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The 36-Kilodalton Embryonic-Type Cytoplasmic Polyadenylation Element-Binding Protein in Xenopus Laevis Is Elra, a Member of the ELAV Family of RNA-Binding Proteins

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The 36-Kilodalton Embryonic-Type Cytoplasmic Polyadenylation Element-Binding Protein in Xenopus Laevis Is Elra, a Member of the ELAV Family of RNA-Binding Proteins University of Massachusetts Medical School eScholarship@UMMS Open Access Articles Open Access Publications by UMMS Authors 1997-10-29 The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins Lin Wu University of Massachusetts Medical School Et al. Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/oapubs Part of the Life Sciences Commons, and the Medicine and Health Sciences Commons Repository Citation Wu L, Good PJ, Richter JD. (1997). The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins. Open Access Articles. Retrieved from https://escholarship.umassmed.edu/oapubs/1448 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in Open Access Articles by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. MOLECULAR AND CELLULAR BIOLOGY, Nov. 1997, p. 6402–6409 Vol. 17, No. 11 0270-7306/97/$04.0010 Copyright © 1997, American Society for Microbiology The 36-Kilodalton Embryonic-Type Cytoplasmic Polyadenylation Element-Binding Protein in Xenopus laevis Is ElrA, a Member of the ELAV Family of RNA-Binding Proteins 1 2,3 1 LIN WU, PETER J. GOOD, AND JOEL D. RICHTER * Department of Molecular Genetics and Microbiology, University of Massachusetts Medical Center, Worcester, Massachusetts 016551; Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland 208922; and Department of Biochemistry and Molecular Biology and Center for Excellence in Cancer Research, Louisiana State University Medical Center, Shreveport, Louisiana 711303 Received 17 June 1997/Returned for modification 28 July 1997/Accepted 15 August 1997 Downloaded from The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3* untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12–27 embryonic-type CPE (eCPE) in their 3* UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. We now report that this 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding mcb.asm.org proteins. The proteins are identical in size, antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. Cl2 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments failed to at UNIV OF MASS MED SCH on August 15, 2008 define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype. The possible functions of ElrA in gastrulation are discussed. Early development in many animals is programmed by precocious polyadenylation during maturation (34, 35). Be- mRNAs inherited by the egg at the time of fertilization. While cause deletion of the masking element results in premature many of these mRNAs are translationally dormant in oocytes, polyadenylation during oocyte maturation, it is likely that the they become activated in a sequence-specific manner at sub- factors that support embryonic polyadenylation are present sequent developmental periods. Several mechanisms are likely and active in maturing oocytes. In studies to identify these to control the translation of these messages, but one that has factors, UV cross-linking in egg extracts has shown that the 36- been studied extensively in Xenopus laevis is poly(A) elonga- and 45-kDa proteins bind to the eCPE (34); however, only the tion (31). Some mRNAs, which encode such proteins as c-Mos, 36-kDa protein is cytoplasmic and, based on parallel competi- cyclin B1, cyclin-dependent kinase 2 (Cdk2), and the early- tion assays for both UV cross-linking and polyadenylation, development-specific histone B4, are polyadenylated and trans- appeared to be functionally important for embryonic polyad- lated during oocyte maturation (27–29, 33, 39). Other mRNAs, enylation (36). however, undergo these processes sometime after fertilization; The products of Xenopus ELGs (embryonic lethal abnormal they encode such proteins as polypeptide chain release factor vision [ELAV]-like genes) are similar to Drosophila ELAV and (Cl1), Cl2 (function unknown), and activin receptor (28, 34– other members of this family in that they contain a short 36). amino-terminal domain followed by two consecutive RNA rec- Both groups of mRNAs contain two cis-acting elements lo- ognition motifs (RRMs), a linker region, and a carboxyl-ter- 9 cated in their 3 untranslated regions (UTRs) that are required minal RRM (32). There are four ELG proteins in Xenopus, for cytoplasmic polyadenylation. One is the virtually ubiqui- named ElrA, ElrB, ElrC, and ElrD, with molecular masses tous hexanucleotide AAUAAA, which is also important for ranging from 36 to 42 kDa (10). The RRMs are highly con- nuclear pre-mRNA cleavage and polyadenylation. The second, served among ElrB, -C, and -D; the RRMs for ElrA are less called the cytoplasmic polyadenylation element (CPE), is a well conserved. In addition, ElrA has shorter amino-terminal U-rich sequence located upstream of the hexanucleotide. The and linker regions. By Northern analysis, ElrA appears to be exact sequence of CPE differs between the two groups of expressed ubiquitously, ElrB is expressed in both maternal and mRNAs mentioned. The maturation-type CPE is UUUUUAU brain tissues, whereas ElrC and -D have a brain-specific ex- (consensus), whereas the embryonic CPE (eCPE) is oligo pression pattern (10). The ElrA sequence and expression pat- (U) (reviewed in reference 31). At least two mRNAs that 12–27 tern are also conserved in mammals (22, 26). Members of the are polyadenylated in embryos also contain a third cis-acting ELAV family interact with an AU-rich element in the 39 UTRs sequence in the 39 UTR: the masking element that prevents of several cytokine and other mRNAs and, at least in the case of human cells, have been implicated in the control of cell * Corresponding author. Mailing address: Department of Molecular growth and differentiation, perhaps by influencing the degra- Genetics and Microbiology, University of Massachusetts Medical Cen- dation of such messages (1, 7, 17, 21, 22, 24). ter, 222 Maple Ave., Shrewsbury, MA 01545. Phone: (508) 842-8921, In this report, we show that the 36-kDa eCPE-binding pro- ext. 340. Fax: (508) 842-3915. E-mail: [email protected]. tein and ElrA are the same protein. Although ElrA specifically 6402 VOL. 17, 1997 CPE-BINDING PROTEIN IN X. LAEVIS 6403 interacts with RNAs harboring the eCPE both in vitro and in fragment was digested with BamHI and ligated into the BamHI site of pPET15b. vivo, an extensive series of experiments failed to demonstrate Capped RNAs were in vitro synthesized by using a T7 message machine (Am- bion) after templates were linearized with ClaI. Then 1 ng of RNA was injected that it has a function in cytoplasmic polyadenylation. However, into each fertilized egg. overexpression of a truncated ElrA in injected embryos results Embryos injected with RNA (two different preparations) were collected at in abnormal development beginning at the gastrula stage, different stages and fixed, embedded in paraffin, sectioned, and stained as de- which suggests that it plays a role in early embryogenesis. scribed by Kelly et al. (18). Whole embryos were fixed in 4% paraformaldehyde and 0.25% glutaraldehyde. After washing and dehydration, they were embedded in paraffin and cut into 10-mm-thick sections. After the paraffin was removed, the MATERIALS AND METHODS tissues were rehydrated and stained with hematoxylin and eosin Y. UV cross-linking and immunoprecipitation. Oocyte and egg extracts were Construction and purification of GST fusion protein. Because His-tagged prepared as described by McGrew and Richter (23) except that the oocytes were mutant ElrA proteins made in bacteria were insoluble, GST fusions were also first manually enucleated. UV cross-linking and immunoprecipitation were car- constructed. To construct GST fusion proteins, XhoI-BipI (blunt ended with ried out as described by Simon et al. (34) and Hake and Richter (12), respec- Klenow enzyme) fragments of His-tagged constructs were isolated and inserted tively. Constructs for an eCPE-containing Cl2 39 UTR (27 U’s) and eCPE- into XhoI and HindIII (blunt ended with Klenow enzyme) sites of vector pGEX- lacking Cl2 39 UTRs (no U’s and 9 U’s) were described by Simon et al. (34). To KG. The fusion proteins were overexpressed in bacteria and were further puri- generate ElrA antiserum, the ElrA open reading frame was amplified by PCR, fied by affinity chromatography on glutathione-agarose as described by Smith and inserted into the pET21D vector, and used to transform bacteria. Following Johnson (37). induction, ElrA protein was isolated from inclusion bodies and further purified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The ElrA band was then excised and injected into rabbits. To generate ElrB antiserum, a PCR fragment that encodes amino acids 161 to 388 of ElrB was RESULTS Downloaded from inserted into pGEX-KG, and the truncated protein was expressed in induced bacteria and purified on glutathione S-transferase (GST)–agarose (Pharmacia) In previous experiments, we identified two Xenopus proteins before injection into rabbits. To generate ElrD antiserum, the complete open with molecular sizes of 36 and 45 kDa that were UV cross- reading frame of ElrD was PCR amplified and inserted into pET15b, and bac- linked to the eCPEs of Cl1, Cl2, and ActR mRNAs (35, 36).
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