Double-stranded RNA 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. We have recently found that 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 inosine. As expected from the fundamental importance to all cells this unwinding/modify activity (RNA duplex unwindase or dsRNA unwindase) is present in every type of cell. Several instances where this activity has been shown to be responsible for modifying a specific target gene system have now been reported. These include a crucial step in Xenopus oocyte maturation (turnover of maternal bFGF mRNA) and the pathogenesis of a severe human neurological disease (subacute sclerosing panencephalitis). The diversity of these examples hints at the wide range of physiological functions that may be dependent on proper functioning of this machinery for handling dsRNA. The goal of our research is to elucidate the normal functions and biological substrates of the dsRNA unwindase. To achieve this goal. we must first completely characterize the enzyme itself. including all of the biochemical parameters and genetic controls that regulate the time, place, and level of its activity. In accord with this goal, our proposed research has two major specific aims. First, we will determine the mechanism and the substrate specificity of the dsRNA unwindase. In particular, we will measure the size limit for the substrate RNAs, the nature of the discrimination between intermolecular and intramolecular dsRNAs, the extent of sequence specificity, if any, for the regions surrounding target adenosine residues, and the polarity or direction of the unwinding/modifying reaction. All of this information is necessary to delimit the range of natural biological substrate for this activity. Second, we will generate the specific probes and tools (antibodies and cDNA) that are essential for determining the time and place of the unwindase expression. To do this, we win complete our purification of this enzyme from bovine liver nuclear extracts. The purified protein will be used to generate antibodies and to determine a partial amino acid sequence. Using these antibodies or oligonucleotide probes deduced from the partial amino acid sequence, first cDNA, then genomic clones coding for the unwindase will be isolated and sequenced. The information gained by using these tools will illustrate how the cell regulates this unwinding/modifying activity. reveal its homology to any other known gene or protein, and thereby direct us to an understanding of its physiological functions.
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