title: Evidence for an RNA polymerization activity in axolotl and Xenopus egg extracts.
abstract: We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE) or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE) from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i) the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii) the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3'dAMP), but sensitive to cordycepin 5'-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii) the RNA polymerization is not a 3' end labelling and that iv) the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp).
authors.affiliation: Université Pierre et Marie Curie, UMR 7622, Paris, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Pelczar
authors.forename: Anne
authors.initials: A
authors.lastname: Woisard
authors.forename: Jean Marc
authors.initials: JM
authors.lastname: Lemaître
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chachou
authors.forename: Yannick
authors.initials: Y
authors.lastname: Andéol
mesh_terms: D053959:Alpha-Amanitin / Q000378:metabolism; D000558:Ambystoma mexicanum / Q000378:metabolism*; D000818:Animals; D017104:Bacteriophage M13 / Q000378:metabolism; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000737:chemistry; D003843:Deoxycytidine Monophosphate / Q000378:metabolism; D019143:Evolution, Molecular; D005260:Female; D009865:Oocytes / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism; D012320:RNA Polymerase III / Q000378:metabolism; D014981:Xenopus
chemical_list: D053959:Alpha-Amanitin; D003838:Deoxyadenine Nucleotides; D003843:Deoxycytidine Monophosphate; C011746:3'-deoxyadenosine 5'-triphosphate; D012313:RNA; D004247:DNA; D012319:RNA Polymerase II; D012320:RNA Polymerase III