MOLECULAR BIOLOGY OF DSRNA ADENOSINE DEAMINASE

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Adenosine deaminases (ADARs) comprise a family of RNA editing enzymes that convert adenosine to inosine within RNA. This activity is phylogenetically conserved and is suspected to have a wide-variety of biological roles. So far, the only known function of ADARs is to deaminate adenosines in codons of selected mRNAs, resulting in the synthesis multiple protein isoforms from a single transcription unit. ADARs are involved in producing functionally important isoforms of hepatitis delta antigen as well as several cellular proteins involved in neurotransmission, including glutamate and serotonin receptors. The long-term goal of this proposal is to determine additional functions for cellular ADARs and to learn the biochemical properties of these enzymes to allow them to fulfill their roles. In vitro biochemical studies will be accompanied by in vivo studies in the nematode Caenorhabditis elegans (C. elegans). For the in vitro studies, site-directed mutagenesis will be used to gain information about the amino acids involved in catalysis and metal binding and inhibitor studies will be used to distinguish between possible catalytic strategies. In vitro studies will also be used to investigate the substrate specificity of the enzyme. To understand how RNA structural features allow ARAR selectivity, deamination will be monitored in RNA substrates designed to mimic structural features of biological substrates, and in addition, in vitro selection experiments will be performed. To reveal uncharacterized biological roles, a recently developed technique to identify inosine-containing RNAs will be used to identify cellular ADAR substrates in C. elegans. Analysis of C. elegans strains in which the worm ADAR genes have been inactivated will supplement these studies.