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J. Biol. Chem., Vol. 279, Issue 33, 34165-34174, August 13, 2004

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The N-Glycosidase Activity of the Ribosome-inactivating Protein ME₁ Targets Single-stranded Regions of Nucleic Acids Independent of Sequence or Structural Motifs^*

Sang-Wook Park, Ramarao Vepachedu¶, Robert A. Owens||, and Jorge M. Vivanco**

From the Department of Horticulture and Landscape Architecture and the ^**Cell and Molecular Biology Program, Colorado State University, Fort Collins, Colorado 80523 and the ^||Molecular Plant Pathology Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 20705

ME₁, a type I ribosome-inactivating protein (RIP), belongs to a family of enzymes long believed to possess rRNA N-glycosidase activity directed solely at the universally conserved residue A4324 in the sarcin/ricin loop of large eukaryotic and prokaryotic rRNAs. We have investigated the effect of modifying the structure of nonribosomal RNA substrates on their interaction with ME₁ and other RIPs. ME₁ was shown to depurinate a variety of partially denatured nucleic acids, randomly removing adenine residues from single-stranded regions and, to a lesser extent, guanine residues from wobble base-pairs in hairpin stems. A defined sequence motif was not required for recognition of non-paired adenosines and cleavage of the N-glycosidic bond. Substrate recognition and ME₁ activity appeared to depend on the physical availability of nucleotides, and denaturation of nucleic acid substrates increased their interaction with ME₁. Pretreatment of mRNA at 75 °C rather than 60 °C, for example, lowered the apparent K_D from 87.1 to 73.9 nM, making it more vulnerable to depurination by RIPs. Exposure to ME₁ in vitro completely abolished the infectivity of partially denatured RNA transcripts of the potato spindle tuber viroid, suggesting that RIPs may target invading nucleic acids before they reach host ribosomes in vivo. Our data suggest that the extensive folding of many potential substrates interferes with their ability to interact with RIPs, thereby blocking their inactivation by ME₁ (or other RIPs).

Received for publication, January 6, 2004 , and in revised form, April 9, 2004.

^* This work was supported by a CAREER Award MCB-0093014 from the National Science Foundation (to J. M. V.) and by the Colorado State University Agricultural Experiment Station (to J. M. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1 and 2.

Present address: Boyce Thompson Institute for Plant Research, Ithaca, NY 14853.

^¶ Present address: National Jewish Medical and Research Center, Denver, CO 80206.

To whom correspondence should be addressed: Dept. of Horticulture and Landscape Architecture and Cell and Molecular Biology Program, Colorado State University, Shepardson Bldg., Rm. 217, Fort Collins, CO 80523. Tel.: 970-491-7170; Fax: 970-491-7745; E-mail: j.vivanco{at}colostate.edu.

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