EDEN-BP-dependent post-transcriptional regulation of gene expression inXenopussomitic segmentation

Abstract

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 knockdown 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.

A strategy to analyze the phenotypic consequences of inhibiting the association of an RNA-binding protein with a specific RNA

“…2C-E). We have shown previously that 90% of the embryos injected with a morpholino designed to knock down CUGBP1 display very similar segmentation defects (Gautier- Courteille et al 2004).…”

“…For EMSAs (Marquis et al 2006), 32 P-labeled RNAs were obtained by T7 in vitro transcription from PCR products using a pGbSu(H) plasmid (Gautier-Courteille et al 2004) with the appropriate primers, or a pGbsEg5 plasmid (Paillard et al 1998), previously linearized with BamH1. Two-fold serial dilutions of recombinant CUGBP1 starting from 400 nM, or threefold serial dilutions of morpholino starting from 500 nM were used.…”

“…It is easy to score by external observation of the embryos. Su(H) mRNA is normally repressed by CUGBP1 and is overexpressed in CUGBP1 knockdown embryos (Gautier-Courteille et al 2004). However, whether Su(H) derepression and segmentation defects are causally related remains an open question, as the knockdown of CUGBP1 may deregulate several mRNA targets of CUGBP1 in addition to Su(H) (Graindorge et al 2008).…”

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

“…Immunodepletion of EDEN-BP from egg extracts abolishes EDENdependent deadenylation activity , which is restored by the addition of recombinant protein . When EDEN-BP was depleted in developing embryos by the injection of specific antibodies or morpholino antisense oligonucleotides, an important disruption of somite segmentation was observed (Gautier-Courteille et al, 2004). Interestingly, a reporter RNA containing human c-Jun ARE, a class III (non-AUUUA-containing) ARE (Chen and Shyu, 1995), is rapidly deadenylated after injection in Xenopus embryos.…”

CUG-BP and 3'UTR sequences influence PARN-mediated deadenylation in mammalian cell extracts

“…Notably, EDEN-BP (Embryo Deadenylation Element-Binding Protein) interacts with a UR-rich motif in the 3'UTR of Xenopus mRNAs and triggers their deadenylation, perhaps by PARN, in developing embryos (Bonnet-Corven et al, 2002;Paillard et al, 1998;Takahashi et al, 2000). EDEN-BP, therefore, plays a vital role in the dynamics of gene expression patterns that are observed after oocyte fertilization (Gautier-Courteille et al, 2004). Homologs to EDEN-BP can be identified in numerous eukaryotes including humans, mice, zebrafish, Drosophila and C. elegans, suggesting that it likely plays a key role in somitic gene expression throughout evolution (Delaunay et al, 2004).…”