LOCALIZATION OF A MOLECULAR-FORM OF INTERFERON-REGULATED RNASE-L IN THE CYTOSKELETON

RNase L (also termed 2-5A-dependent RNase) is a crucial enzyme involve d in the molecular mechanism of interferon (IFN) action, Activated by 2',5'-oligoadenylate oligomers (2-5A), this enzyme controls the regula tion of RNA stability in IFN-treated or virus-infected mammalian cells . Knowledge of RNase location within cells may provide additional info rmation about its function. Previous work located RNase as a detergent -soluble molecule in nuclei and cytoplasm. In this study, we demonstra te that this enzyme was also present in a detergent-insoluble fraction associated with proteins of the cytoskeleton, A cellular fractionatio n procedure was used to prepare the cytoskeleton, which was shown to c ontain 2-5A binding activity not due to cytoplasmic contaminants, In c ontrast to the cytoplasmic fraction, which contained RNase L with a 2- 5A-accessible site, the insoluble RNase molecular form of the cytoskel eton could not be assayed by the classic radiobinding method or the co valent UV cross-linking procedure, which only detects the 2-5A binding site in an open position, that is, free of 2-5A or with an unmasked 2 -5A site, The 2-5A binding site present in the cytoskeleton was comple tely masked and not directly accessible to its 2-5A activator. This pa rticular molecular form of RNase can be detected after a specific dena turing-renaturing treatment of the cytoskeleton, which separates the R Nase from cytoskeletal proteins, unmasking the 2-5A site, The cytoskel etal RNase was no longer present at this site when cells were stimulat ed for a short time with 12-O-tetradecanoylphorbol-13-acetate (TPA), O ur data suggest the existence of a pathway that targets the RNase to a nother subcellular location, To explore the issue further, we examined in vitro the ability of calcium and phospholipid-dependent protein ki nase C (PKC) to catalyze significant phosphorylation of the RNase.