Characterization of mRNA endonucleases

Endonucleases are key effecters of mRNA degradation, particularly for mRNAs whose turnover rates are regulated by extracellular stimuli. The rapid cle arance of mRNA degradation products in vivo and the need to selectively ide ntify mRNA endonucleases in the presence of many other cellular ribonucleas es make the study of these enzymes particularly challening. We have success fully purified and cloned one such enzyme, termed polysomal RNase 1, or PMR -1. Presented here are protocols either developed in our laboratory or adap ted from the work of others that we have used successfully in characterizin g PMR-1. We first describe methods to determine whether a particular mRNA i s degraded in vivo through an endonuclease-initiated mechanism, and then pr esent approaches for developing an in vitro mRNA degradation system. Next w e describe experiments one should perform to optimize reaction conditions, determine cofactor requirements for an endonuclease, map in vitro cleavage sites, and characterize endonucleolytic cleavage products. Finally we descr ibe kinetic parameters one should evaluate in characterizing the enzymology of mRNA endonucleases, with particular concern focused on the relative sel ectivity of these enzymes for cleavage at preferred sites within target mRN As. (C) 1999 Academic Press.