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 ?-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3dAMP), 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).
Following fertilization in amphibian, early cleavage stages are maternally controlled at a post-transcriptional level before initiation of zygotic transcriptions at the mid blastula transition (MBT). We document the expression levels of the axolotl Awnt-1, Awnt-5A and Awnt-5B genes as well as the adenylation states of their corresponding mRNAs from the end of oogenesis until the tailbud stages. Awnt-1/-5A RNAs are stable until MBT then degraded before gastrulation. Awnt-5B RNAs are degraded at fertilization and zygotically expressed after MBT with high level expression from gastrulation. Estimation of the poly(A) tail lengths reveals no direct link between deadenylation and degradation periods for each Awnt transcript. To investigate the molecular mechanisms involved in Awnt-1/-5A/-5B RNAs stability, synthetic full-length or 3 untranslated region (UTR) Awnt RNAs progressively deleted from their 3 end were microinjected in axolotl oocytes, unfertilized and fertilized eggs. We identified degrading and stabilizing sequences in the 3UTR whose activities depend on the cellular context and are also modulated by the 5UTR and coding sequence within each RNA. Using axolotl nuclear extracts from stage VI oocytes, we further produced evidence of destabilizing factors targeting the Awnt-5B RNAs. Altogether, these results show that oocyte maturation and late cleavages following MBT are two important periods when axolotl Wnt RNAs are highly regulated.
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