MOLECULAR BIOLOGY OF AN RNA UNWINDING/MODIFYING ACTIVITY

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A previously uncharacterized double-stranded RNA (dsRNA) unwinding activity has been reported to exist in Xenopus laevis, Caenorhabditis elegans and a number of mammalian tissue culture cells. The activity exhibits an unusual mechanism of unwinding: during the reaction adenosine residues are covalently modified to inosine residues. The covalent modification results in the substitution of a stable AU base-pair by the considerably less stable IU base-pair; thus the dsRNA substrates are permanently unwound. The long term goal of the proposed research is to determine the biological function of the unwinding/modifying activity. Although the role of the modification may be to unwind the dsRNA, the discovery that the activity covalently modifies its RNA substrate raises the possibility that the primary function of the activity is to modify rather than unwind. In regards to the long term goal, studies will focus on determining the natural biological substrate(s) of the unwinding/modifying activity. Given the ubiquity of double-stranded regions (intramolecular as well as intermolecular) within cellular RNAs, many potential substrates can be imagined. Experiments that will narrow the range of possibilities will be performed. The substrate specificity of the unwinding/modifying activity will be defined in more detail, utilizing previously developed in vitro assays. In addition, cellular RNAs will be analyzed to determine if a particular subset of RNAs is more likely to contain the biological substrate(s). Sensitive HPLC methods, as well as anti-inosine antibody, will be used to assay various populations of RNAs for the presence of inosine and, using an antibody to the unwinding/modifying activity, the ability of specific RNAs to interact with the activity will be tested. At later stages of the proposed research, microinjection techniques will be utilized to alter the expression of the unwinding/modifying activity in Xenopus oocytes and early embryos, and to monitor phenotypic changes, in particular changes in cellular RNAs, that may occur during altered expression. The proposal includes protocols for purifying the unwinding/modifying activity, raising antibodies to the protein and cloning the cDNA.