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

International audienceBACKGROUND 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

Post-transcriptional regulation in Xenopus embryos: role and targets of EDEN-BP.

International audienceEDEN (embryo deadenylation element)-dependent deadenylation is a regulatory process that was initially identified in Xenopus laevis early embryos and was subsequently shown to exist in Drosophila oocytes. Recent data showed that this regulatory process is required for somitic segmentation in Xenopus. Inactivation of EDEN-BP (EDEN-binding protein) causes severe segmentation defects, and the expression of segmentation markers in the Notch signalling pathway is disrupted. We showed that the mRNA encoding XSu(H) (Xenopus suppressor of hairless), a protein central to the Notch pathway, is regulated by EDEN-BP. Our data also indicate that other segmentation RNAs are targets for EDEN-BP. To identify new EDEN-BP targets, a microarray analysis has been undertaken

Ptbp1 and Exosc9 knockdowns trigger skin stability defects through different pathways

AbstractIn humans, genetic diseases affecting skin integrity (genodermatoses) are generally caused by mutations in a small number of genes that encode structural components of the dermal–epidermal junctions. In this article, we first show that inactivation of both exosc9, which encodes a component of the RNA exosome, and ptbp1, which encodes an RNA-binding protein abundant in Xenopus embryonic skin, impairs embryonic Xenopus skin development, with the appearance of dorsal blisters along the anterior part of the fin. However, histological and electron microscopy analyses revealed that the two phenotypes are distinct. Exosc9 morphants are characterized by an increase in the apical surface of the goblet cells, loss of adhesion between the sensorial and peridermal layers, and a decrease in the number of ciliated cells within the blisters. Ptbp1 morphants are characterized by an altered goblet cell morphology. Gene expression profiling by deep RNA sequencing showed that the expression of epidermal and genodermatosis-related genes is also differentially affected in the two morphants, indicating that alterations in post-transcriptional regulations can lead to skin developmental defects through different routes. Therefore, the developing larval epidermis of Xenopus will prove to be a useful model for dissecting the post-transcriptional regulatory network involved in skin development and stability with significant implications for human diseases

Rôles de la protéine de liaison aux ARN, CELF1, dans les fonctions testiculaires

La CELF1 est une protéine de liaison aux ARNm ubiquitaire et multifonctionnelle, intervenant dans les régulations post-transcriptionnelles de l'expression génétique. Les souris mâles dont le gène Celf1 a été invalidé (Celf1 / ) présentent une hypofertilité associée à des défauts de la spermiogenèse, l'élongation des cellules post-méiotiques. Nous montrons ici que ces défauts apparaissent dès la première vague de la spermatogenèse chez l'animal prépubère, et sont associés à un retard du développement des souris Celf1 / . Chez l'adulte, les mâles présentent une chute de la testostéronémie. L'élongation des spermatides rondes étant fortement dépendante de la testostérone, nous avons supposé que les défauts de spermiogenèse étaient dus à cette hypotestostéronémie. Nous avons pu valider cette hypothèse par une supplémentation en testostérone des souris Celf1 / . Nous avons montré que l'hypotestostéronémie des souris (Celf1 / ) est associée à une surexpression du gène Cyp19al, codant pour l'aromatase, un enzyme qui convertit les androgènes en oestrogènes. Or la CELF1 interagit in vitro avec l'ARNm chez la souris sauvage. Ces données indiquent que la CELF1 réprime l'expression de l'aromatase en déstabilisant son ARNm chez la souris sauvage, et que l'hypotestostéronémie observée chez les souris Celf1 / est due au moins en partie à une perte de cette répression. L'aromatase étant fortement exprimée dans les cellules de Leydig, nous avons supposé que cette dérégulation a lieu principalement dans ces cellules. Nous avons donc réalisé une inactivation conditionnelle de Celf1 dans les cellules de Leydig pour le confirmer.CELF1 is an ubiquitous and multifunctional RNA-binding protein, involved in the epost-transcriptional regulations of the genetic expression. Male mice that are inactivated for the Celf1 gene (Celf1 / ) display a hypofertility associated with defects of the spermiogenesis, the elongation of post-meiotic cells. We show here that these defects appear from the first wave of the spermatogenesis in the prepubescent animal, and are associated to a delay of the development of Celf1 / mice. At the adult, males present a decreased testosterone level. The elongation of the round spermatid being strongly dependent on the testosterone, we supposed that the defects of spermiogenesis are due to this hypotestosteronemia. We validated this hypothesis by a supplementation in testosterone of Celf1 / mice. We showed that the decreased testosterone level of Celf1 / mice is associated with an overexpression of the Cyp19al gene, encoding for the aromatase, an enzyme that converted androgens into estrogens. CELF1 interacts in vivo with the Cypl19al mRNA. These data indicate that CELF1 represses the expression of the aromatase by destabilizing its mRNA to the wild-type mice, and that the hypotestosteronemia in Celf1 / mice is due at least in part to a loss of this repression. The aromatase being strongly expressed in the Leydig cells, we supposed that this deregulation takes place mainly in these cells. We thus generated a conditional inactivation of Celf1 / in the Leydig cells to confirm it.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Étude du rôle de la protéine de liaison aux ARN CUGBP1 dans le développement des vertébrés

Les régulations post-transcriptionnelles sont indispensables au contrôle de l expression génique. Les protéines de liaison aux ARNm font parties des acteurs des ces régulations. Au laboratoire, nous nous intéressons à la protéine CUGBP1, connue pour son rôle dans la régulation de la stabilité des ARNm et de l'épissage alternatif. L'inactivation du gène /Cugbp1/ chez la souris a montré son implication dans la spermatogenèse. Chez les souris /Cugbp1/-/- la spermatogenèse s'arrête, et les souris sont stériles. Nous avons pu montré que ces souris produisent moins de testostérone que les souris sauvages. Nous ne connaissons pas les causes moléculaires de cette chute qui pourrait expliquer cette stérilité. Chez l'amphibien xénope, une inhibition de la CUGBP1 provoque des défauts de segmentation somitique. Ce processus repose sur l'oscillation de l'expression de certains gènes, "l'horloge", et sur des gradients de signalisation permettant la définition d'un front de détermination au-delà duquel les oscillations s'arrêtent et les frontières se forment. Nous avons élaboré un outil permettant d'inhiber spécifiquement l'interaction de la CUGBP1 avec l'ARNm de Su(H), indispensable à la signalisation Notch. Cette inhibition conduit à une surexpression de Su(H) et à une absence de segmentation, la répression de l'ARNm Su(H) par la CUGBP1 est donc nécessaire à la segmentation somitique. Dans un second temps, nous avons étudié les conséquences de la "dé-répression" de Su(H). Impliquée jusqu'alors dans les oscillation de gènes nous avons montré que la voie Notch est également responsable du "couplage" des voies FGF et acide rétinoïque qui permet le bon positionnement du front. Ce rôle pourrait être conservé chez tous les vertébrés.Post-transcriptional controls of gene expression play key roles in cell life. These controls require RNA-binding proteins such as CUG-BP1 which is involved in the regulation of alternative splicing and mRNA stability. To elucidate the role of CUG-BP1 in mammalian development, mice inactivated for this/ /gene were obtained by homologous recombination. Most /Cugbp1/^-/- males exhibited impaired fertility due to a partial to total arrest of spermatogenesis. We have shown that testosterone production was strongly reduced in these males. The molecular causes for this decrease are unknown but it could explain the male sterility. In /Xenopus leavis/, inhibiting CUGBP1 function led to severe defects in somitic segmentation. Somitic segmentation relies on the oscillating expression of a subset of genes, the clock , and on gradients of signalling proteins that finely position the determination front. We have designed a new tool, an antisense oligonucleotide that masks the binding site of the RNA-BP CUGBP1 on Su(H), a CUG-BP1 mRNA target that encodes a key component of the Notch signalling. This masking derepressed Su(H) mRNA and lead to Su(H) overexpression and a concomitant loss of somatic segmentation, probably due to a deregulation of Notch signalling already known to be involved in gene expression oscillations. Here we show that Notch signalling controls the crosstalk between the RA and FGF pathways, allowing the correct positioning of the front. This new role of Notch signalling in somitic segmentation could be conserved among vertebrates.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Post-transcriptional controls - adding a new layer of regulation to clock gene expression.

International audienceLiving organisms undergo biochemical, physiological and behavioral cycles with periods ranging from seconds to years. Cycles with intermediate periods are governed by endogenous clocks that depend on oscillating gene expression. Here we illustrate the modalities and specific functions of post-transcriptional control of gene expression (exerted on pre-mRNAs and mRNAs) in biological clocks through two examples: the circadian clock and the vertebrate somite segmentation clock, an embryonic clock with a period far below a day. We conclude that both constitutive and cyclic post-transcriptional controls underpin clock function

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

Targeted inactivations of RNA-binding proteins (RNA-BPs) can lead to huge phenotypical defects. These defects are due to the deregulation of certain mRNAs. However, we generally do not know, among the hundreds of mRNAs that are normally controlled by one RNA-BP, which are responsible for the observed phenotypes. Here, we designed an antisense oligonucleotide (“target protector”) that masks the binding site of the RNA-BP CUG-binding protein 1 (CUGBP1) on the mRNA Suppressor of Hairless [Su(H)] that encodes a key player of Notch signaling. We showed that injecting this oligonucleotide into Xenopus embryos specifically inhibited the binding of CUGBP1 to the mRNA. This caused the derepression of Su(H) mRNA, the overexpression of Su(H) protein, and a phenotypic defect, loss of somitic segmentation, similar to that caused by a knockdown of CUGBP1. To demonstrate a causal relationship between Su(H) derepression and the segmentation defects, a rescue experiment was designed. Embryonic development was restored when the translation of Su(H) mRNA was re-repressed and the level of Su(H) protein was reduced to a normal level. This “target protector and rescue assay” demonstrates that the phenotypic defects associated with CUGBP1 inactivation in Xenopus are essentially due to the deregulation of Su(H) mRNA. Similar approaches may be largely used to uncover the links between the phenotype caused by the inactivation of an RNA-BP and the identity of the RNAs associated with that protein

A gene regulation network controlled by Celf1 protein-rbpj mRNA interaction in Xenopus somite segmentation.

International audienceSomite segmentation is impaired in Xenopus celf1 morphant embryos. The Celf1 RNA-binding protein targets bound mRNAs for rapid degradation, and antisense approaches demonstrated that segmentation defects in celf1 morphants were due to a derepression of rbpj mRNA. Rbpj protein is a key player of Notch signalling. Because segmentation involves complex cross-talk between several signalling pathways, we analysed how rbpj derepression impacted these pathways. We found that rbpj derepression stimulated the Notch pathway. Notch positively controlled the expression of cyp26a, which encodes a retinoic acid (RA)-degrading enzyme. Thus, rbpj derepression led to cyp26a overexpression and RA attenuation. It also repressed fgf8, consistent with an inhibition of FGF signalling. Pharmacological inhibition of the FGF pathway repressed cyp26a, but rbpj derepression was sufficient to restore cyp26a expression. Hence, while it was known that the FGF pathway antagonized RA signalling through expression of cyp26a, our results suggest that Rbpj mediates this antagonism. Furthermore, they show that the post-transcriptional repression exerted by Celf1 on rbpj mRNA is required to keep cyp26a expression under the control of FGF signalling. We conclude that rbpj repression by Celf1 is important to couple the FGF and RA pathways in Xenopus segmentation

Inactivation of the Celf1 gene that encodes an RNA-binding protein delays the first wave of spermatogenesis in mice.

BACKGROUND: The first wave of spermatogenesis in mammals is characterized by a sequential and synchronous appearance of germ cells in the prepubertal testis. Post-transcriptional controls of gene expression play important roles in this process but the molecular actors that underlie them are poorly known. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the requirement for the RNA-binding protein CELF1 during the first wave of spermatogenesis in mice. Mice inactivated for Celf1 gene were not viable on pure genetic backgrounds. On a mixed background, we observed by histology and gene profiling by RT-qPCR that the testes of inactivated prepubertal mice were characterized by several features. (i) Spermiogenesis (differentiation of post-meiotic cells) was blocked in a subset of prepubertal inactivated mice. (ii) The appearance of the different stages of germ cell development was delayed by several days. (iii) The expression of markers of Leydig cells functions was similarly delayed. CONCLUSIONS/SIGNIFICANCE: Celf1 disruption is responsible for a blockage of spermiogenesis both in adults and in prepubertal males. Hence, the spermiogenesis defects found in Celf1-inactivated adults appear from the first wave of spermiogenesis. The disruption of Celf1 gene is also responsible for a fully penetrant delayed first wave of spermatogenesis, and a delay of steroidogenesis may be the cause for the delay of germ cells differentiation