Serotonin-2C receptor pre-mRNA editing in rat brain and in vitro by splicesite variants of the interferon-inducible double-stranded RNA-specific adenosine deaminase ADAR1

The interferon-inducible RNA-specific adenosine deaminase (ADAR1) is an RNA editing enzyme implicated in the site-selective deamination of adenosine t o inosine in cellular pre-mRNAs. The pre-mRNA for the rat serotonin-2C rece ptor (5-HT2CR) possesses four editing sites (A, B, C, and D), which undergo A-to-I nucleotide conversions that alter the signaling function of the enc oded G-protein-coupled receptor. Measurements of 5-HT2CR pre-mRNA editing i n vitro revealed site-specific deamination catalyzed by ADAR1, Three splice site variants, ADAR1-a, -b, and -c, all efficiently edited the A site of 5 -HT2CR pre-mRNA, but the D site did not serve as an efficient substrate for any of the ADAR1 variants. Mutational analysis of the three double-strande d (ds) RNA binding motifs present in ADAR1 revealed a different relative im portance of the individual dsRNA binding motifs for deamination of the A si te of 5-HT2CR and synthetic dsRNA substrates. Quantitative reverse transcri ption-polymerase chain reaction analyses demonstrated that the 5-HT2CR pre- mRNA was most highly expressed in the choroid plexus of rat brain. However, ADAR1 and the related deaminase ADAR2 showed significant expression in all regions of the brain examined, including cortex, hippocampus, olfactory bu lb, and striatum, where the 5-HT2CR pre-mRNA was extensively edited.