Anne Couturier

Cloning by differential screening of a Xenopus cDNA that encodes a kinesin-related protein.

By differential screening of a Xenopus egg cDNA library, we selected nine clones (Eg1 to Eg9) corresponding to mRNAs which are deadenylated and released from polysomes soon after fertilization. The sequence of one of these clones (Eg5) revealed that the corresponding protein has the characteristic features of a kinesin-related protein. More specifically, Eg5 was found to be nearly 30% identical to a kinesin-related protein encoded by bimc, a gene involved in nuclear division in Aspergillus nidulans.

Changes in the polyadenylation of specific stable RNA during the early development of Xenopus laevis

The distribution of four specific RNA species between the poly(A)+ and poly(A)- fractions has been studied during the first hours of Xenopus laevis development, before the mid-blastula transition (MBT). Two of these specific RNA species correspond to clones selected by differential hybridization from a Xenopus egg cDNA library. Another corresponds to Xenopus c-raf mRNA and the last one to RNA revealed by a mouse ornithine decarboxylase probe. We show that two of these RNAs are adenylated after fertilization and remain in the poly(A)+ population. During the same period, the other two RNAs are deadenylated and these new poly(A)- RNAs remain stable at least until the MBT. These results show (i) that polyadenylation of specific RNA species occurs after fertilization in Xenopus and (ii) that, in the absence of transcription, adenylation and deadenylation can occur simultaneously in the fertilized egg.

M-phase MELK activity is regulated by MPF and MAPK

Caroline Badouel, Roman Körner, Marie Frank-Vaillant, Anne Couturier, Erich A. Nigg and Jean-Pierre TassanThe protein kinase MELK is implicated in the control of cell proliferation, cell cycleand mRNA splicing. We previously showed that MELK activity is correlated with itsphosphorylation level, is cell cycle dependent, and maximal during mitosis. Here we report onthe identification of T414, T449, T451, T481 and S498 as residues phosphorylated inXenopus MELK (xMELK) in M-phase egg extract. Phosphorylations of T449, T451, T481are specifically detected during mitosis. Results obtained in vivo showed that MPF andMAPK pathways are involved in xMELK phosphorylation. In vitro, MPF and MAPK directlyphosphorylate xMELK and MPF phosphorylates xMELK on T481. In addition,phosphorylation by MPF and MAPK enhances MELK activity in vitro. Taken together ourresults indicate that MELK phosphorylation by MPF and MAPK enhance its activity duringM-phase.

Characterization of the poly(A) binding proteins expressed during oogenesis and early development of Xenopus laevis

During vertebrate oogenesis and early embryogenesis, gene expression is governed mainly by translational control. The recruitment of Poly(A) Binding Protein (PABP) during poly(A) tail lengthening appears to be the key to translational activation during this period of development in Xenopus laevis. We showed that PABP1 and ePABP proteins are both present during oogenesis and early development. We selected ePABP as an eRF3 binding protein in a two‐hybrid screening of a X. laevis cDNA library and demonstrated that this protein is associated with translational complexes. It can complement essential functions of the yeast homologue Pab1p. We discuss specific expression patterns of the finely tuned PABP1 and ePABP proteins.

Cell‐cycle‐dependent cortical localization of pEg3 protein kinase in Xenopus and human cells

Background information. Protein kinase pEg3 belongs to the evolutionarily conserved KIN1/PAR‐1/MARK family, whose members are involved in a variety of functions, including cell polarity, microtubule stability, intracellular signalling and the cell cycle. Activity and phosphorylation of pEg3 are cell‐cycle dependent and rise to maximum levels during mitosis. pEg3 was shown to interact with and phosphorylate phosphatase CDC25B, and to potentially control cell‐cycle progression. Subcellular localization of pEg3 was investigated in Xenopus and human cultured cells.

pEg6, a Spire family member, is a maternal gene encoding a vegetally localized mRNA in Xenopus embryos

Background information. In Xenopus, during oocyte maturation and the segmentation period, cell cycle progression is independent of new transcription, but requires de novo translation. This suggests that the completion of oocyte maturation and then the rapid cell division period is controlled exclusively at a post‐transcriptional level by specific gene products. To isolate these maternal genes, a differential screening of a Xenopus egg cDNA library was performed. Several cDNAs were isolated which correspond to mRNA polyadenylated in eggs and deadenylated in embryos, and these constitute the founders members of the Eg family of mRNAs.

The von Hippel–Lindau tumour suppressor gene: uncovering the expression of the pVHL172 isoform

Background:The von Hippel–Lindau (VHL) gene encodes two mRNA variants. Variant 1 encodes two protein isoforms, pVHL213 and pVHL160, that have been extensively documented in the literature. Variant 2 is produced by alternative splicing of exon 2 and encodes a pVHL isoform of 172 amino acids with a theoretical molecular weight of 19 kDa (pVHL172), the expression of which has never been demonstrated so far due to the absence of suitable antibodies.Methods:We have generated an anti-pVHL monoclonal antibody (JD-1956) using pVHL172 recombinant protein. We tested the antibody against exogenous or endogenous expressed proteins in different cell lines. We identified the pVHL172 using a silencing RNA strategy. The epitope of the antibody was mapped using a peptide array.Results:We efficiently detected the three different isoforms of pVHL in cell lines and tumorigenic tissues by western blotting and immunohistochemistry and confirmed for the first time the endogenous expression of pVHL172.Conclusions:The endogenous expression of the three isoforms and particularly the pVHL172 has never been shown before due to a lack of a highly specific antibody since none of the available commercial antibodies distinguish the three isoforms of pVHL in cells or in both normal and cancerous human tissues. Evidence of pVHL172 expression emphasises the need to further study its implication in renal tumorigenesis and VHL disease.

Expression of RNA isolated from the water-shunting complex of a sap-sucking insect increases the membrane permeability for water in Xenopus oocytes

The highly specialized membranes of the filter chamber found in the digestive tract of some homopteran insects could represent a favorable material for characterizing water channels. In order to demonstrate that membrane proteins of this epithelial complex serve as water channels, we have investigated the membrane permeability for water in Xenopus oocytes injected with RNA isolated from the filter chamber. Volumes of oocytes injected with filter chamber RNA were increased by 15% following a 16-min osmotic shock, while volumes of oocytes injected with RNA from midgut not of filter chamber or with water were increased only by 8.5 and 10%, respectively. This significant difference in oocyte swelling leads us to conclude that RNA isolated from the filter chamber contains mRNA coding for water channel proteins.

Xenopus c-raf proto-oncogene: cloning and expression during oogenesis and early development.

Summary— We have isolated and characterized a cDNA which contains the entire coding sequence of Xenopus laevis raf protein. raf mRNA is identified as a member of the class of maternal RNAs. It is already relatively abundant at the beginning of oogenesis and is stable at least until the midblastula transition. The RNA is also detected later during embryogenesis in particular in gastrula, neurula, tailbud and feeding tadpole. We have also found the RNA in several adult tissues (skin, testis, stomach, intestine) at different levels.

Investigation of the functional properties and subcellular localization of alpha human and rainbow trout estrogen receptors within a unique yeast cellular context.

Estrogens are steroid hormones that play a pivotal role in growth, differentiation and function of reproductive and non-reproductive tissues, mediated through estrogen receptors (ERs). Estrogens are involved in different genomic and non-genomic cell signaling pathways which involve well-defined subcellular ER localizations. Thus, ER activity results from complex interplays between intrinsic binding properties and specific subcellular localization. Since these two factors are deeply intricate, we carried out, in a unique yeast cell context, a comparative study to better understand structure/function/subcellular distribution relationships. This was carried out by comparing two ERs: the human ER α subtype (hERα) and the short form of the α isoform of the rainbow trout ER (rtERαS). Their distinct binding properties to agonist and antagonist ligands and subcellular localizations were characterized in Saccharomyces cerevisiae yeast cells. An unexpected partial agonistic effect of ICI 182-780 was observed for rtERαS. Concomitant to distinct binding properties, distinct subcellular localizations were observed before and after ligand stimulation. Due to the unique cell context, the link between ERs intrinsic binding properties and subcellular localizations is partly unveiled and issues are hypothesized based on the role of cytoplasmic transient complexes which play a role in the ER cytoplasmic/nuclear partition, which in turn is critical for the recruitment of co-regulators in the nucleus.