RNA editing in Drosophila is modulated by alternative splicing and by self-editing. L.P. Keegan 1, A. Gallo 1, J. Brindle 1, G.M. Ring 1, A. Weathers-Lowin 2, R. Reenan 2, M. O'Connell 1. 1) Dept Chromosome Biol, MRC Human Genetics Unit, Edinburgh, Scotland; 2) University of Connecticut Health Center, Genetics, 263 Farmington Ave, Farmington, CT 06030.
Drosophila has a single CNS-expressed Adar (Adenosine Deaminase acting on RNA) gene on the X chromosome encoding the RNA editing enzyme that edits codons in pre-mRNAs encoding ion channel subunits (1,2). To understand the regulation of this process ADAR protein isoforms that are expressed at different stages during development have been overexpressed, purified, and assayed for editing activity in vitro on an RNA substrate we have defined from the cacophony transcript. Rescue of the Adar mutant phenotype has also been measured using the GAL4-UAS system for cDNA expression.
In vivo rescue is quantitated using open field locomotion tests to measure restoration of walking activity in male Adar mutants. Male Adar mutant flies are viable but severely defective in walking, flying and mating. ADAR comprises two dsRNA binding domains and a deaminase domain. Embryos and larvae express an alternatively spliced form of ADAR with an increased spacing between the dsRNA binding domains that is less active in vitro and less effective in rescuing the Adar walking defect. Transcription of Adar increases in adults and transcripts undergo an RNA editing event that we have also reconstructed in vitro. Unusually, the sequences required for self-editing reside in the exon rather than in a flanking intron. Self editing leads to expression of an ADAR isoform in which a conserved serine in the catalytic domain is changed to glycine. This isoform has drastically reduced editing activity in vitro, suggesting that self-editing is a mechanism for negative autoregulation.
(1) Keegan et al. Nature Reviews Genetics (2001), 2, 869-878
(2) Palladino et al. Cell, (2000), 102, 437-449.