A Rapid New Assay to Detect RNA Editing Reveals Antipsychotic-Induced Changes in Serotonin-2C Transcripts

ABSTRACT We report the development of a new assay as an alternative to direct DNA sequencing to measure RNA-edited variation in tissue. The new assay has been validated and is accurate, cheaper, more rapid, and less labor-intensive than DNA sequencing. We also outline the statistical modeling required for analyses of the hierarchical, clustered RNA-editing data generated in these studies. Using the new technique, we analyzed the effects of long-term antipsychotic medication on serotonin-2C receptor (5-HT 2C R) RNA editing in rat brain. Our hypothesis that a drug with high affinity for 5-HT 2C R, such as clozapine, would alter its RNA-editing profile was not confirmed. Whereas haloperidol, a typical antipsychotic drug that is primarily a dopamine receptor antagonist, reduced 5-HT 2C VNV isoform frequency and the level of RNA editing at the D site, risperidone and not the prototype atypical antipsychotic drug clozapine increased the frequency of 5-HT 2C VNV and D-site editing. Our data emphasize that caution is required in the interpretation of RNA-editing data in studies of psychiatric disorders, because these studies usually include subjects who received long-term exposure to medication. This newly established method will facilitate high-throughput investigations of RNA editing in disease pathology and in the pharmacological activity of drugs. The revelation that the human genome comprises between 30,000 and 40,000 protein-coding genes was unexpected, because such few genes are unlikely to explain the functional diversity between humans and less complex organisms. These interspecies differences could arise from post-transcriptional processes such as RNA editing and alternative splicing, which allow a single gene to generate several protein variants. The current study focuses on RNA editing. The recent detection of 1637 potential new substrates for RNA editing Because of their relatively recent discovery, substrates of RNA editing have not been extensively characterized. Adenine-to-inosine RNA editing in mammalian brain has been identified in ionotropic receptors (such as the GluR2 subunit of the ␣-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor) and in the serotonin (5-HT)-2C receptor (R), which is coupled to GTP-binding protein. Discrepancies between genomic DNA and cDNA sequences led to the discovery of nucleotide changes caused by the activity of ADAR enzymes. In the fully edited 5-HT 2C R, three amino acid codons in the pre-mRNA are changed so that the sequence coding for IRNPI becomes VRGP