Nucleic Acids Research

We have surveyed 14 different toxic and nontoxic ribosome-inactivating proteins from plants for the ability to act Ol the RNA of the eucaryotic 60 S ribosomal subunit. All of these proteins act to introduce a specific modification into 26-28 S RNA which renders the RNA sensitive to cleavage by aniline. Sequence analysis of the 5'-termini of the fragments produced by ricin and saporin following aniline cleavage indicate that both proteins possess identical specificity. Our observations support the conclusion of Endo and Tsurugi (J. Biol. Chem. 262, 8128-8130, 1987) that ricin is a specific N-glycosidase and we have located the site of this cleavage by direct sequence analysis. Our results further suggest that all plant ribosomeinactivating proteins function as specific N-glycosidases with the same specificity. INTRODUCTION: Proteins capable of inactivating the eucaryotic ribosome, RIPs (ribosome.inactivating proteins), are widely distributed in the plant kingdom (reviewed in refs. 1-3). These proteins exist either as single chain (Type 1 RIPs) or as two chain (Type 2 RIPs) proteins. The latter proteins are potent toxins (4), the best known example of which is ricin. The B-chains of these toxins bind to galactose-containing receptors on the cell surface and allow the Achains to enter the cytoplasm where they inactivate ribosomes. The more numerous Type 1 RIPs are much less toxic to intact cells because they lack the -surface binding component. All known Type 1 RIPs and the A-chains of Type 2 RIPs act catalytically to inactivate the 60 S ribosomal subunit. This inactivation occurs in the absence of cofactors and it is thus presumed that RIPs are hydrolytic enzymes (5). However, despite a great deal of interest and investigation, the target(s) of the RIPs within the ribosome and the mechanism(s) of RIP action have until recently eluded discovery. A major breakthrough in this area was recently reported by Endo et al. (6,7). These investigators observed that the ©) I R L Press Limited, Oxford, England. 1349 Nucleic Acids Research toxins ricin, abrin and modeccin introduce a lesion at a specific location in 28 S RNA of rat liver ribosomes concomitant with the release of adenine. This lesion renders the phosphodiester backbone of RNA susceptible to anilinecatalyzed hydrolysis. The properties of this "aniline-labile site" and the release of adenine suggested that ricin is a specific N-glycosidase. The observations we report in this paper extend the findings of Endo and coworkers and further suggest that N-glycosidase activity is the general mechanism of plant RIP action. MATERIALS AND METHODS: The proteins dodecandrin, cloned ricin A-chain, a-sarcin and viscumin were gifts from J.D. Robertus (University of Texas), L.L. Houston (Cetus Corp.), I.C. Wool (University of Chicago) and P. Ziska (Berlin, DDR), respectively. Pokeweed antiviral protein was purchased from Worthington. All other RIPs were prepared as described in the references cited in Table 1. Rabbit reticulocyte lysate was prepared by the method of Pelham and Jackson (18). Yeast lysates were prepared by mechanical breakage (19). Both types of lysates were stored frozen at -700C. In order to analyze the effect of a-sarcin and RIPs on rRNA, lysates were incubated with these proteins for 20 min at 370C. The reactions were terminated by the addition of sodium dodecylsulfate to a final concentration of 0.5% and RNA was prepared by phenol extraction. In order to cleave the "aniline-labile site", RNA was dissolved in 1 M aniline-acetate (pH 4.5) and the solution was incubated for 10 min at 0°C. At the conclusion of the incubation, the solution was extracted twice with ether and the RNA was precipitated with ethanol. For both analysis and purification of fragments, the RNA was electrophoresed (20) on 5% acrylamide gels (4.85% acrylamide, 0.15% bis-acrylamide, 7 M urea, 90 mM Tris-Borate pH 8.3). Electrophoresis was at 1200 V until the bromophenol dye had migrated 30 cm. For the results shown in Fig. 1, the gel described above was stained with silver (21). In order to prepare fragments for 5'-terminal labeling, the RNA was visualized by U.V. shadowing (22) and the fragments were eluted by diffusion. Labeling of the 5'-termini of the fragments with 32p was performed with T4 polynucleotide kinase (23). Terminal base analysis of labeled RNA was performed by P1 nuclease digestion (24) followed by PEI cellulose thinlayer chromatography with 1 M LiCl. Enzymatic sequence analysis (23) was performed on 20% acrylamide-7M urea gels. The thinlayer chromatograms and sequencing gels were autoradiographed overnight.