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Molecular and Cellular Biology, October 2005, p. 9028-9039, Vol. 25, No. 20
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.20.9028-9039.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Xenopus ELAV Protein ElrB Represses Vg1 mRNA Translation during Oogenesis

Lucy J. Colegrove-Otero,{dagger} Agathe Devaux,{dagger} 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
 
Xenopus laevis Vg1 mRNA undergoes both localization and translational control during oogenesis. We previously characterized a 250-nucleotide AU-rich element, the Vg1 translation element (VTE), in the 3'-untranslated region (UTR) of this mRNA that is responsible for the translational repression. UV-cross-linking and immunoprecipitation experiments, described here, revealed that the known AU-rich element binding proteins, ElrA and ElrB, and TIA-1 and TIAR interact with the VTE. The levels of these proteins during oogenesis are most consistent with a possible role for ElrB in the translational control of Vg1 mRNA, and ElrB, in contrast to TIA-1 and TIAR, is present in large RNP complexes. Immunodepletion of TIA-1 and TIAR from Xenopus translation extract confirmed that these proteins are not involved in the translational repression. Mutagenesis of a potential ElrB binding site destroyed the ability of the VTE to bind ElrB and also abolished translational repression. Moreover, multiple copies of the consensus motif both bind ElrB and support translational control. Therefore, there is a direct correlation between ElrB binding and translational repression by the Vg1 3'-UTR. In agreement with the reporter data, injection of a monoclonal antibody against ElrB into Xenopus oocytes resulted in the production of Vg1 protein, arguing for a role for the ELAV proteins in the translational repression of Vg1 mRNA during early oogenesis.


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INTRODUCTION
 
Posttranscriptional control is vital during early development to allow both spatial and temporal regulation of gene expression. Maternal mRNAs accumulate in storage particles during oogenesis, and individual transcripts are activated at specific points during the developmental process to allow the appropriate synthesis of proteins essential for the correct development of the embryo. The regulation of posttranscriptional processes also enables the spatial regulation of gene expression. Localization of an mRNA to a particular region of the egg or developing embryo allows the restriction of the resulting protein product, engendering asymmetry. The results of mRNA localization are often accentuated by translational control so that the regulated RNA is not translated unless correctly localized.

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
 
Preparation of plasmid constructs. Synthesis of the pLUC-1200, pLUC-VU, pLUC-VU1, pLUC-VTE, pRLUC, and pGEM-VTE constructs was described previously (42). The pLUC-VUmut, pLUC-VTEmut, and pGEM-VTEmut constructs were synthesized by site-directed mutagenesis of pLUC-VU, pLUC-VTE, and pGEM-VTE, respectively. Two complementary oligonucleotides consisting of the sequences 5'-GTGGTATTTAAATAATTCTACGTAGCTAAAAAATATATTTTTAGGC-3' and 5'-GCCTAAAAATATATTTTTTAGCTACGTAGAATTATTTAAATACCAC-3' were utilized for site-directed mutagenesis by the QuikChange site-directed mutagenesis protocol (Stratagene). The mutagenic oligonucleotides introduced a SnaBI site to allow screening for mutated constructs.

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
 
The ELAV proteins ElrA and ElrB cross-link to the VTE. We reported previously that the VTE (depicted in Fig. 1) interacts specifically with four different proteins, p36, p42, p45, and p60, as shown by UV-cross-linking analysis (42). These proteins did not interact with the VLE or the first third of the Vg1 3'-UTR (VU1 [Fig. 1]), which does not repress translation. In addition, their binding depended on the presence of the AU-rich sequences in the 5' and 3' portions of the VTE that were required for translational repression (42), suggesting that these proteins are functionally relevant. Wilhelm et al. similarly saw two proteins of 38 and 45 kDa interacting specifically with the translational control element (60). A number of proteins, such as TIA-1/TIAR (20), AUF1 (34), and the ELAV proteins (15, 45), had previously been characterized as binding to AREs. Two of the Xenopus ELAV homologues, ElrA and ElrB, are expressed in ovary and are of appropriate size, 36 and 45 kDa, to correspond to two of the cross-linking proteins (19). TIA-1 and TIAR, two closely related proteins, both with isoforms of 43 and 50 kDa, were also of interest as they had previously been shown to play a part in the translational repression of the tumor necrosis factor alpha (46) and COX-2 mRNAs (12).



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  		FIG. 1. Depiction of the constructs utilized. The inserts shown were cloned into the SacI and BamHI sites of the firefly luciferase vector (pLUC) and pGEM1 to give the appropriate constructs. The –1200 insert consisted of a 1.2-kb fragment derived from the vector pSP64. The other inserts shown were derived from the Vg1 3'-UTR, and their relative positions within the 3'-UTR are depicted. The asterisk shows the position of the ElrB consensus binding motif that is mutated in the VUmut and VTEmut constructs.

To determine whether ElrA and ElrB do interact with the VTE, immunoprecipitation of UV-cross-linking reactions with total ovary extract and 32P-labeled VTE RNA was performed using a monoclonal antibody against the human homologue of ElrA, HuR (16). It can be seen, by autoradiography, that immunoprecipitation with this antibody results in the precipitation of the three specifically interacting proteins, p36, p42, and p45, but not of the 69- and 56/58-kDa proteins (Fig. 2A, left panel). The 69- and 56/58-kDa proteins had previously been identified as Vg1RBP and FRGY2, respectively, and also interact with the VLE (42). Labeled VLE RNA was used as a control, and as expected, no material cross-linking to this sequence is immunoprecipitated by the anti-HuR antibody. To determine which of the immunoprecipitated proteins interact directly with the antibody, the corresponding samples were subjected to Western blotting with the anti-HuR antibody. The right panel in Fig. 2A shows that both p36 and p45 are recognized directly by the antibody. As the ELAV homologues are highly conserved, it is likely that the anti-HuR antibody interacts with both the orthologue, ElrA, and the paralogue, ElrB. This was confirmed by the use of antipeptide antibodies directed specifically against ElrA and ElrB (Fig. 2B). Immunoprecipitations were performed from oocyte lysate using anti-HuR or preimmune or immune sera from rabbits injected with peptides specific to ElrA or ElrB. These samples were then subjected to Western blotting with anti-HuR. While anti-HuR immunoprecipitates two proteins recognized by the same antibody upon blotting, anti-ElrB precipitates only the larger protein of 45 kDa and anti-ElrA precipitates only the smaller protein of 36 kDa. These results confirm that the proteins of 36 and 45 kDa that interact with the VTE correspond to the Xenopus ELAV proteins ElrA and ElrB, respectively. Further Western analysis revealed that p42 corresponds to a degradation product of ElrB (data not shown). The 45-kDa protein remaining in the VTE supernatant is not recognized by the anti-HuR antibody, implying that a second uncharacterized 45-kDa protein binds to the VTE.



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  		FIG. 2. ElrA and ElrB and TIA-1 and TIAR interact with the VTE. (A) The left panel shows immunoprecipitation, performed using an antibody against HuR, of UV-cross-linking reactions containing 32P-labeled VTE probe or VLE probe as a control. The proteins were detected by autoradiography. The three lanes for each RNA show the original UV-cross-linking reaction (UV), the immunoprecipitated sample (IP), and the proteins remaining in the supernatant (SN). The proteins FRGY2 and Vg1RBP, previously identified by immunoprecipitation (42), are shown in the UV sample. Molecular size markers are shown in kilodaltons. The right panel shows a Western blot of the VTE samples with anti-HuR. (B) Western analysis with anti-HuR ({alpha}-HuR) of immunoprecipitations from stage II lysate, performed using antipeptide antibodies directed specifically against ElrB ({alpha}-ElrB) or ElrA ({alpha}-ElrA). Lanes labeled {alpha}-ElrB PI and {alpha}-ElrA PI show immunoprecipitations performed using the relevant preimmune sera. IgG, immunoglobulin G. (C) Immunoprecipitation of UV-cross-linking reactions with labeled VTE and VLE, performed using antibodies directed against TIA-1 ({alpha}-TIA-1) and TIAR ({alpha}-TIAR). The immunoprecipitated proteins (IP) and proteins remaining in the supernatant (SN) are shown in each case. Molecular size markers are shown in kilodaltons.

To detect whether TIA-1 and TIAR are capable of interacting with the VTE, further immunoprecipitation of the UV-cross-linking reactions was performed using polyclonal antibodies against TIA-1 and TIAR. Figure 2C shows that it is possible to detect immunoprecipitated protein cross-linked to the VTE, but not to the VLE, with both anti-TIA-1 and anti-TIAR antibodies. However, as these proteins are not readily visible in the initial UV-cross-linking reaction, they appear to represent weakly interacting proteins or interacting proteins that are low in abundance.

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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  		FIG. 3. Characterization of ElrA, ElrB, TIA-1, and TIAR from Xenopus oocytes. (A) Western analysis of lysates prepared with oocytes from the six stages of oogenesis (I through VI shown at top), performed using anti-HuR ({alpha}-HuR), anti-TIAR ({alpha}-TIAR), and anti-Vg1 ({alpha}-Vg1) antibodies and anti-Xp54 ({alpha}-Xp54) antibody as a control. (B) Western analysis of nuclear (N) and cytoplasmic (C) extracts, performed using anti-HuR and anti-TIAR, with anti-PARN ({alpha}-PARN) as a control for the integrity of the nuclear and cytoplasmic fractions. (C) Western analysis of fractions of stage III extract obtained from a Superose 6 HR 10/30 gel filtration column. Gel filtration was also performed with extract which had been pretreated with RNase A. Size estimations were obtained from a calibration curve obtained using thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa). Lanes T show samples of nonfiltrated extract.

The distribution of the proteins between the cytoplasm and nucleus was examined using cytoplasmic and nuclear extracts derived from stage III oocytes. Western analysis of these extracts with the anti-HuR antibody revealed that ElrA and ElrB are cytoplasmic (Fig. 3B, upper panel). The same distribution is seen in stage VI oocytes (data not shown). TIA-1/TIAR were also found to be present only in the cytoplasm (Fig. 3B, middle panel). An antibody against the nuclear deadenylase PARN (8) was used to confirm the integrity of the nuclear and cytoplasmic fractions.

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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  		FIG. 4. Immunodepletion of Xenopus translation extract. (A) Western analysis of mock-depleted and anti-HuR-depleted translation extract, using anti-HuR as the primary antibody. (B) Western analysis with anti-TIAR of mock-depleted and anti-TIAR-depleted translation extract. (C) Three different luciferase reporter RNAs (LUC-1200, LUC-VU, and LUC-VU1) were assayed in the mock-depleted and anti-TIAR-depleted translation extracts. (D) RT-PCR, of total RNA extracted from immunoprecipitations from stage II oocytes carried out using the indicated antibodies, was performed with primers specific to the Vg1 3'-UTR. The expected size of the fragment is 420 bp. Lanes M contain molecular size markers. Abbreviations: SN, proteins remaining in the supernatant; Prot. A-Seph., protein A-Sepharose alone; Prot. A-Seph. + {alpha}-HuR, protein A-Sepharose beads preincubated with anti-HuR antibody; Prot. G-Seph., protein G-Sepharose alone; Prot. G-Seph. + {alpha}-TIAR, protein G-Sepharose beads preincubated with anti-TIAR antibody; IgG, immunoglobulin G; Ab, antibody; PI, immunoprecipitations using the relevant preimmune sera; Temp., template; RT, reverse transcriptase; Fluc, firefly luciferase; Rluc, Renilla luciferase.

The efficiency of translation of the in vitro-transcribed reporter RNAs, LUC-1200, LUC-VU, and LUC-VU1, in the TIA-1/TIAR depleted extract was then examined. These RNAs were described previously (42) and are depicted in Fig. 1. LUC-VU comprises the firefly luciferase open reading frame followed by the full-length Vg1 3'-UTR, while LUC-1200 is a control RNA consisting of the firefly luciferase open reading frame followed by a fragment of the pSP64 plasmid equal in length to the Vg1 3'-UTR (1.2 kb). LUC-VU1 is a further control that contains the first third of the Vg1 3'-UTR, previously shown not to repress translation (42). The RNAs were transcribed in vitro and trace labeled with 35S-UTP to allow quantitation. The ability of the RNAs to be translated to the same extent was confirmed by translation in rabbit reticulocyte lysate (data not shown), as we had previously described that the Vg1 3'-UTR has no repressive effect in this lysate (42). It can be seen, in Fig. 4C, that immunodepletion of TIA-1 and TIAR from the extract has no effect on the repression of luciferase translation by the Vg1 3'-UTR, comparing LUC-VU to the LUC-1200 and LUC-VU1 controls. This correlates with the observation that TIA-1/TIAR homologues, but no ELAV homologues, can be detected in rabbit reticulocyte lysate (data not shown), in which repression by the VTE is not observed (42). Therefore, TIA-1/TIAR are not involved in the VTE-dependent repression in vitro.

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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  		FIG. 5. Mutation of a putative ElrB binding site markedly reduces ElrB binding to the VTE. (A) An alignment of the 5' AU-rich regions from X. tropicalis (T; EST TEgg016d06 5' [http://www.sanger.ac.uk/cgi-bin/BLAST/submitblast/x_tropicalis]), X. borealis (B; AFO41844), and X. laevis (L; M18055) is shown. Conserved nucleotides are underlined in the X. laevis sequence. A conserved motif highlighted in bold corresponds to the ElrB homologue binding site described by Gao et al. (17). This motif is mutated to the italicized sequence in the VUmut and VTEmut constructs. (B) UV-cross-linking reactions performed with either 32P-labeled VTE or VTEmut are shown. Lane M, molecular size markers are shown in kilodaltons. (C) Band shift assays were performed using 32P-labeled VTE or VTEmut and total ovary extract. Anti-HuR ({alpha}-HuR) antibody was added to determine whether the shifted complexes contained ElrA or ElrB.

In order to examine the role of this motif in ElrB binding, the sequence AUUUGUUUUUA was mutated to CUACGUAGCUA in the context of the VTE (VTEmut) or the entire Vg1 3'-UTR (VUmut). Figure 5B shows direct UV cross-linking to the VTE mutant sequence. While the level of FRGY2 cross-linking remains unchanged between the VTE and VTE mutant probes, the amounts of ElrB and ElrA cross-linking are dramatically reduced. The residual 45-kDa cross-linking protein does not correspond to ElrB, as it cannot be immunoprecipitated by anti-HuR antibody (see Fig. 7A). We note that the amount of Vg1RBP cross-linking to the VTEmut is greater than that binding to the VTE, but binding of this protein to the VTE is observed under normal circumstances (Fig. 2A).



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  		FIG. 7. Multiple copies of the ElrB binding site enable ElrB binding and translational repression. (A) UV-cross-linking reactions were performed with 32P-labeled VTE, VU1, VTEmut, and 5xEBS RNAs (UV). The right side of the panel shows immunoprecipitation of these UV-cross-linking reactions, performed using anti-HuR antibody (IP). Molecular size markers are given in kilodaltons. (B) Luciferase reporter RNAs were injected into stage VI oocytes, and the resulting luciferase activities were assayed. (C) Luciferase reporter RNAs were assayed in Xenopus translation extract. Both panels B and C show results representative of at least three independent experiments. (D) The stability of the reporter RNAs was measured in extract. The top panel shows a comparison of 32P-labeled LUC-1200 and LUC-5xEBS RNAs extracted from translation extract at the given times and separated by agarose gel electrophoresis. The bottom panel shows ethidium bromide staining of rRNA as a loading control. No degradation was observed during the time course of the experiment. (E) Xenopus translation extract was preincubated with increasing amounts of 5xEBS competitor RNA. The preincubated aliquots of extract were then utilized for translation assays with the reporter RNAs shown, and the resulting luciferase activities were measured. The amounts of 5xEBS RNA added were in molar excess, as shown, over the reporter RNAs. Fluc, firefly luciferase; Rluc, Renilla luciferase.

The destruction of ElrB binding is further shown by a band shift assay. Addition of ovary extract to labeled VTE RNA results in a shift of the probe on a nondenaturing gel due to the formation of complexes with proteins in the lysate (Fig. 5C, left panel). In the presence of the anti-HuR antibody, a further shift or supershift is observed due to the interaction of the antibody with complexes containing either ElrA or ElrB. The use of control antihemagglutinin and antiphosphotyrosine antibodies did not result in a supershift (data not shown). When the VTEmut RNA is used (Fig. 5C, right panel), however, while it does interact with proteins in the lysate (as evidenced by retardation of the RNA), these proteins cannot include ElrA or ElrB, as no supershift is observed upon addition of the anti-HuR antibody. Thus, mutation of the consensus ElrB binding site disrupts the ability of the VTE RNA to bind ElrB and ElrA.

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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  		FIG. 6. Mutation of the ElrB binding site abolishes translational repression by the VTE. (A) Luciferase reporter mRNAs were injected into stage VI oocytes, and the resulting luciferase activities were assayed. (B) The same reporter RNAs were analyzed with Xenopus translation extract. Both panels A and B show results representative of at least three independent experiments. (C) The stability of the reporter RNAs was measured in extract. The top panel shows a comparison of 32P-labeled LUC-1200, LUC-VU, and LUC-mut RNAs extracted from translation extract at the given times and separated by agarose gel electrophoresis. The bottom panel shows ethidium bromide staining of rRNA as a loading control. Fluc, firefly luciferase; Rluc, Renilla luciferase.

From Fig. 6C, it can be seen that the mutation in the ElrB binding site does not affect the stability of the RNA relative to the control (LUC-1200) or the wild-type (LUC-VU) sequence. 32P-labeled RNAs, at the same concentrations as those used for the luciferase assays, were incubated in extract for the given times. The RNAs were then extracted and analyzed by agarose gel electrophoresis. Some degradation was observed during the course of the experiment, but this was equivalent for all three mRNAs and the LUC-VUmut RNA was not preferentially stabilized. Therefore, mutation of the ElrB binding motif, even in the context of the full-length Vg1 3'-UTR, destroys the ability of the RNA to repress translation, indicating that ElrB is required for repression.

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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  		FIG. 8. Injection of anti-HuR antibody alleviates Vg1 translational repression. (A) Immunoprecipitation of [35S]methionine-labeled stage III oocytes using anti-Vg1 ({alpha}-Vg1) antibody. Prior to labeling, the oocytes were injected with either phosphate-buffered saline (PBS) buffer or an equal amount of either a control antibody (Ab) or anti-HuR ({alpha}-HuR) antibody. Molecular size markers are shown in kilodaltons on the left. (B) Lysates prepared from 35S-labeled oocytes were subjected to SDS-PAGE and autoradiography. Each lane represents one oocyte equivalent. This experiment was performed twice, with similar results.


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DISCUSSION
 
The ELAV proteins have previously been described as playing a part in the regulation of both RNA stability and translation. Posttranscriptional control by these proteins has been implicated in neuronal (2) and adipocyte (23) differentiation, cell cycle control (3, 30, 35), and apoptosis (32). We now describe a role for the Xenopus ELAV homologue ElrB in the translational repression of a localized RNA during oogenesis.

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 {alpha} 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
 
We thank Joan Steitz, Mike Wormington, and Daniel Kessler for providing anti-HuR, anti-PARN, and anti-Vg1 antibodies; Peter Good, Muriel Perron, and Veronique Kruys for plasmids; and Jenny Pain for helpful comments on the manuscript. We also thank Rachel Allison for assistance with the gel filtration.

This work was supported by a Wellcome Trust project grant.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom. Phone: 01223 333673. Fax: 01223 766002. E-mail: nms{at}mole.bio.cam.ac.uk. Back

{dagger} Authors contributed equally. Back


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Molecular and Cellular Biology, October 2005, p. 9028-9039, Vol. 25, No. 20
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.20.9028-9039.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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