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

Identification of CUG-BP1/EDEN-BP target mRNAs in Xenopus tropicalis

The early development of many animals relies on the posttranscriptional regulations of maternally stored mRNAs. In particular, the translation of maternal mRNAs is tightly controlled during oocyte maturation and early mitotic cycles in Xenopus. The Embryonic Deadenylation ElemeNt (EDEN) and its associated protein EDEN-BP are known to trigger deadenylation and translational silencing to several mRNAs bearing an EDEN. This Xenopus RNA-binding protein is an ortholog of the human protein CUG-BP1/CELF1. Five mRNAs, encoding cell cycle regulators and a protein involved in the notch pathway, have been identified as being deadenylated by EDEN/EDEN-BP. To identify new EDEN-BP targets, we immunoprecipitated EDEN-BP/mRNA complexes from Xenopus tropicalis egg extracts. We identified 153 mRNAs as new binding targets for EDEN-BP using microarrays. Sequence analyses of the 3′ untranslated regions of the newly identified EDEN-BP targets reveal an enrichment in putative EDEN sequences. EDEN-BP binding to a subset of the targets was confirmed both in vitro and in vivo. Among the newly identified targets, Cdk1, a key player of oocyte maturation and cell cycle progression, is specifically targeted by its 3′ UTR for an EDEN-BP-dependent deadenylation after fertilization

Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N—terminal RRMs of CELF1

CUGBP, Elav-like family member 1 (CELF1) is an RNA binding protein with important roles in the regulation of splicing, mRNA decay and translation. CELF1 contains three RNA recognition motifs (RRMs). We used gel retardation, gel filtration, isothermal titration calorimetry and NMR titration studies to investigate the recognition of RNA by the first two RRMs of CELF1. NMR shows that RRM1 is promiscuous in binding to both UGU and CUG repeat sequences with comparable chemical shift perturbations. In contrast, RRM2 shows greater selectivity for UGUU rather than CUG motifs. A construct (T187) containing both binding domains (RRM1 and RRM2) was systematically studied for interaction with tandem UGU RNA binding sites with different length linker sequences UGU(U)xUGU where x = 1–7. A single U spacer results in interactions only with RRM1, demonstrating both steric constraints in accommodating both RRMs simultaneously at adjacent sites, and also subtle differences in binding affinities between RRMs. However, high affinity co-operative binding (Kd ~ 0.4 µM) is evident for RNA sequences with x = 2–4, but longer spacers (x ≥ 5) lead to a 10-fold reduction in affinity. Our analysis rationalizes the high affinity interaction of T187 with the 11mer GRE consensus regulatory sequence UGUUUGUUUGU and has significant consequences for the prediction of CELF1 binding sites

Expression of a Dominant Negative CELF Protein In Vivo Leads to Altered Muscle Organization, Fiber Size, and Subtype

CUG-BP and ETR-3-like factor (CELF) proteins regulate tissue- and developmental stage-specific alternative splicing in striated muscle. We previously demonstrated that heart muscle-specific expression of a nuclear dominant negative CELF protein in transgenic mice (MHC-CELFΔ) effectively disrupts endogenous CELF activity in the heart in vivo, resulting in impaired cardiac function. In this study, transgenic mice that express the dominant negative protein under a skeletal muscle-specific promoter (Myo-CELFΔ) were generated to investigate the role of CELF-mediated alternative splicing programs in normal skeletal muscle.Myo-CELFΔ mice exhibit modest changes in CELF-mediated alternative splicing in skeletal muscle, accompanied by a reduction of endomysial and perimysial spaces, an increase in fiber size variability, and an increase in slow twitch muscle fibers. Weight gain and mean body weight, total number of muscle fibers, and overall muscle strength were not affected.Although these findings demonstrate that CELF activity contributes to the normal alternative splicing of a subset of muscle transcripts in vivo, the mildness of the effects in Myo-CELFΔ muscles compared to those in MHC-CELFΔ hearts suggests CELF activity may be less determinative for alternative splicing in skeletal muscle than in heart muscle. Nonetheless, even these small changes in CELF-mediated splicing regulation were sufficient to alter muscle organization and muscle fiber properties affected in myotonic dystrophy. This lends further evidence to the hypothesis that dysregulation of CELF-mediated alternative splicing programs may be responsible for the disruption of these properties during muscle pathogenesis

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