4] Polysomal ribonuclease 1

Abstract

Publisher Summary This chapter describes methods and approaches to purify and characterize polysomal ribonuclease 1 (PMR-1). It is the first mRNA endoribonuclease to be purified and cloned. Because PMR-1 was found in association with mRNA-containing complexes, the first steps in its analysis required facile approaches for the isolation of messenger ribonucleoprotein (mRNP) and polysome complexes. Furthermore, PMR-1 selectively associates with membrane-bound polysomes. Two approaches are employed to test whether PMR-1 (or another endonuclease) is specific for single-stranded RNA, or whether it can cleave double-stranded RNA. PMR-1 associates with substrate mRNA in vivo in an RNP complex. Two methods were used to recover these complexes; selection of poly(A)-containing complexes with oligo(dT)-cellulose, and immunoprecipitation of PMR-1-containing complexes with a polyclonal antibody to PMR-1. There is convincing evidence of the appearance of PMR-1 cleavage sites in albumin mRNA coincident with the estrogen-induced initiation of albumin mRNA decay. Thus, the protocols developed for this mRNA endonuclease should be generally applicable to the characterization of other mRNA endonucleases as they are identified.