The major aim of the propsed research is to understand the function of the 2',5'-Oligo(A) polymerase and 2-5A activated endonuclease (RNase L) present in nuclei of HeLa cells. The hypothesis to be tested is that these enzymatic activities are involved in the processing of heterogeneous nuclear RNA (hnRNA). Initial experiments with isolated nuclei will determine whether double-stranded regions of nuclear RNA are capable of activating the polymerase. Synthesis of 2-5A will be measured with a sensitive competitive binding assay. If synthesis is detected, I will determine whether 2-5A is present in the nuclei of intact cells. To accomplish this, cells will be fractionated by non-aqueous techniques and isolated nuclei will be extracted with TCA; 2-5A will be quantitated with the binding assay. Once the presence of 2-5A in the nuclei of intact cells has been established, experiments designed to assess a possible role of the polymerase and RNase L in processing will be performed. Cloned cDNAs to individuals mRNAs will be used as specific probes to monitor RNA processing in isolated nuclei. These probes will initially be used to identify high molecular weight precursors to mRNA on Northern blots. The disappearance of hybridizable material corresponding to precursors will then be used as an assay of processing under conditions either enhancing or inhibiting the synthesis of 2-5A. Finally, similar assays will be performed in the presence of analogs of 2-5A which specifically inhibit RNase L.
NIH ExPorter
NIH ExPorter