Protection of cytoplasmic double-stranded RNA from adenosine deaminases that act on RNA in Xenopus laevis

Adenosine deaminases that act on RNA (ADARs) are a family of enzymes that promote the conversion of adenosine to inosine within double stranded (ds) regions of RNA. The mechanism for adenosine to inosine conversion is a hydrolytic deamination of the N6 exocyclic amino group of adenosine. ADAR activity is present in all metazoans that have been examined including mammals, amphibians, D. melanogaster and C. elegans. This dissertation describes studies of ADAR activity during early development in Xenopus laevis, specifically during meiotic maturation, the period when a stage VI oocyte matures to form an egg. I show that ADAR activity is located in the nucleus of stage VI oocytes and is released into the cytoplasm upon nuclear breakdown during meiotic maturation. I also demonstrate the presence of a factor in the cytoplasm of Xenopus oocytes that protects exogenous dsRNA from ADAR activity released into the cytoplasm at meiotic maturation. Characterization of this factor in vitro demonstrated that protection from ADAR activity is specific for dsRNA. Accordingly, this factor was named cytoplasmic dsRNA binding protein (cyto-dsRBP). These data suggest that endogenous, cytoplasmically located, dsRNA in oocytes will also be protected from ADAR activity during meiotic maturation. Experiments were performed to explore this possibility. In Xenopus the basic fibroblast growth factor (bFGF) gene is transcribed bidirectionally resulting in the production of both a sense and antisense transcript. A 900 bp region of complementarity exists between the sense and antisense mRNAs at each of their 3' ends. Previous data suggested that the complementary region within the mRNAs is hybridized in Xenopus stage VI oocytes. I show that the majority of the bFGF mRNA population isolated after meiotic maturation remained predominantly unmodified, even though ADAR activity is released into the cytoplasm at this time. These data suggest that the bFGF mRNA in the cytoplasm of Xenopus oocytes is protected from ADAR activity released at maturation, consistent with cyto-dsRBP. However, a small percentage of the bFGF mRNA population isolated from stage VI oocytes, as well as from matured oocytes, contained sequence changes consistent with ADAR deamination. The implications of the minor amounts of deaminated bFGF mRNA that I observed are discussed.