Reenan 9728737 Adenosine to inosine (A to I) RNA editing has emerged as a novel mechanism for regulating protein activity through alterations in a protein's primary amino-acid sequence. Mammalian RNA editases, such as DRADA and RED1, have been identified and shown to modify mRNA by hydrolytic deamination of certain adenosine residues to inosine. This reaction occurs via a double-stranded (ds) RNA intermediate. The goal of this research is to examine the process of A to I RNA editing in a model genetic system, Drosophila melanogaster. Preliminary results from this laboratory have identified RNA editing sites in transcripts of the para locus, which encodes a voltage-gated sodium channel. In addition, a candidate editase gene has been cloned from Drosophila, DRED-1 (Drosophila RNA editase-1). Experiments will be performed to address the nature of para RNA editing sites through in vivo and in vitro experiments using purified DRED-1 protein and synthetic para RNA substrates. Site-directed mutagenesis will be used to identify cis-acting regulatory sequences necessary for efficient RNA editing. In order to address whether the DRED-1 protein is essential for viability, site directed transposon mutagenesis of the DRED-1 locus will be performed. These experiments, in addition to determining the effect of DRED-1 knockouts, will address the necessity of DRED-1 activity for editing of para. Another approach, targeted tissue specific expression of a dominant negative DRED-1 protein, will address the effects of negating DRED-1 activity in selected tissues. Lastly, the investigator will utilize immunolocalization of the DRED-1 protein to Drosophila salivary gland polytene chromosomes to identify new targets for the process of RNA editing. The process whereby information encoded by genes is used to direct the synthesis of proteins is crucial to the proper development and function of all organisms. This research project will examine an important phenomenon called RNA editing, in which the infor mation in messenger RNAs, which are copies from genes, is changed or edited, resulting in a change in the final protein product. The changes in protein structure resulting from messenger RNA editing, can have a profound influence on a protein's function. This research will utilize a very powerful experimental system, the fruit fly Drosophila melanogaster, to dissect the role of RNA editing in the function of a complex organism, examining effects ranging from development to adult behavior.
