The conversion of adenosine to inosine by RNA editing represents an increasingly important mechanism for generating diversity in neurotransmitter receptor expression. Such RNA modifications have been shown to alter both the ion permeation and electrophysiological properties of ligand-gated ion channels and to modulate the efficacy of receptor G-protein interactions. Recent studies in our laboratory have demonstrated that ADAR2, a double-stranded RNA-specific adenosine deaminase involved in the editing of glutamate and serotonin receptor transcripts, can selectively modify its own pre-mRNA, suggesting a negative autoregulatory mechanism by which ADAR2 can modulate its own expression level to prevent editing at aberrant sites. The long-term objectives of these studies are to define the cellular processes by which such RNA editing events can modulate neurotransmitter receptor expression and function in the central nervous system. To identify the cis-active regulatory sequences necessary for ADAR2 editing, a tissue culture model system will be utilized that exhibits RNA processing patterns analogous to those observed in vivo. Analyses of RNA from cells transfected with a variety of mutant ADAR2 minigene constructs will serve as the primary methodology for these mapping studies. An in vitro RNA editing system, utilizing purified, recombinant ADAR proteins, will also be used as a mapping technique and to further define the ADAR proteins responsible for site-specific modifications of ADAR2 pre-mRNA. The effect of ADAR2 protein levels on the specificity of adenosine modification will be examined using an inducible tissue culture system and an in vitro editing system to test whether over expression of ADAR2 can lead to the modification of known ADAR2 substrates(GluR-Band 5-HT2CR) at aberrant sites competition studiesbetweenADAR2 pre-mRNA and otherADAR2 substrates will be performed to determine if such competition represents a cellular mechanism for maintaining appropriate ADAR2 protein levels. To examine the molecular and phenotypic consequences of ADAR2 misregulation, genetically modified strains of mice will be generated in which ADAR2 levels have been altered by over expression or by selective ablation of ADAR2 auto editing. Multiple, independent mouse lines will be assessed for gross alterations in animal phenotype, changes in the level of ADAR2 expression/activity, alterations in editing patterns for previously identified ADAR2 substrates and increases in mRNA-derived inosine content. It is anticipated that these studies will provide new insights concerning the cellular processes modulatingADAR2 expression as well as the effects that such modulation confer upon the expression of neurotransmitter receptor isoforms.
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