Differential RNA-binding activity of the hnRNP G protein correlated with the sex genotype in the amphibian oocyte

A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein. Three isoforms of this protein with a differential binding affinity for a specific RNA probe were identified in the P. walt oocyte. In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome. This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals

Etude moléculaire et biochimique des interactions de la protéine hnRNP G avec l ARN

La protéine hnRNP G est impliquée dans l épissage alternatif de plusieurs précurseurs d ARN messager. Pour mieux comprendre la spécificité de cette protéine, j ai étudié ses interactions avec les ARNs in vitro et in vivo. Un motif d ARN structuré dans une tige-boucle qui contient la séquence GGAAA a été identifié in vitro comme une cible potentielle d hnRNP G. Cet ARN m a permis d identifier un nouveau domaine d interaction avec l ARN dans la structure de l hnRNP G, situé dans sa région C-terminale et désigné C-ter RBD .Le modèle des chromosomes en écouvillon chez l amphibien m a permis de montrer qu un nouveau domaine situé au centre de la structure de la protéine et distinct de ses domaines d interaction avec l ARN est responsable de son recrutement au niveau des transcrits naissants des chromosomes. Ce domaine a été désigné Nascent transcripts Targeting Domain (NTD). Cette étude montre que l hnRNP G s associe avec la majorité des ARNs transcrits par l ARN polymérase II tout en montrant un recrutement plus important au niveau de quelques boucles des chromosomes. En particulier, l association de l hnRNP G avec les transcrits du bivalent sexuel ZW chez Pleurodeles waltl, montre un recrutement hétéromorphe au niveau de certains sites de ce bivalent.Au cours de ma thèse, j ai participé à la mise en évidence d une activité différentielle de liaison à l ARN liée au génotype sexuel, pour les isoformes de l hnRNP G dans l ovocyte de P. waltl. Les résultats suggèrent la présence d une famille multigénique de l hnRNP G répartie sur un autosome et sur les chromosomes sexuels. Cette étude contribue à la caractérisation de l hnRNP G et à la compréhension de ses fonctions.PARIS-BIUP (751062107) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Les mécanismes cellulaires de la gonadogenèse chez l'amphibien xenopus tropicals (étude histologique et immunohistologique)

Dans le cadre des efforts qui visent à introduire Xenopus tropicalis comme un nouveau modèle en biologie du développement et de la reproduction, nous avons effectué une étude histologique, cytologique et immunohistologique de la gonadogenèse, afin de comprendre le processus de la différenciation sexuelle chez cette espèce. Nous avons montré que la gonadogenèse progresse selon un gradient antéro postérieur. En l absence de marqueurs génétiques du sexe, ce gradient nous a permis de définir les différentes étapes de la différenciation histologique des gonades. Nous avons montré que la structure corticomédullaire, présente dans les gonades des deux sexes chez la plupart des vertébrés, n est visible que dans la gonade femelle chez X. tropicalis. Afin d édifier une table de développement gonadique, nous avons établi la correspondance entre les stades gonadiques que nous avons défini avec ceux du développement larvaire. Cette table pourrait ainsi servir à étudier les effets des différents facteurs sur la différenciation sexuelle.Nous avons ensuite analysé par hybridation in situ et par immunohistologie l expression de SOX9, un gène conservé chez les vertébrés et dont le rôle dans la différenciation du sexe chez les amniotes est bien connu. Nos résultats montrent que SOX9 n est pas nécessaire pour déclencher les premiers événements de la différenciation sexuelle des gonades chez X. tropicalis ; en revanche, comme ceci a été décrit chez les amniotes, SOX9 aurait conservé un rôle dans les stades plus tardifs de la différenciation des gonades mâles. Par contre, contrairement aux amniotes, nos résultats montrent que SOX9 jouerait aussi un rôle au cours de l ovogenèse. Un tel rôle est discuté en relation avec l évolution des tétrapodes.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Tips and tricks for preparing lampbrush chromosome spreads from Xenopus tropicalis oocytes.

International audienceDue to their large size and fine organization, lampbrush chromosomes (LBCs) of amphibian oocytes have been for decades one of the favorite tools of biologists for the analysis of transcriptional and post-transcriptional processes at the cytological level. The emergence of the diploid Xenopus tropicalis amphibian as a model organism for vertebrate developmental genetics and the accumulation of sequence data made available by its recent genomic sequencing, strongly revive the interest of LBCs as a powerful tool to study genes expressed during oogenesis. We describe here a detailed protocol for preparing LBCs from X. tropicalis oocyte and give practical advice to encourage a large number of researchers to become familiar with these chromosomes

Precocious detection on amphibian oocyte lampbrush chromosomes of subtle changes in the cellular localisation of the Ro52 protein induced by in vitro culture.

International audienceSubterminal lampbrush loops of one of the 12 bivalents of the oocyte karyotype of Pleurodeles waltl (Amphibian, Urodele) underwent prominent morphological changes upon in vitro culture. These loops exhibited a fine ribonucleoprotein (RNP) granular matrix, which evolved during culture into huge structures that we have named 'chaussons' (slippers). This phenomenon involved progressive accumulation of proteins in the RNP matrix without protein neosynthesis. One of these proteins, which translocated into the nucleus during the culture, was identified as a homolog of the human Ro52 E3 ubiquitin ligase. RNA polymerase III was also found to accumulate on the same loops. These results suggest that the subterminal loops of bivalent XII act as a storage site for the components of a nuclear machinery involved in the quality control of RNA synthesis and maturation in response to cellular stress. They also emphasise the considerable value of the lampbrush chromosome system for a direct visualisation of modifications in gene expression and open the question of a nuclear accumulation of Ro52 in human or animal oocytes cultured in vitro for assisted reproductive technologies (ART)

Working map of the lampbrush chromosomes of Xenopus tropicalis: A new tool for cytogenetic analyses.

International audienceThe amphibian Xenopus tropicalis, whose genome has been recently sequenced, has become an important model organism for vertebrate developmental genetics. The development of cytogenetic tools in this new model organism should contribute to an understanding of the organization of the amphibian genome and the mapping of a variety of loci of interest. In this respect, oocyte lampbrush chromosomes are particularly useful for the localization of genomic sequences expressed during oogenesis. We have constructed a working map of X. tropicalis lampbrush chromosomes, which allows the 10 bivalents of the oocyte karyotype to be readily identified by distinctive combinations of specific landmark structures composed of lateral loops, spheres, and granules. We have also established the patterns of RNA Pol III sites over the chromosomes by immunofluorescence using antibodies directed against two Pol III subunits. Specific staining patterns were found for each chromosome, which constitute a supplementary tool for their identification. Developmental Dynamics 238:1492-1501, 2009. (c) 2009 Wiley-Liss, Inc

Early aspects of gonadal sex differentiation in Xenopus tropicalis with reference to an antero-posterior gradient.

International audienceIn an effort to contribute to the development of Xenopus tropicalis as an amphibian model system, we carried out a detailed histological analysis of the process of gonadal sex differentiation and were able to find evidence that gonadal differentiation in X. tropicalis follows an antero-posterior gradient. Although the main reason for the presence of a gradient of sex differentiation is still unknown, this gradient enabled us to define the early events that signal ovarian and testicular differentiation and to identify the undifferentiated gonad structure. Given the various advantages of this emerging model, our work paves the way for experiments that should contribute to our understanding of the dynamics and mechanisms of gonadal sex differentiation in amphibians. J. Exp. Zool. 309A, 2008. (c) 2008 Wiley-Liss, Inc

Sex-specific expression of SOX9 during gonadogenesis in the amphibian Xenopus tropicalis.

International audienceTo investigate the role of SOX9 gene in amphibian gonadogenesis, we analyzed its expression during male and female gonadogenesis in Xenopus tropicalis. The results showed that in both sexes SOX9 mRNA and protein were first detectable after metamorphosis when the gonads were well differentiated and remained present until the adult stage. In the testis, SOX9 expression was restricted to the nucleus of Sertoli-like cells, similarly to what has been observed in other vertebrates suggesting a conserved role in vertebrate testicular differentiation. In the ovary, in sharp contrast with what has been observed in all vertebrates examined so far, the SOX9 protein was localized in the cytoplasm of previtellogenic oocytes before being translocated into the nucleus of vitellogenic oocytes suggesting an unexpected role during oogenesis. These results suggest that the SOX9 gene may not be a sex-determining gene in X. tropicalis and may play different functions in testicular and ovarian differentiation. Developmental Dynamics 237:2996-3005, 2008. (c) 2008 Wiley-Liss, Inc

Novel domains in the hnRNP G/RBMX protein with distinct roles in RNA binding and targeting nascent transcripts

The heterogenous nuclear ribonucleoprotein G (hnRNP G) controls the alternative splicing of several pre-mRNas. While hnRNP G displays an amino terminal RNA recognition motif (RRM), we find that this motif is paradoxically not implicated in the recruitment of hnRNP G to nascent transcripts in amphibian oocytes. In fact, a deletion analysis revealed that targeting of hnRNP G to active transcription units depends on another domain, centrally positioned, and consisting of residues 186–236. We show that this domain acts autonomously and thus is named NTD for nascent transcripts targeting domain. Furthermore, using an RNA probe previously characterized in vitro as an RNA that interacts specifically with hnRNP G, we demonstrate a new auxiliary RNA binding domain (RBD). It corresponds to a short region of 58 residues positioned at the carboxyl terminal end of the protein, which recognizes an RNA motif predicted to adopt an hairpin structure. The fact that the NTD acts independently from both the RRM and the RBD strongly suggests that the initial recruitment of hnRNP G to nascent pre-mRNAs is independent of its sequence-specific RNA binding properties. Together, these findings highlight the modular organization of hnRNP G and offer new insights into its multifunctional roles