Characterization of mRNA Endonucleases Daniel R. Schoenberg 1 and Kristopher S. Cunningham Department of Pharmacology and The Comprehensive Cancer Center, Ohio State University College of Medicine, Columbus, Ohio 43210 –1239 Endonucleases are key effectors of mRNA degradation, par- ticularly for mRNAs whose turnover rates are regulated by extracellular stimuli. The rapid clearance of mRNA degrada- tion products in vivo and the need to selectively identify mRNA endonucleases in the presence of many other cellular ribo- nucleases make the study of these enzymes particularly chal- lenging. We have successfully purified and cloned one such enzyme, termed polysomal RNase 1, or PMR-1. Presented here are protocols either developed in our laboratory or adapted from the work of others that we have used success- fully in characterizing PMR-1. We first describe methods to determine whether a particular mRNA is degraded in vivo through an endonuclease-initiated mechanism, and then present approaches for developing an in vitro mRNA degrada- tion system. Next we describe experiments one should per- form to optimize reaction conditions, determine cofactor re- quirements for an endonuclease, map in vitro cleavage sites, and characterize endonucleolytic cleavage products. Finally we describe kinetic parameters one should evaluate in character- izing the enzymology of mRNA endonucleases, with particular concern focused on the relative selectivity of these enzymes for cleavage at preferred sites within target mRNAs. © 1999 Academic Press The synthesis of a protein product is the ultimate outcome of gene expression, and posttranscriptional regulation of the half-life of any given mRNA pro- vides the cell with a highly flexible mechanism to control the ultimate expression of that gene. The complex processes that impact on posttranscrip- tional control are the subject of a recent monograph (1). In this paper we deal with how one evaluates the contribution of messenger ribonucleases, specifically endoribonucleases, to the process of mRNA decay in vertebrate cells and tissues. The paper by Jeff Ross in this issue presents approaches to study exonucle- ases involved in mRNA decay (1a). The term messenger RNase was coined by Ross and Kobs (2) to denote RNases that catalyze the degradation of mRNA. Whereas exonucleases are the predominant messenger RNases in yeast (3), vertebrate cells use both exonucleases and endonu- clease to catalyze mRNA decay (4). Endonucleases appear to be particularly important for initiating mRNA decay in response to extracellular stimuli (4). More is known about endonucleolytic decay path- ways than about the endonucleases themselves. In many cases endonuclease-initiated decay does not involve prior deadenylation. This is a useful crite- rion to evaluate when determining whether the degradation of a particular mRNA uses an endonuclease-based pathway. Some examples of mRNAs degraded by endonuclease-dependent path- ways are insulin-like growth factor 2 (5– 8), gro (9, 10), c-myc (11–13), transferrin receptor (14), avian apo-very low density lipoprotein II (apo-VLDL II) (15, 16), Xenopus maternal homeobox mRNAs (17, 18), and Xenopus serum albumin (19 –21). In- terested individuals may wish to examine these pa- pers because many of the methods presented below are used in these citations. This paper is organized roughly in the order of experiments one would follow when evaluating an endonuclease involved in mRNA decay. We first de- scribe experiments one would use to determine whether the mRNA decay phenomenon under study uses an endonucleolytic cleavage pathway. This is followed by approaches to determine the in vivo cleavage sites of the candidate endonuclease in the target mRNA. The latter provide information that is 1 To whom correspondence should be addressed at the Depart- ment of Pharmacology, Ohio State University, 333 West 10th Avenue, Columbus, OH 43210 –1239. Fax: (614) 292–7232. E- mail: schoenberg.3@osu.edu. METHODS: A Companion to Methods in Enzymology 17, 60 –73 (1999) Article ID meth.1998.0708, available online at http://www.idealibrary.com on 60 1046-2023/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.