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

Abstract

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 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 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 mRNAs inherited by the egg at the time of fertilization. While many of these mRNAs are translationally dormant in oocytes, they become activated in a sequence-specific manner at subsequent developmental periods. Several mechanisms are likely to control the translation of these messages, but one that has been studied extensively in Xenopus laevis is poly(A) elongation (31). Some mRNAs, which encode such proteins as c-Mos, cyclin B1, cyclin-dependent kinase 2 (Cdk2), and the earlydevelopment-specific histone B4, are polyadenylated and translated during oocyte maturation (27-29, 33, 39). Other mRNAs, however, undergo these processes sometime after fertilization; they encode such proteins as polypeptide chain release factor (Cl1), Cl2 (function unknown), and activin receptor (28,(34)(35)(36).Both groups of mRNAs contain two cis-acting elements located in their 3Ј untranslated regions (UTRs) that are required for cytoplasmic polyadenylation. One is the virtually ubiquitous hexanucleotide AAUAAA, which is also important for nuclear pre-mRNA cleavage and polyadenylation. The second, called the cytoplasmic polyadenylation element (CPE), is a U-rich sequence located upstream of the hexanucleotide. The exact sequence of CPE differs between the two groups of mRNAs mentioned. The maturation-type CPE is UUUUUAU (consensus), whereas the embryonic CPE (eCPE) is oligo (U) 12-27 (reviewed in reference 31). At least two mRNAs that are polyadenylated in embryos also contain a third cis-acting sequence in the 3Ј UTR: the masking element that prevents precocious polyadenylation during maturation (34, 35). Because deletion of the maskin...

Xenopus Cold-inducible RNA-binding Protein 2 Interacts with ElrA, the Xenopus Homolog of HuR, and Inhibits Deadenylation of Specific mRNAs

“…1C). Previous experiments revealed that both xCIRP2 and ElrA proteins are present predominantly in the oocyte cytoplasm (25,26). Xenopus oocyte lysates were prepared and subjected to immunoprecipitation with anti-xCIRP2 antibodies.…”

“…Among them, upon UV irradiation, cytoplasmic fractions of both proteins are increased and are involved in stabilization of specific mRNAs (24,32). Third, both ElrA and xCIRP2 proteins are predominantly cytoplasmic in Xenopus oocytes (25,26). As a large stockpile of maternal mRNA is accumulated in the oocyte cytoplasm, ElrA and xCIRP2 may play roles in the control of poly(A) lengths of these mRNAs.…”

“…Many studies have established that HuR up-regulates gene expression by stabilizing ARE-containing mRNAs in vitro and in vivo (21)(22)(23)(24). Xenopus homolog of HuR, ElrA, was identified as an RNA-binding protein that binds to a cis-acting element, the embryonic CPE or eCPE, required for cytoplasmic polyadenylation occurring during early embryogenesis (25).…”

Transitioning from egg to embryo: Triggers and mechanisms of egg activation

“…For instance, in Xenopus U-rich or C-rich CPEs initiate polyadenylation upon fertilization (Simon et al, 1992;Paillard et al, 2000). The mechanism by which the protein complexes that bind to CPEs accomplish polyadenylation is not known for activation (Paillard et al, 2000;Wu et al, 1997), but is better understood during oocyte maturation. During maturation, CPEs ultimately bind cytoplasmic polyadenylation specificity factor (CPSF), which recruits poly(A) polymerase to the 3ЈUTR (Mendez et al, 2000).…”

Position and sequence requirements for poly(A) length regulation by the poly(A) limiting element

“…With the exception of histone mRNA, most vertebrate mRNAs possess 39 poly(A) tails ranging from 100 to 200ϩ residues in length+ Poly(A) addition is one of the last steps in pre-mRNA processing, occurring prior to splicing of the terminal intron and nuclear export (Bauren & Wieslander, 1994)+ Based on the exon definition model, the 39 processing complex substitutes for a 59 splice site to define the end of the terminal exon (Berget, 1995), and there is good evidence linking 39 processing to splicing of the terminal intron (Lou et al+, 1996;Cooke et al+, 1999;Custodio et al+, 1999)+ This interaction between splicing and 39 processing plays a key role in regulating gene expression, particularly in cases involving exon selection linked to selection of alternative 39 ends (Lou et al+, 1999)+ Pre-mRNA 39 processing is regulated by cis-acting elements in the message body (reviewed in Zhao et al+, 1999)+ Upstream elements, or USEs, in HIV-1 proviral RNA (Gilmartin et al+, 1995), adenovirus late RNA (Sittler et al+, 1995), SV40 late RNA (Schek et al+, 1992), and C2 complement gene (Moreira et al+, 1998) can act as enhancers to stimulate 39 processing of their respective RNAs+ Conversely, U1A binding to a pair of U1A binding sites adjacent to AAUAAA in U1A pre-mRNA blocks poly(A) addition by inhibiting poly(A) polymerase without affecting the initial cleavage step (Gunderson et al+, 1994)+ Elements upstream of AAUAAA can also regulate poly(A) tail length+ Poly(A) length regulation has been best characterized for mRNAs that undergo deadenylation or poly(A) addition during various stages of egg maturation and embryonic development (reviewed in Richter, 1999)+ This form of regulation is cytoplasmic, and involves the interaction of the cytoplasmic polyadenylation element binding protein (CPE-BP) with a U-rich cytoplasmic polyadenylation element (CPE) upstream of AAUAAA (Wu et al+, 1997)+ Embryospecific deadenylation is controlled by a distinct upstream element, EDEN, through its interaction with EDEN-BP (Paillard et al+, 1998)+ The deadenylation ef-ficiency of the EDEN element is enhanced by the presence of upstream AUU repeats, suggesting an interplay between multiple cis-acting elements in regulating deadenylation (Audic et al+, 1998)+ In the course of studying the estrogen regulation of mRNA stability in Xenopus, we found that albumin and several other serum protein mRNAs have an unusually short, 17-nt poly(A) tail (Schoenberg et al+, 1989;Pastori et al+, 1991)+ Using an intron-specific primer in an RT-PCR assay for poly(A) tail length (Salles et al+, 1999), we subsequently identifi...…”