocyte lysates. Lysates were prepared in each case from ovaries that had been collagenased for 3 h and then washed in modified Barth's solution (MBS). Xenopus translation extract was prepared from selected stage VI oocytes as described previously (42). Total ovary extract for UV cross-linking was prepared in a similar manner (42), while staged oocyte lysates were prepared according to the method of Mowry (39a).
To obtain nuclear and cytoplasmic extracts, oocytes of the appropriate stage were enucleated in J buffer (10 mM HEPES, pH 7.2, 70 mM KCl, 1 mM MgCl2, 2 mM dithiothreitol, 5% glycerol, 1x Complete protease inhibitor [Roche]). The nuclei and cytoplasmic carcasses were homogenized by pipetting in a volume of buffer equivalent to 5 µl/nucleus or cytoplasm. The extracts were centrifuged for 5 min in a microfuge, and the supernatants were utilized for subsequent procedures.
UV-cross-linking and competition assays. UV-cross-linking reactions were carried out as described previously (57). One hundred thousand counts per minute or the appropriate molar equivalent of 32P-labeled RNA was utilized per reaction, together with 1 to 2 µl total ovary extract. For competition assays, 35 fmol 32P-labeled VTE was used for each reaction, together with a molar excess of 6.25-, 12.5-, 25-, 50-, and 100-fold cold competitor, in the presence of 1 µg rRNA and 50 µg heparin. The competitor was preincubated with the lysate for 10 min before addition of the probe.
Band shift assays. Reactions were set up as for UV cross-linking, with 10 fmol 32P-labeled RNA and 2 µl total ovary extract, and incubated for 15 min at room temperature. Where appropriate, antibody was added to the reaction and a further 5-min incubation was performed. After the addition of 2 µl 30% glycerol, samples were analyzed by electrophoresis through a 5% nondenaturing gel and exposed to Fuji Super RX X-ray film.
Translation of luciferase RNAs in vitro and in vivo. Injection of luciferase reporter RNAs into stage VI oocytes and subsequent assay of the resulting luciferase activity were performed as described previously (42), using five pools of five oocytes for each RNA. In general, firefly luciferase activity was expressed as a ratio relative to Renilla luciferase activity. The average ratio was calculated from the five samples, discarding the highest and lowest values obtained. The results are generally shown as a percentage of the translation of the LUC-1200 control. Each experiment was repeated at least three times with oocytes from different frogs, and a representative experiment is shown.
In vitro translations were also performed in Xenopus translation extract as described previously (42).
Measurement of RNA stability. Reactions were prepared in the same manner as for in vitro translation assays, with the exception that 32P-labeled RNAs were utilized. Samples were incubated at 23°C, removed at appropriate times, frozen on dry ice, and then analyzed as described previously (42).
Immunodepletion of translation extract. Protein A- or protein G-Sepharose was preincubated with total ovary extract for 2 h on ice and then washed with EB100 (50 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2). Protein A-Sepharose was then incubated with anti-HuR (Santa Cruz Biotechnology, Inc.) and protein G-Sepharose with anti-TIAR (Santa Cruz Biotechnology, Inc.) antibodies. After being washed further with EB100, the resins were added to translation extract and incubated for 4 h on ice with periodic agitation. After gentle centrifugation to remove the resin, the extract was recovered. The beads were washed in EB100 and resuspended in protein sample buffer for analysis by Western blotting.
Immunoprecipitation and Western blotting. For immunoprecipitation of UV-cross-linked proteins, 15 UV-cross-linking reactions were performed and pooled in a 20-fold excess of NET buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 0.25% gelatin, 0.02% azide). The pooled reactions were incubated with protein A-Sepharose beads preincubated with the appropriate antibody. After incubation at 4°C for 2 h, the beads were washed with NET buffer and bound proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Immunoprecipitation of non-cross-linked lysate was performed in a similar fashion by using lysate prepared by homogenization of appropriately staged oocytes in NET buffer, followed by two rounds of centrifugation for 5 min in a microfuge to remove yolk protein.
Immunoprecipitation from labeled oocytes was performed in the following manner. Prior to injections into oocytes, anti-HuR and anti-His (QIAGEN) antibodies were dialyzed against phosphate-buffered saline to remove azide and concentrated using Slide-A-Lyzer concentrating solution (Pierce). Antibody concentrations were confirmed by SDS-PAGE and Coomassie blue staining. Stage III oocytes were injected with 0.3 ng antibody or an equal volume of buffer. The injected oocytes were incubated in MBS for 2 h prior to the addition of an appropriate volume of 0.02 MBq/µl [35S]methionine in MBS and further incubation overnight. Lysate was prepared from 50 labeled oocytes by homogenization in NET buffer, followed by two rounds of centrifugation for 5 min to remove yolk protein. The resulting lysate was then incubated for 2 h at 4°C with protein A-Sepharose beads preincubated with a monoclonal anti-Vg1 antibody (clone D5; kind gift of Dan Kessler) (53).
For Western blotting, SDS-PAGE was followed by semidry transfer of the proteins to an Immobilon-P transfer membrane (Millipore). After overnight blocking in 2% milk, incubations with the primary and secondary antibodies were both performed for 1 h at room temperature. Anti-HuR and anti-TIAR antibodies were both used at a dilution of 1 in 2,000. Monoclonal anti-Vg1 antibody was used at a dilution of 1 in 1,000.
Antipeptide antibodies against ElrA and ElrB were raised in rabbits by Eurogentec, using peptides selected and synthesized by the company (A. Devaux, L. J. Colegrove-Otero, and N. Standart, unpublished data).
Gel filtration. A Superose 6 HR 10/30 column (Amersham Biosciences) was equilibrated with eluent buffer (34.2 mM Na2HPO4, 15.8 mM NaH2PO4, 150 NaCl). To calibrate the column, a protein standard sample containing thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), ovalbumin (43 kDa), and chymotrypsinogen (25 kDa) was utilized and a calibration curve was plotted. Two hundred microliters of stage III extract was loaded onto the column, and 0.3-ml fractions were collected. The proteins in the fractions were analyzed by SDS-PAGE and Western blotting. RNase-treated extract was incubated with 167 µg/ml RNase A for 10 min at 25°C before loading on the column.
RT-PCR of immunoprecipitated RNA. Immunoprecipitated samples were incubated with 200 µg/ml proteinase K (Sigma) for 30 min at 50°C and then extracted twice with phenol-chloroform. The resulting RNA was precipitated, and one-half was used in a reverse transcription (RT) reaction containing 0.5 µg random primers (Promega) and 200 U Superscript II reverse transcriptase (Invitrogen). Twenty-four cycles of PCR were then performed using primers OLO121 and OLO123, described previously (42).
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RESULTS |
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Expression of ElrB during oogenesis correlates with the translational repression of Vg1 mRNA. To examine the patterns of expression of ElrA and ElrB and TIA-1 and TIAR during oogenesis, lysate samples were prepared from oocytes in each of the six individual stages of oogenesis. Samples corresponding to two oocyte equivalents for each stage were electrophoresed and analyzed by Western blotting with the anti-HuR and anti-TIAR antibodies. Anti-TIAR detects both Xenopus TIAR and TIA-1 (data not shown). Figure 3A shows that ElrA and ElrB have significantly different expression profiles. While the level of ElrA per oocyte gradually increases as oogenesis progresses, ElrB is predominantly expressed in the early stages of oogenesis and its levels then decline in the later stages. TIAR shows a pattern similar to that of ElrA, increasing during oogenesis. Western blotting of the staged oocyte lysates with anti-Vg1 antibody confirms that the Vg1 protein is only expressed in the later stages of oogenesis in a pattern complementary to ElrB (10, 53). The helicase Xp54 is a loading control known to be present at equal levels throughout oogenesis (31). The amounts of ElrA and ElrB in stage III and stage VI oocytes were estimated by comparative Western blotting with recombinant ElrA and ElrB (data not shown). It was found that the anti-HuR antibody has a significantly higher affinity for ElrA than for ElrB, which is unsurprising as the antibody is directed against the human ElrA homologue (16). This results in an underrepresentation, by Western blotting, of the amount of ElrB present in the later stages of oogenesis. ElrB is present at concentrations of approximately 25 ng/oocyte in stage III and 5 ng/oocyte in stage VI, while ElrA is found at around 2.5 ng/oocyte during stage III and 7.5 ng/oocyte during stage VI.
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In order to determine the distribution of the proteins in complexes in the cell, gel filtration of a stage III lysate, either pretreated or not with RNase A, was performed using a Superose 6 HR 10/30 column and fractions were analyzed by Western blotting. Use of the anti-HuR antibody revealed that ElrA and ElrB are only detectable in very large complexes, peaking at approximately 2,000 kDa (Fig. 3C). The association of the proteins with these complexes is completely disrupted by the addition of RNase A. In striking contrast, TIA-1/TIAR show a different distribution. The majority of the protein exists as a monomer or in small complexes, although a very minor proportion is involved in a large complex with a peak greater than 1,500 kDa. The large complexes are not sensitive to RNase, implying that TIA-1/TIAR and ElrA/ElrB are not found within the same RNA-dependent complexes in Xenopus oocytes. The distribution of these proteins is very similar to that observed by Lal et al. on examining the centrifugation of cytoplasmic material from HeLa cells through sucrose gradients (33). While HuR migrated with the polysomal fractions, TIAR was present at the top of the gradient in the uncomplexed fractions.
These results show that ElrB, in particular, is present at the correct time and location to participate in the translational control of the Vg1 mRNA. Vg1 is repressed during stages I to III of oogenesis, when ElrB is present at its greatest concentration. In addition, ElrA and ElrB are purely cytoplasmic in Xenopus oocytes, making them available to repress translation, even though HuR, for example, has been shown to shuttle between the nucleus and cytoplasm and is predominantly nuclear in HeLa and mouse L929 cells (15). Both proteins are present in large complexes that could potentially be involved in translational control.
TIA-1 and TIAR are not involved in the translational repression of Vg1 mRNA. We had previously shown that translational repression by the VTE could be reproduced in Xenopus in vitro translation extract (42). To determine whether the identified cross-linking proteins are functionally involved in the repression, immunodepletion of the translation extract was attempted. Translation extract was incubated with either protein A-Sepharose beads preincubated with anti-HuR antibody or protein G-Sepharose beads preincubated with anti-TIAR antibody. Mock depletions were performed with either protein A-Sepharose alone or protein G-Sepharose alone. Samples of the bound and supernatant fractions were taken and analyzed by Western blotting. Figure 4A shows that the vast majority of ElrA and ElrB present, when incubated with the anti-HuR-coated beads, remains in the supernatant. It was, however, possible to immunodeplete TIA-1 and TIAR from the extract (Fig. 4B). Further attempts at immunodepletion of ElrA and ElrB were performed using protein G-coated magnetic beads (Dynal), higher concentrations of anti-HuR, and multiple rounds of depletion, without success (data not shown). It is likely that the presence of ElrA and ElrB in large complexes, as shown by gel filtration (Fig. 3C), results in the inaccessibility of these proteins to the antibody and the inability to immunodeplete.
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To determine whether the proteins interact with Vg1 mRNA in vivo, RNA was recovered from immunoprecipitations of stage II oocyte lysates performed using either the antipeptide antibodies against ElrA and ElrB or anti-HuR or anti-TIAR and subjected to semiquantitative RT-PCR with primers specific for the Vg1 3'-UTR. Vg1 mRNA can be detected in immunoprecipitations using all three anti-ELAV antibodies, although anti-ElrA precipitates a lesser proportion of the RNA (Fig. 4D). No Vg1 mRNA is detectable in immunoprecipitations performed with the preimmune sera. Vg1 mRNA is also undetectable in the anti-TIAR immunoprecipitation both with stage II oocytes as shown and with stage VI oocytes (data not shown), which contain a higher level of TIA-1/TIAR. Therefore, the ELAV proteins do interact with Vg1 mRNA in Xenopus oocytes, whereas TIA-1 and TIAR, consistent with the immunodepletion data, do not show an interaction in vivo.
Mutation of a putative ElrB binding site within the VTE substantially reduces ElrB binding. Gao et al. (17) described the characterization of a consensus sequence for binding by the human ElrB homologue Hel-N1. This comprises the sequence A/GU/AUUUA/GUUUU/AA/G. Examination of the 5' AU-rich portion of the VTE revealed the presence of a similar sequence located between bases 1933 and 1943 of the Vg1 cDNA sequence (Fig. 5A), while no related sequence is found elsewhere in the Vg1 3'-UTR. Comparison of the 3'-UTR sequences of Xenopus tropicalis and Xenopus borealis Vg1 mRNAs revealed that the sequence is conserved between these related species, implying a functional significance (Fig. 5A).
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Reduction in ElrB binding correlates with relief of translational repression. To examine the effect of the reduction in ElrB binding on the ability of the VTE to repress translation, LUC-1200, LUC-VU, LUC-VU1, LUC-VTE, LUC-VUmut, and LUC-VTEmut RNAs, transcribed in vitro, were injected into stage VI oocytes and the resulting luciferase activities were assayed. The details of these RNAs are depicted in Fig. 1. As observed previously (42), LUC-VU and LUC-VTE are translationally repressed relative to the LUC-1200 and LUC-VU1 controls (Fig. 6A). With LUC-VUmut and LUC-VTEmut, in which only the ElrB consensus binding site has been mutated, repression has been completely relieved. The same results were obtained after injection into stage III oocytes (data not shown). Figure 6B shows that the same results are obtained with LUC-VU and LUC-VUmut in the in vitro extract.
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Multiple copies of the ElrB binding site support both ElrB binding and translational repression. Mutation of the consensus ElrB binding site destroyed the repression of translation by the VTE. To test if recruitment of ElrB by this sequence supports translational repression, multiple copies of the ElrB binding motif were cloned downstream of the luciferase coding region or into pGEM to allow transcription for UV-cross-linking competition studies.
The ability of the five copies of the binding motif to bind ElrB was analyzed by UV cross-linking. In Fig. 7A, cross-linking of p36, p42, and p45 is observed with the RNA consisting solely of five copies of the ElrB binding site (5xEBS). In addition, these proteins are all immunoprecipitated by the anti-HuR antibody, confirming their identities as ElrA and ElrB. It can be noted that the intensity of the p45 cross-linking band is stronger for the VTE RNA than for the 5xEBS sequence, whereas the intensities of the immunoprecipitated 45-kDa proteins are similar. This is due to the unidentified 45-kDa protein, which interacts with the VTE sequence and remains in the supernatant of the immunoprecipitation reaction (Fig. 2A) but is not bound by the 5xEBS sequence. The VU1 sequence, which does not repress translation, shows no interaction with ElrA or ElrB, while as previously noted, VTEmut binds only the unidentified 45-kDa protein. The ability of this sequence to repress translation was then examined both by injection into stage VI oocytes (Fig. 7B) and by translation in an in vitro extract (Fig. 7C). In oocytes, the five copies of the binding motif repress translation even more effectively than either the full-length Vg1 3'-UTR or the VTE. In vitro, a similar level of repression is observed with all three sequences. The effect of 5xEBS on the amount of protein produced occurs at the level of translation rather than RNA stability, as the 32P-labeled LUC-1200 and LUC-5xEBS RNAs undergo only minimal degradation during the time course of the experiment (Fig. 7D). Therefore, recruitment of ElrB by multiple copies of the ElrB binding motif allows translational repression of the reporter RNA.
To confirm that the factors responsible for repression by the VTE are the same as those binding to the 5xEBS motif, a competition experiment was performed in the context of the translation extract. The extract was preincubated with a 10-, 50- or 100-fold excess of 5xEBS RNA over the reporter firefly luciferase RNAs. After the addition of the reporter RNAs, in vitro translation was performed in the usual manner. In Fig. 7E, it can be seen that addition of the 5xEBS competitor relieves repression of the LUC-VTE mRNA relative to that of the controls. Relief of repression of the LUC-VU mRNA does occur but not to the same extent as with the shorter mRNA. This may imply that there are additional factors involved in the translational control, which cannot be competed off by the 5xEBS motif and which interact more strongly in the context of the full-length Vg1 3'-UTR than with the VTE alone.
Injection of anti-HuR antibody into stage III oocytes enhances the translation of Vg1 mRNA. Translational repression of reporter RNAs containing the VTE appears to be dependent on their ability to bind ElrB, while copies of the ElrB binding site alone will repress translation. To determine whether ElrB directly affects the translational control of the Vg1 mRNA in vivo, anti-HuR antibody was injected into stage III oocytes and the oocytes were incubated in the presence of [35S]methionine to label newly synthesized proteins. Stage III oocytes were utilized, as the endogenous Vg1 mRNA is still largely repressed at this stage (10, 53) (Fig. 3A). The injection of antibodies into Xenopus oocytes to examine the function of RNA binding proteins involved in translational control has been previously described (21, 52). Lysates prepared from the labeled oocytes were then immunoprecipitated using anti-Vg1 antibody. Figure 8A shows that in either uninjected oocytes or oocytes injected with buffer or a control mouse monoclonal antibody, the levels of Vg1 expression are virtually undetectable, as expected for this repressed protein. However, after injection with anti-HuR antibody, labeled Vg1 protein is detectable, implying that the anti-HuR antibody has relieved the translational repression. The constant band found in all four anti-Vg1-containing lanes is unrelated to Vg1, as it is not recognized on a Western blot (data not shown) and acts as a loading control. The antibody does not enhance translation in general, as the overall incorporation of [35S]methionine is not increased in the anti-HuR-injected oocytes (Fig. 8B). Therefore, disrupting the activity of ElrB in vivo results in the relief of Vg1 translational repression.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Repression of the Vg1 mRNA, which is localized to the vegetal cortex of the Xenopus oocyte, is mediated by an element in the 3'-UTR, the VTE (42). ElrB is the major specific protein interacting with the VTE, as shown by UV cross-linking. Repression by the VTE correlates with its ability to bind ElrB, as the mutation of a sequence fitting the Hel-N1 (human ElrB homologue) consensus binding site (17) abolishes both ElrB binding and translational repression. This was further confirmed by the observation that multiple copies of this binding motif are sufficient both to bind ElrB and to repress translation. The involvement of ElrB in the control of Vg1 translation in vivo has been shown by the ability of anti-HuR antibody to alleviate the translational repression of the endogenous Vg1 RNA.
The TIA-1 proteins are the best-established ARE binding proteins that act as translational silencers (12, 46). It is possible to detect an interaction of TIA-1 and TIAR with the VTE, but they are not represented in the major UV-cross-linking proteins. Immunodepletion of Xenopus translation extract confirmed that these proteins are not required for repression by the VTE, highlighting the complexity of control through AU-rich elements.
The conserved motif present in the 5' region of the VTE appears to bind ElrA and ElrB, as mutation of this sequence affects their ability to cross-link to the VTE and multiple copies of the sequence interact with both proteins. We cannot rule out the possibility that ElrA also plays a part in the translational repression. However, given the pattern of expression of these two proteins, ElrB is likely to have the major role as it is expressed at significantly higher levels during the period when the Vg1 mRNA is repressed. We have shown that ElrA and ElrB display distinct differences. For example, while ElrB undergoes RNA-mediated self-oligomerization, ElrA is unable to form such higher-order structures, perhaps giving rise to key functional differences between the proteins (Devaux, Colegrove-Otero, and Standart, unpublished data). At present, we have no information as to the relative location of these proteins within the oocyte, with the exception that they are cytoplasmic. It will be of interest to determine whether ElrA and ElrB are localized to different regions of the oocyte and whether this localization changes during oogenesis. For example, given the differing expression patterns of ElrA and ElrB during oogenesis, there is a possibility that while ElrB acts to repress Vg1 translation, ElrA behaves as an activator and is perhaps localized to the vegetal cortex.
Our observations concerning the ability of ElrB to repress translation correlate with those of Kullmann et al. (30). They identified an internal ribosomal entry site in the 5'-UTR of the p27 mRNA, which encodes a Cdk inhibitory protein, and reported that the ELAV homologues HuR and HuD are both capable of binding to the p27 5'-UTR. Overexpression of these proteins in HeLa cells resulted in a decrease in internal ribosomal entry site-dependent translation, while knockdown of the proteins by RNA interference dramatically induced p27 synthesis. Antic et al. (2) and Jain et al. (23), however, have reported enhancement of translation of their mRNAs of interest due to overexpression of Hel-N1. Antic et al. (2) showed that overexpression of Hel-N1 in human teratocarcinoma cells resulted in neurite formation together with increased expression of NF-M protein, a neuronal marker. The levels of NF-M mRNA remained unaltered, but a substantial recruitment of the RNA to the heavy polysomal fractions was observed, presumably due to an increase in translation initiation. Similar results were obtained upon overexpression of Hel-N1 in 3T3-L1 preadipocytes (23). This resulted in a 10-fold increase in the expression of the GLUT1 glucose transporter, usually expressed in differentiated adipocytes. Again, this increase in expression correlated with a shift of the GLUT1 mRNA to the heavy polysomal fractions. In the same fashion, Mazam-Mamczarz et al. (35) and Lal et al. (32) reported that HuR stimulates the translation of both p53 and the anti-apoptotic factor prothymosin 
mRNAs. The synthesis of both of these proteins is increased in cells overexpressing HuR and decreased in cells where levels of HuR have been knocked down by RNA interference.
While these results may appear contradictory, RNA binding proteins that can repress or activate translation under different conditions have been described previously. The cytoplasmic polyadenylation binding protein (CPEB) interacts with cytoplasmic polyadenylation elements in the 3'-UTRs of mRNAs regulated by cytoplasmic polyadenylation during early development (21). These mRNAs are translationally activated by the lengthening of their poly(A) tails upon the maturation of oocytes to eggs (reviewed in reference 49). CPEB plays a key part in this translational activation (21, 52). Prior to maturation, however, CPEB acts as a repressor of translation (11, 38, 54). The switch triggering the conversion of CPEB from a repressor to an activator appears to be phosphorylation of the protein and a resulting alteration in its binding partners (37, 48, 55). Therefore, it is possible to envisage that ElrB could act, in an analogous manner, as either a translational repressor or activator depending on the status of the cell. Indeed, in the case of a localized RNA such as Vg1, ElrB may first act as a repressor during localization and then switch to activate translation, perhaps by interacting with a prelocalized factor at the target site.
Future studies will need to address the mechanism by which ElrB, and potentially other members of the family, can repress translation. The Y-box protein YB-1 has been shown both to act as a translational repressor and to enhance RNA stability (14). It functions by disrupting the interaction of the cap binding protein eukaryotic initiation factor 4E (eIF4E) with the cap structure found at the 5' end of most eukaryotic mRNAs (14). eIF4E binding to the cap usually promotes translation initiation by recruiting the initiation factor eIF4G and subsequently the 40S ribosome (47). As degradation of most mRNAs is triggered by decapping (44), binding of YB-1 at or near the cap structure can affect both translation and RNA degradation. We have shown that repression by the VTE is dependent on the presence of a cap but not of a poly(A) tail (42). Therefore, it is possible that ElrB could act in a similar fashion to YB-1 to both stabilize and translationally repress its target mRNAs. Other factors that bind to the 3'-UTRs of translationally regulated mRNAs appear to act via intermediary proteins. For example, CPEB interacts with maskin, which in turn binds eIF4E to prevent it from interacting with eIF4G, thus disrupting translation initiation (51). Cup, which has been shown to bind to the translational repressors Bruno and Smaug, also disrupts the eIF4E-eIF4G interaction by binding to the eIF4G binding site on eIF4E (40, 41, 61). Analysis of the ElrB binding partners will be necessary to determine if it is recruiting analogous proteins to the RNA.
The ELAV proteins were originally described as having a stabilizing effect on unstable RNAs containing AREs (15, 23, 45). During oogenesis and early embryogenesis, AREs do promote the deadenylation of ARE-containing mRNAs but do not have a destabilizing effect (56). Whereas under most conditions, deadenylation of mRNAs results in decapping and subsequent degradation (44), in oocytes, the decapping activity is absent (63) and deadenylation results in translational repression (49, 59) rather than RNA decay. This allows the study of additional functions of proteins such as ElrB in isolation from their involvement in RNA stability. A recent study has highlighted the global link between RNA stabilization and translational repression. Kawai et al. (25) used cDNA arrays to monitor changes in RNA stability and translational status of mRNAs in HeLa cells subjected to endoplasmic reticulum stress. They observed a significant enrichment of translationally repressed transcripts among those mRNAs, which were stabilized upon endoplasmic reticulum stress. While these data are obviously related to a specific set of conditions, if this observation proves to be more generally applicable, the ELAV proteins could potentially provide a key link between these two phenomena.
In the future, it will be interesting to determine whether other RNAs which are localized or regulated during early development employ a similar mechanism of translational repression and whether such control by the ELAV proteins is a more widely utilized strategy.
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ACKNOWLEDGMENTS |
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This work was supported by a Wellcome Trust project grant.
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FOOTNOTES |
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REFERENCES |
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Authors contributed equally.