Adenosine deaminases that act on RNA (ADARs) convert
adenosine to inosine in double-stranded regions of RNA.
ADAR activity is in the nucleus in Xenopus laevis
stage VI oocytes, and released into the cytoplasm at oocyte
maturation. We previously demonstrated that a cytoplasmic
double-stranded RNA (dsRNA) binding factor(s), cyto-dsRBP,
protects microinjected dsRNA from the ADAR released at
maturation. Here we describe experiments to determine whether
an endogenous dsRNA, the duplex formed between sense and
antisense transcripts of basic fibroblast growth factor
(bFGF), is protected in a similar manner. Consistent with
the presence of cyto-dsRBP, we observed that the majority
of bFGF RNA was not deaminated, before or after maturation.
However, a minor fraction of the bFGF RNA was deaminated
whether the RNA was isolated from stage VI oocytes or matured
oocytes. Since ADAR activity is in the nucleus in stage
VI oocytes, our results suggest that a fraction of the
bFGF RNAs are hybridized in the nucleus and are ADAR substrates.
Adenosine deaminations result in A-to-G changes in cDNAs,
so we quantified the fraction of modified molecules using
restriction-enzyme assays of RT-PCR products. Caveats due
to recombination during RT-PCR are discussed.