Analysis of splicing patterns by pyrosequencing

Several different mRNAs can be produced from a given pre-mRNA by regulated alternative splicing, or as the result of deregulations that may lead to pathological states. Analysing splicing patterns is therefore of importance to describe and understand developmental programs, cellular responses to internal or external cues, or human diseases. We describe here a method, Pyrosequencing Analysis of Splicing Patterns (PASP), that combines RT–PCR and pyrosequencing of PCR products. We demonstrated that: (i) Ratios of two pure RNAs mixed in various proportions were accurately measured by PASP; (ii) PASP can be adapted to virtually any splicing event, including mutually exclusive exons, complex patterns of exon skipping or inclusion, and alternative 3′ terminal exons; (iii) In extracts from different organs, the proportions of RNA isoforms measured by PASP reflected those measured by other methods. The PASP method is therefore reliable for analysing splicing patterns. All steps are done in 96-wells microplates, without gel electrophoresis, opening the way to high-throughput comparisons of RNA from several sources

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

Identification of new subgroup of HSP70 in Bythograeidae (hydrothermal crabs) and Xanthidae

International audienceCrabs of the Bythograeidae family (Crustacea: Brachyura: Bythogreoidea) are the only endemic crab family living in hydrothermal fields. The hydrothermal environment is characterized by unique ecological parameters, such as the high temperature gradient around the hydrothermal chimney (2-350 °C), a fluid environment containing high levels of metals and numerous gases. The 70-kDa Heat Shock Protein (HSP70) group is the most-studied HSP, because it is ubiquitous, and a strong positive correlation has been found between the amounts of HSP70 produced in response to stress, and the ability of the organism to withstand stressful conditions. The 70-kDa heat shock protein genes from Bythograeids (species analyzed: Bythograea thermydron, Cyanagraea praedator and Segonzacia mesatlantica) were characterized. Our results revealed that Bythograeidae possess genes which are similar with those present in Xanthids (coastal crabs). The deduced protein sequences displayed motifs distinct from those in the other crustacean HSC70/HSP70s available in the databases. Phylogenetic analysis showed that these members of HSP70 family identified in Bythograeidae and Xanthidae constitute a new subgroup within this family

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

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 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

The expression of Leydig cells markers is delayed in prepubertal <i>Celf1</i>^−/− mice.

<p>We quantified by real-time RT-PCR the relative amounts of the indicated mRNAs in +/+ (blue diamonds) and −/− (orange squares) testes at the indicated prepubertal ages. NA (not affected) and A (affected) refer to 42 dpp −/− mice with and without elongated spermatids respectively based on histological analyses. <b>A</b>, <i>Lhr. </i><b>B</b>, The main pathway of steroidogenesis in rodents and the corresponding enzymes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046337#pone.0046337-Eacker1" target="_blank">[37]</a>. <i>Hsd3b1</i> and <i>Hsd3b6</i> have different expression patterns but encode enzymes with similar activities <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046337#pone.0046337-OShaughnessy1" target="_blank">[22]</a>. Results are expressed as the means of 3–5 animals for each age and genotype. Error bars are standard deviations. We used a Student’s t-test to statistically compare the different genotypes of identical ages, and we show the p-values below 0.1 above the corresponding symbols.</p