Oligomerization of EDEN-BP is required for specific mRNA deadenylation and binding.

BACKGROUND INFORMATION: mRNA deadenylation [shortening of the poly(A) tail] is often triggered by specific sequence elements present within mRNA 3' untranslated regions and generally causes rapid degradation of the mRNA. In vertebrates, many of these deadenylation elements are called AREs (AU-rich elements). The EDEN (embryo deadenylation element) sequence is a Xenopus class III ARE. EDEN acts by binding a specific factor, EDEN-BP (EDEN-binding protein), which in turn stimulates deadenylation. RESULTS: We show here that EDEN-BP is able to oligomerize. A 27-amino-acid region of EDEN-BP was identified as a key domain for oligomerization. A mutant of EDEN-BP lacking this region was unable to oligomerize, and a peptide corresponding to this region competitively inhibited the oligomerization of full-length EDEN-BP. Impairing oligomerization by either of these two methods specifically abolished EDEN-dependent deadenylation. Furthermore, impairing oligomerization inhibited the binding of EDEN-BP to its target RNA, demonstrating a strong coupling between EDEN-BP oligomerization and RNA binding. CONCLUSIONS: These data, showing that the oligomerization of EDEN-BP is required for binding of the protein on its target RNA and for EDEN-dependent deadenylation in Xenopus embryos, will be important for the identification of cofactors required for the deadenylation process.

EDEN-BP-dependent post-transcriptional regulation of gene expression in Xenopus somitic segmentation.

EDEN-BP is a Xenopus RNA-binding protein that triggers deadenylation [poly(A) tail shortening], and thereby translational repression and degradation, of a subset of maternal mRNAs soon after fertilization. We show here that this factor is expressed in the presomitic mesoderm of older embryos, the site where somitic segmentation takes place. Inhibiting EDEN-BP function using either antisense morpholino oligonucleotides or neutralizing antibodies leads to severe defects in somitic segmentation, but not myotomal differentiation. This is associated with defects in the expression of segmentation markers belonging to the Notch signalling pathway in the presomitic mesoderm. We show by a combination of approaches that the mRNA encoding XSu(H), a protein that plays a central role in Notch signalling, is regulated by the EDEN-BP pathway. Accordingly, XSu(H) is overexpressed in EDEN-BP knock-down embryos, and overexpressing XSu(H) causes segmentation defects. We finally give data indicating that, in addition to XSu(H), other segmentation RNAs are a target for EDEN-BP. These results show that EDEN-BP-dependent post-transcriptional regulation of gene expression is required for the process of somitic segmentation.

EDEN-dependent translational repression of maternal mRNAs is conserved between Xenopus and Drosophila.

Translational control is a key level in regulating gene expression in oocytes and eggs because many mRNAs are synthesized and stored during oogenesis for latter use at various stages of oocyte maturation and embryonic development. Understanding the molecular mechanisms that underlie this translational control is therefore crucial. Another important issue is the evolutionary conservation of these mechanisms--in other words the determination of their universal and specific aspects. We report here a comparative analysis of a translational repression mechanism that depends on the EDEN (embryo deadenylation element) element. This small cis-acting element, localized in the 3' untranslated region of c-mos and Eg mRNAs, was shown to be involved in a deadenylation process. We demonstrate here that in Xenopus embryos, mRNAs that contain an EDEN are translationally repressed. Next, transgenic flies were used to study the effect of the EDEN motif on translation in Drosophila oocytes. We show that this element also causes the translational repression of a reporter gene in Drosophila demonstrating that the EDEN-dependent translational repression is functionally conserved between Xenopus and Drosophila.

c-Jun ARE targets mRNA deadenylation by an EDEN-BP (embryo deadenylation element-binding protein)-dependent pathway.

In mammalian cells, certain mRNAs encoding cytokines or proto-oncogenes are especially unstable, because of the presence of a particular sequence element in their 3'-untranslated region named ARE (A/U-rich element). AREs cause this instability by provoking the rapid shortening of the poly(A) tail of the mRNA. The deadenylation of mRNAs mediated by AREs containing repeats of the AUUUA motif (class I/II AREs) is conserved in Xenopus embryos. Here, we first extend these observations by showing that c-Jun ARE, a representative of class III (non-AUUUA) AREs, also provokes the deadenylation of a reporter RNA in Xenopus embryos. Next, by immunodepletion and immunoneutralization experiments, we show that, in Xenopus, the rapid deadenylation of RNAs that contain the c-Jun ARE, but not an AUUUA ARE, requires EDEN-BP. This RNA-binding protein was previously shown to provoke the rapid deadenylation of certain Xenopus maternal RNAs. Finally, we show that CUG-BP, the human homologue of EDEN-BP, specifically binds to c-Jun ARE. Together, these results identify CUG-BP as a plausible deadenylation factor responsible for the post-transcriptional control of c-Jun proto-oncogene mRNA in mammalian cells.