Double-stranded RNA (dsRNA) is produced within cells by several processes that have an important impact on cell physiology. Examples include mRNA processing, switching off gene expression by endogenous antisense RNA, and replication of RNA viruses. Cells have evolved a novel machinery for dealing with these molecules, a dsRNA specific activity that generates base-pair mismatches, and thus unwinds dsRNA, by converting many adenosine residues to inosinne. Although the in vivo role of this dsRNA adenosine deaminase or DRADSA has remained obscure since its discovery in 1987, it is most likely to affect the fate of the RNA duplex formed between sense and antisense RNAs and to interfere with the translation of the mRNAs modified by DRADA. Precise knowledge of the fate of dsRNA in vivo or the molecular mechanism for suppression of targeted genes translation is prerequisite for applicaiton of antisense RNA technology to develop therapeutics designed to block viral infection or to treat tumors. The goal of this project is to better understand the effect of DRADA on the antisense RNA strategy. We have recently succeeded in isolating DRADA clones. We will conduct in-depth investigation on the effect of the DRADA on the sense and antisense RNA duplex and its fate using DRADA expression constructs and purified recombinant DRADA proteins available in our laboratory.
Two specific aims are sought: 1. The effect of DRADA on the stability of the dsRNA will be investigated by transfecting a DRADA conditional expression construct into a cell line in which the expression of the antisense c-fos RNA can be manupulated conditionally. In addition, the effect of DRADA on the translation of the antisense RNA targeted endogenous c-fos mRNA will be investigated. 2. Using purifed recombinant DRADA proteins, we will examine in vitro whether DRADA will attack the RNA strand of RNA:DNA, RNA:phosphorothioate modified DNA, and RNA: O-methylated-RNA duplexes. By combining the informaiton from all of these experiments, we aim to increase our understanding of the effect of DRADA on dsRNA and, thereby, the success of the antisense RNA strategy.
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