REGIONAL DIFFERENCES IN THE EXTENT OF RNA EDITING OF THE GLUTAMATE-RECEPTOR SUBUNITS GLUR2 AND GLUR6 IN RAT-BRAIN

The extent of RNA editing of the glutamate receptor subunits GluR2 and GluR6 was studied by using a newly developed method based on the rest riction analysis of the subunit-specific polymerase chain reaction (PC R) product with the enzyme Bbv 1. Total RNA was isolated from followin g brain regions: cortex, striatum, hippocampus, thalamus, hypothalamus , cerebellum pons/medulla oblongata and white matter. RNA was transcri bed into cDNA, which was used as template for PCR. PCR was run with Gl uR2- and GluR6-specific primers to amplify a product across the edited region. The PCR products were analysed with the restriction enzyme Bb v 1 and gel electrophoresis of the restriction digest. Bbv 1 recognize s the sequence GCAGC which is identical with the sequence of the PCR p roduct originating from unedited GluR2 or GluR6 mRNA. Thus, this enzym e splits the non-edited PCR product into two fragments while leaving t he edited PCR product intact. After electrophoresis of the restriction digest and photographing gels, optical density of bands was quantifie d with image analysis. For quantification calibration curves were made with PCR products from constructs originating from edited and non-edi ted GluR6 mRNA. GluR2 mRNA was completely edited in all brain structur es studied. Editing of GluR6 mRNA, in contrast, was high in gray matte r structures (above 90%) but considerably lower in the pons/medulla ob longata (66%) and white matter (55%). It is, therefore, suggested that editing of GluR2 and GluR6 mRNA is performed by different enzymatic a ctivities. Studying RNA editing of glutamate receptor subunits will ex tend knowledge about the role of calcium fluxes through non-NMDA gluta mate receptor ion channels.