Cytoplasmic Polyadenylation of Activin Receptor mRNA and the Control of Pattern Formation inXenopusDevelopment

Abstract

The activin receptor, a transmembrane serine-threonine kinase, is a key component necessary for pattern formation in early Xenopus development. This protein interacts with members of the transforming growth factor beta family and stimulates cells of the marginal zone to differentiate along the mesodermal pathway. In large part, this function of the activin receptor has been inferred from observations of phenotypes induced by injected mRNA encoding wild-type or mutant forms of the protein. Naturally occurring activin receptor mRNA is maternally inherited and contains within its 3' untranslated region an embryonic-type cytoplasmic polyadenylation element (CPE), an oligouridylic acid sequence that promotes cytoplasmic polyadenylation and resultant translational activation. Based on the presence of this element, we predicted in a previous report that activin receptor mRNA expression in embryos might be regulated by cytoplasmic polyadenylation (Simon and Richter, Mol. Cell. Biol. 14, 7867-7875, 1994). In this study, we have tested this hypothesis and show that not only do endogenous and injected activin receptor mRNAs undergo cytoplasmic polyadenylation during embryogenesis, but also that this process is necessary for stimulating translation and inducing the morphological defects observed by mRNA overexpression. The activin receptor CPE is bound by a Mr 36 x 10(3) protein in vitro, and competition for this factor between mRNAs in vivo inhibits activin receptor mRNA polyadenylation. This competition may be responsible for the lack of mesoderm formation observed in such injected embryos. These data suggest that cytoplasmic polyadenylation controls differentiation and pattern formation in early Xenopus development.

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

“…In previous experiments, we identified two Xenopus proteins with molecular sizes of 36 and 45 kDa that were UV crosslinked to the eCPEs of Cl1, Cl2, and ActR mRNAs (35,36). However, only the 36-kDa protein was cytoplasmic and therefore seemed the most likely one to be involved in mRNA polyadenylation in embryos.…”

“…To determine whether the 36-kDa UV cross-linked protein was the one that reacted with anti-ElrA antibody, we took advantage of our previous observation that only the 36-kDa protein is cytoplasmic (36). Thus, extracts were prepared from enucleated oocytes and used for UV cross-linking as described above.…”

“…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).…”

“…Because deletion of the masking element results in premature polyadenylation during oocyte maturation, it is likely that the factors that support embryonic polyadenylation are present and active in maturing oocytes. In studies to identify these factors, UV cross-linking in egg extracts has shown that the 36-and 45-kDa proteins bind to the eCPE (34); however, only the 36-kDa protein is cytoplasmic and, based on parallel competition assays for both UV cross-linking and polyadenylation, appeared to be functionally important for embryonic polyadenylation (36).…”

Activity-dependent polyadenylation in neurons

“…The beads were washed in buffer containing 25% formamide at 50 C, which removes nonspecific RNAs and those with short poly(A) tails, usually less than 50 nt. Formamide-containing buffer at 60 C is then passed through the column, which elutes RNAs with poly(A) tails greater than 55 bases (Simon et al 1996, based on Palatnik et al 1981).…”