2′,5′-Oligoadenylate-dependent RNAse located in nuclei: biochemical characterization and subcellular distribution of the nuclease in human and murine cells

Abstract

A cellular fractionation procedure allowed the rapid preparation of membraneless nuclei which contained a 2′,5′-oligoadenylate (2-5A)-binding activity which was not due to cytoplasmic contaminants. Purified nuclei prepared from human lymphocytic leukaemia cells and mouse fibroblasts were found to contain 20-22% of the total cellular enzyme. In contrast with the cytoplasmic enzyme which was only present in a 2-5A-free form, 75% of the 2-5A-binding activity was found in the nuclei after a denaturing-renaturing procedure as the 2-5A-binding site was masked. Although the purification of nuclei from mouse fibroblasts was less effective, it appeared that, in confluent and growing cells, 50% and 75% respectively of the 2-5A-binding site was masked. Additional findings obtained by partial proteolysis and two-dimensional gel analysis provided definitive data on the nuclear location of this enzyme. Study of the nuclear 2-5A-dependent RNAase with a 2-5A-masked site could lead to an understanding of the molecular pathway involved in single-stranded RNA stability.