Identification of new targets of Drosophila pre-mRNA adenosine deaminase

Adenosine deaminase acting on RNA (ADAR) in Drosophila and mammals has recently become the target of numerous investigations. It is now clear that this protein has a number of functions in the nervous system. Indeed, the mutation of ADAR in Drosophila (dADAR) results in many pathological and physiological changes, such as sensitivity to hypoxia and neuronal degeneration. To understand the full scope of dADAR function, it is crucial to identify new dADAR targets. A polyclonal antibody against inosine was developed and used to enrich inosine-containing mRNAs. The efficiency of immunoaffinity purification was confirmed for the Q/R editing site of GluR-B pre-mRNA that has been edited by ADAR2 to generate inosines at the editing site. This approach was applied to enrich inosine-containing mRNAs from total mRNAs of wild-type and dADAR mutant flies, respectively. The enriched mRNA portion was then amplified and hybridized with Drosophila cDNA arrays. With this method, over 500 mRNAs were identified as potential dADAR targets by showing a higher amount in the enriched mRNA portion from wild-type flies than from dADAR mutant flies. The occurrence of A-to-G conversion in these mRNAs was further analyzed by comparing over 7,000 Drosophila cDNAs sequences with their genomic sequences. A final list of 62 candidates was generated from the overlap of the two approaches. Twelve genes from the final list were further examined by sequencing the RT-PCR products of these genes from wild-type and dADAR mutant flies. Seven of the 12 genes were proven to have A-to-G changes in the wild-type but not in mutant flies. We conclude that the combination of immunoaffinity enrichment of inosine-containing mRNA, DNA microarrays, and sequence comparison could facilitate the discovery of new dADAR substrates, which in turn allows us to better understand the targets of dADAR and the biological function of A-to-I RNA editing in flies.