103 research outputs found
mRNP3 and mRNP4 are phosphorylatable by casein kinase II in Xenopus oocytes, but phosphorylation does not modify RNA-binding affinity
AbstractmRNP3 and mRNP4 (also called FRGY2) are two mRNA-binding proteins which are major constituents of the maternal RNA storage particles of Xenopus laevis oocytes. The phosphorylation of mRNP3â4 has been implicated in the regulation of mRNA masking. In this study, we have investigated their phosphorylation by casein kinase II and its consequence on their affinity for RNA. Comparison of the phosphopeptide map of mRNP3â4 phosphorylated in vivo with that obtained after phosphorylation in vitro by purified Xenopus laevis casein kinase II strongly suggests that casein kinase II is responsible for the in vivo phosphorylation of mRNP3â4 in oocytes. The phosphorylation occurs on a serine residue in a central domain of the proteins. The affinity of mRNP3â4 for RNA substrates remained unchanged after the treatment with casein kinase II or calf intestine phosphatase in vitro. This suggests that phosphorylation of these proteins does not regulate their interaction with RNA but rather controls their interactions with other proteins
Caractérisation du rÎle d'Unr, une protéine de liaison à l'ARN, dans les cellules souches embryonnaires murines
Le gĂšne unr (upstream of N-ras) code une protĂ©ine de liaison Ă l ARN, Unr, qui rĂ©gule la stabilitĂ© et la traduction d ARN messagers cibles. L invalidation du gĂšne unr chez la souris conduit Ă une lĂ©talitĂ© embryonnaire Ă mi-gestation (10,5 jours post-coitum, jpc). Unr est donc essentielle pour le dĂ©veloppement de la souris. Le phĂ©notype le plus frappant des embryons unr-/- est leur petite taille qui est dĂ©jĂ visible Ă 8,5jpc. Ce phĂ©notype pourrait reflĂ©ter un problĂšme prĂ©coce de prolifĂ©ration/diffĂ©renciation au cours du dĂ©veloppement qu il est possible d Ă©tudier dans les cellules souches embryonnaires (ES). Les cellules ES sont dĂ©rivĂ©es des cellules pluripotentes des embryons au stade blastocyste (3,5jpc). Les cellules ES peuvent s auto-renouveler c est Ă dire prolifĂ©rer indĂ©finiment sous forme non diffĂ©renciĂ©e ce qui correspond Ă leur Ă©tat pluripotent ou diffĂ©rencier en tous les types cellulaires (lignages) adultes dĂ©rivĂ©s des feuillets primordiaux (endoderme dĂ©finitif, mĂ©soderme et ectoderme) ce que dĂ©fini la pluripotence. Ces deux propriĂ©tĂ©s des cellules ES conditionnent leur devenir et dĂ©finissent leur identitĂ©. Nous avons remarquĂ© que les cellules ES unr-/- ont tendance Ă diffĂ©rencier spontanĂ©ment alors qu elles sont cultivĂ©es dans des conditions qui les maintiennent dans un Ă©tat non diffĂ©renciĂ© et prolifĂ©ratif (Ă©tat pluripotent). En routine, les cultures de cellules ES unr-/- contiennent environ 25% de cellules morphologiquement diffĂ©renciĂ©es. Nos travaux montrent en effet, qu elles diffĂ©rencient en endoderme primitif. Nous avons reproduit ce phĂ©notype dans une autre lignĂ©e des cellules ES de fond gĂ©nĂ©tique diffĂ©rent par dĂ©plĂ©tion stable d Unr. La restauration de l expression d Unr dans les cellules ES unr-/- limite fortement leur engagement en diffĂ©renciation. Unr contribue donc au maintient de l Ă©tat pluripotent des cellules ES en prĂ©venant leur diffĂ©renciation spontanĂ©e vers le lignage endodermique primitif (Epr). Ce tissu au cours du dĂ©veloppement va former l Ă©pithĂ©lium de la poche embryonnaire ou sac vitellin. Nos donnĂ©es prĂ©liminaires montrent que les cellules ES en absence d Unr maintiennent tout de mĂȘme leur capacitĂ© de diffĂ©renciation multi-lignages (endoderme dĂ©finitif, mĂ©soderme et ectoderme) quand celle-ci est induite. Ensuite, nous nous sommes intĂ©ressĂ©s au(x) mĂ©canisme(s) d action d Unr. Nous avons fait l hypothĂšse qu Unr pourrait directement agir en rĂ©gulant positivement des gĂšnes qui inhibent la diffĂ©renciation des cellules ES en Epr ou en rĂ©gulant nĂ©gativement des gĂšnes qui l induisent. Nous avons identifiĂ© le gĂšne gata6 comme cible potentielle d Unr. Une augmentation modĂ©rĂ©e de l expression du facteur de transcription Gata6 dans les cellules ES conduit Ă une autorĂ©gulation positive du gĂšne gata6 et induit la diffĂ©renciation des cellules ES en Epr. Nos donnĂ©es suggĂšrent qu Unr pourrait directement dĂ©stabiliser les ARNms Gata6 dans les cellules ES afin de prĂ©venir leur diffĂ©renciation spontanĂ©e en Endoderme primitif.Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein with cold shock domains, involved in regulation of messenger RNA stability and translation. Unr is essential to mouse development since Embryos deficient for Unr die at mid-gestation. Here we report that unr knockout ES cells maintained under growth conditions that sustain self-renewal spontaneously differentiate toward the primitive endoderm (PrE) lineage. This phenotype was reproduced in another ES line (E14tg2a) after shRNA-induced Unr depletion. Moreover, Unr rescue in Unr-deficient ES cells limits their PrE differentiation engagement. However, Unr is dispensable for multilineage differentiation, as shown by knockout ES cells capacity to produce differentiated teratomas. We further investigated the molecular mechanisms underlying the differentiation of unr-/- ES to primitive endoderm, and found that Unr acts downstream of Nanog. Our data also show Gata6 mRNAs are more stable in Unr-deficient ES cells as compared to wild-type ES cells. We propose that the possible repression by Unr of this key inducer of PrE differentiation at a post-transcriptional level may contributes to the stabilization of ES cells pluripotent state.BORDEAUX2-Bib. Ă©lectronique (335229905) / SudocSudocFranceF
Identification de nouveaux gÚnes de sensibilité aux agents antitumoraux par sélection d'éléments génétiques suppresseurs (modulation de la sensibilité cellulaire aux dommages de l'ADN par des protéines Arginine N-Methyltransférases)
PARIS-BIUSJ-ThĂšses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocCACHAN-ENS (940162301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF
Bleomycin resistance in mammalian cells expressing a genetic suppressor element derived from the SRPK1 gene
The genetic suppressor element (GSE) approach allows identification of genes essential for certain cell phenotypes. To identify genes controlling the cell response to cytotoxic agents, a normalized retroviral library of randomly fragmented cDNAs from the Chinese hamster cell line DC-3F was screened for GSEs conferring resistance to bleomycin. One of these GSEs, GSE(BLM), conferring an approximately 2-fold bleomycin resistance in DC-3F cells, displayed 98% identity with an amino acid sequence located in the functional domain of human SRPK1. Using GSE(BLM) as a probe, we cloned a cDNA with a nucleotide sequence that was 76.7% identical to that of human SRPK1, whereas the corresponding amino acid sequence was 92.6% identical to that of this enzyme. When GSE(BLM), inserted in the retroviral vector pLNCX, was transduced in HeLa cells, its expression resulted in a 5-10-fold bleomycin resistance, which was abolished when these cells were further transfected with SRPK1 cDNA. In our experimental conditions, DC-3F or HeLa cells expressing GSE(BLM) did not show any detectable cross-resistance to other cytotoxic agents with various mechanisms of action. GSE(BLM), which is sense oriented in the vector, is likely to be translated in a peptide active as a dominant-negative inhibitor of SRPK1. SRPK1 is a protein serine kinase that regulates the activity of RS-proteins (arginine-serine-rich proteins), a group of nuclear factors controlling various physiological processes
Synthesis of Nâ(9HâXanthenâ9âyl)aminoalkanamide and Nâ(9HâThioxanthenâ9âyl)aminoalkanamide Derivatives and their in vitro Evaluation as Potential Intercalators and Antitumor Drugs
A series of new Nâ(9Hâxanthenâ9âyl)aminoalkanamide and Nâ(9Hâthioâxanthenâ9âyl)aminoalkanamide derivatives was synthesized and evaluated as potential intercalators by measuring their DNA binding affinity. They were also tested for cytotoxic activity against L1210. The results suggest that the cytotoxicity of these molecules was not due to an intercalating mechanism. Copyright © 1994 WILEYâVCH Verlag GmbH & Co. KGaA, Weinhei
Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.
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