Title

dADAR, a Drosophila RNA editing enzyme, modifies ion channel transcripts and is required for normal behavior and nervous system integrity

Date of Completion

January 2000

Keywords

Biology, Molecular|Biology, Neuroscience|Biology, Genetics

Degree

Ph.D.

Abstract

Although A-to-I RNA editing activity was discovered in mammals a decade ago, little is known about the biological function of editing in metazoans. Many RNA editing sites have been characterized in para and cac transcripts. I have cloned and characterized a Drosophila RNA editing enzyme that acts on RNA, dADAR. Embryonic in situ hybridization shows dADAR expression in the developing nervous system coincident with cac and para expression. Molecular analysis of dADAR demonstrated extensive alternative splicing and alternative promoter usage. Semi-quantitative RT-PCR was used to determine the relative contribution of the two promoters throughout development. Additionally, I found and characterized an evolutionarily conserved and developmentally regulated RNA editing site in the dADAR transcript itself, the S/G site. The editing efficiency at the S/G site was shown to correlate negatively with alternative exon 3a inclusion. Interestingly, exon 3a containing transcripts are only detected from one of the two dADAR promoters. I hypothesize that dADAR S/G editing reflects an autoregultory function of dADAR. ^ Using a site-selected mutagenesis approach, I have identified aphenotypic transposon insertions into the dADAR locus. By remobilizing a P element and screening F2 progeny for lethal mutations linked to dADAR, 13 mutations were obtained. All of these mutations fail to complement each other phenotypically and molecularly, and all of the phenotypes are rescued by a duplication containing the dADAR locus. Semi-quantitative RT-PCR and assays of non-specific A-to-I activity, a measure of ADAR activity, indicate that these represent null alleles. Twenty-five specific A-to-I RNA editing sites have been reported in transcripts of three ion channel genes in Drosophila. I have demonstrated that none of these sites are edited in dADAR animals. Together these data suggest that dADAR is the major, and possibly only, RNA editing enzyme in Drosophila. ^ Interestingly, dADAR mutant animals develop into morphologically normal flies. However, dADAR flies are severely behaviorally compromised and develop progressive neurodegeneration of the central brain and optic lobes, which correlates with an increased frequency of seizures and tremors. These data suggest that RNA editing is primarily needed to fine-tune behavior and maintain nervous system integrity. ^