Pre-mRNA editing is a recently discovered process that leads to the production of a variety of protein isoforms from a single gene. The A-to-I RNA editing, a distinct mechanism of mRNA editing in mammalian cells has been previously identified by our laboratory. Specifically, using HeLa cells we have shown that glutamate receptor subunit B (gluR-B) mRNA can undergo site specific deamination at an adenosine residue which converts adenosine to inosine (A-to-I), a critical change required to maintain receptor function. Two RNA-dependent adenosine deaminases (A-to-I RNA editase) have been identified in HeLa as well as a variety of other mammalian cells. In agreement with these observations, numerous inosine-containing mRNA (I-mRNA) were detected in different tissues as well. The presence of A- to-I RNA editase and I-mRNA in mammalian cells suggests that A- to-I RNA editing play an important role in gene expression and function. The broad, long-term objective of this proposal is to understand the mechanism and function of A-to-I RNA editing. Three specific aims are proposed as a basis for this research plan. First, to characterize I-mRNA in mammalian cells using the modified affinity purification approach to be established. Specifically, I-mRNA from HeLa cells will be enriched and identified. The cDNA of I-mRNA will be analyzed to determine the A-to-I editing site. The effect of the identified I-mRNA on protein identity, mRNA stability and structure will be studied in vitro and in vivo. Second, we will identify the site-specific interactions in the editase-RNA complex. Specifically, the conditions required for editase-RNA complex formation will be determined and intra-complex interactions between the editase and substrate and the catalytic core will be studied by site-specific UV cross-linking and base modifications. The possibility that the catalytic and RNA binding domains are functionally independent will be examined. Also, A-to-I RNA editase with a different RNA binding domain will be characterized, and protein factors associated with A-to-I RNA editase will be studied. Third, we will study the functions of A-to-I RNA editase in HeLa cells. Specifically, dominate-negative mutant and neutralizing antibody will be produced to establish a HeLa cell line in which editase activity can be quantitatively controlled. By using this cell line, the hypothesis that the dsRNA-dependent protein kinase (PKR) is mediated by the dsRNA editing activity of A-to-I RNA editase will be examined. The overall cellular effects of gene expression by A-to-I RNA editase and the I-mRNA decay will be studied. The proposed experiments will provide fundamental information regarding the role of A-to-I pre-mRNA editing in mammalian gene expression and regulation that could be used to combat disease.