The N-methyl-D-aspartate (NMDA) receptor, an excitatory neurotransmitter receptor in the brain, is an important site of action of ethanol. Following chronic ethanol treatment in vivo and in vitro, NMDA receptor number and function are upregulated, with a concomitant increase in R1 and R2B polypeptide levels in vitro. Similar ethanol treatment in vitro increases R1 mRNA half-life from 16 h to more than 24 h (Kumari and Ticku, 1998a) indicating that post-transcriptional mechanisms operate to augment NMDA receptor number in cortical neurons exposed to chronic ethanol treatment (50 mM, 5 days). More recently, we observed that de novo protein synthesis is required for ethanol-induced stabilization of R1 mRNA (Kumari and Ticku, 1998b), suggesting that ethanol-induced unknown protein factor(s) mediate this effect. Long term plans of this project are to elucidate the post-transcriptional mechanisms involved in the stabilization of NMDA R1 mRNA in fetal cortical neurons exposed to chronic ethanol treatment. Hypothesis to be tested in this proposal are (1) to identify specific RNA sequences (or cis-acting regulatory elements) of the R1 mRNA; and, (2) the nature of ethanol-induced cytoplasmic protein(s) (or trans-acting factors) that interact with cis- acting RNA regulatory sequences. These objectives will be achieved by (a) examining whether ethanol induces transcription of a stable R1 splice-variant; (b) delineating the cis -acting regulatory region(s) within the primary sequence of the R1 mRNA using cell-free mRNA decay assay and cell transfections; (c) dissecting the cis-acting sequences within the regulatory region defined above using mutants created by nested deletion, linker scanning and base substitution coupled to RNA gel shift assays and cell transfections, and finally (d) identifying the nature of trans-acting factor(s) by UV cross-linking and Northwestern analysis. A more thorough understanding of the pertinent molecular mechanisms through which ethanol modulates NMDA R1 mRNA stability may permit the design of novel therapeutic approaches to alcohol-related diseases.
