L-glutamate is the major excitatory neurotransmitter in the mammalian brain and serves to mediate both fast excitatory responses and the long-term changes in synaptic efficacy underlying learning and memory. Recent studies have indicated that the electrophysiological function of the non- NIMDA subtypes of inotropic glutamate receptors may be modulated by a novel RNA modification process referred to as RNA editing; the long-term objectives of th proposed research are to define the cellular mechanisms involved in the regulation of glutamate receptor subunit expression in the central nervous system. We propose to precisely identify the cis-active regulatory sequences responsible for dictating the subunit-specific pattern of RNA processing for mRNAs endoding subunits (GluR-B, GluR-5, GluR-6) of the non-NIMDA subtype of glutamate receptor. These studies will take advantage of tissue culture model systems which exhibit RNA processing patterns analogous to those observed in vivo. Analyses of RNA from the rat C6 glioma cell line, transfected with a variety of mutant GluR transcription units, will serve as the primary methodology for these mapping studies. Development of an in vitro RNA editing reaction utilizing nuclear extracts from C6 and HeLa cells will also be used as a mapping technique by testing the ability of in vitro transcribed RNA transcripts to be accurately modified. Isolation and characterization of the cellular machinery involved in GluR-B RNA editing will be based upon an in vitro RNA editing system in which crude nuclear extracts from editing-competent cell lines will be incubated with a variety of non-NMDA receptor-derived RNA substrates. This in vitro assay system will serve as a direct biochemical approach by which any added factor preparation may be assessed for its ability to affect RNA editing patterns and will allow for biochemical purification of the cellular machinery involved in such post-transcriptional processing reactions. Determination of the biochemical mechanism involved in GluR-B RNA editing will take advantage of an in vitro RNA editing system in which substrate RNAs may be specifically labeled with a variety of radionuclide and nucleotide analogs to determine the fate of the specific label subsequent to RNA modification. These studies will serve to distinguish between the possible mechanisms used by various cellular enzymatic pathways to substitute one nucleic acid base for another in the post-transcriptional modification of GluR-B transcripts. It is anticipated that characterization of the mechanisms regulating the RNA editing of non-NMDA glutamate receptor subunits may provide insights into the cellular processes involved in glutamate-mediated neurotoxicity as well as provide information regarding the post-transcriptional mechanisms by which multiple mRNAs are produced from complex transcription units.
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