Characterization of two murine (2′‐5′)(A)_n‐dependent endonucleases of different molecular mass

Abstract

RNase L is considered as the major if not unique target of (2'-5')(A)n and therefore as an important intracellular mediator of interferon action. It behaves as an 185-kDa species in various cell extracts when analyzed by gel filtration. SDS/PAGE analysis of the polypeptides covalently labelled with a (2'-5')(A)4-3'-[32P]pCp probe reveals a single 80-kDa species, thus attesting a multimeric form of the 185-kDa protein. At variance with such data, mouse spleen extracts reveal an additional 40-kDa polypeptide with (2'-5')(A)n-dependent ribonucleolytic activity. This seemingly new form of RNase L migrates as a 40-kDa polypeptide when analyzed under native or denaturing conditions. It bears some structural similarity with the larger-molecular-mass RNase L as revealed by partial proteolysis. It is probably not generated through proteolytic degradation of the 185-kDa RNase L during extract preparation, although its physiological significance is unknown. Indeed various protease inhibitors do not significantly alter the ratio of 40-kDa and 185-kDa (2'-5')(A)n-dependent ribonucleases; moreover, the (2'-5')(A)n-binding capacity of the 40-kDa polypeptide is more stable than that of the 185-kDa one.