EDITING OF PRE MRNA IN NEUROLOGICAL CELL LINES

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DESCRIPTION (From the Applicant's Abstract): ADAR2 is an Adenosine Deaminase that acts on RNA, i.e. an RNA editase that can deaminate specific adenine (A) residues in specific pre-mRNAs to produce inosines (I). Because the ribosome reads an inosine as a guanosine (G), these deaminations cause codon changes that can result in changes in the amino acid sequence of the associated protein. Biologically important transcripts that undergo such deaminations include pre-mRNAs for several glutamate receptor subunits and for a serotonin receptor. In many of these cases the amino acid change has profound effects on the protein function. The frequency of deamination is developmentally regulated and rarely reaches 100 percent. Thus, this type of RNA editing is a mechanism for creating subtle and critical protein sequence diversity. Measurements of the inosine content of brain mRNA and the expression level of ADAR2 in brain regions suggest that there are numerous additional, as yet unidentified, ADAR2 substrates. Given the critical roles played by glutamate and serotonin receptors in neurotransmission, the overall role of mRNA editing in brain development and function may be profound. Alterations or modifications of editing patterns caused by polymorphism or mutation will be good candidates for involvement in neurological abnormalities. The goal of this pilot project is to evaluate the prevalence and relevance of ADAR2 mRNA editing by the identification and characterization of novel substrates expressed in neurologically derived human cell lines. To do this we will use a new procedure to isolate inosine-containing mRNA molecules from human neuroblastoma and NT2 cell lines transformed to stably overexpress human ADAR2 cDNA. Inosine-containing molecules will be cloned, sequenced, and analyzed by RT-PCR for patterns in A to I changes. Each ADAR2 substrate will be mapped in human and mouse. Definition of new substrates for RNA editing will define new sources of protein diversity and regulation of gene expression, and will identify new candidates for neurological disease and developmental anomalies.