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Title: THE N-GLYCOSIDASE ACTIVITY OF THE RIBOSOME-INACTIVATING PROTEIN ME1 TARGETS SINGLE-STRANDED REGIONS OF NUCLEIC ACIDS INDEPENDENT OF SEQUENCE OR STRUCTURAL MOTIFS

Author
item PARK, SANG-WOOK - CO STATE UNIV FT COLLINS
item VEPACHEDU, RAMARAO - CO STATE UNIV FT COLLINS
item Owens, Robert
item VIVANCO, JORGE - CO STATE UNIV FT COLLINS

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/9/2004
Publication Date: 8/1/2004
Citation: Park, S., Vepachedu, R., Owens, R.A., Vivanco, J.M. 2004. The n-glycosidase activity of the ribosome-inactivating protein me1 targets single-stranded regions of nucleic acids independent of sequence or structural motifs. Journal of Biological Chemistry. 279:34165-34174.

Interpretive Summary: Viroids are the smallest known agents of infectious disease - small, circular RNA molecules that lack the coat protein characteristic of most conventional viruses yet are able to multiply and cause disease in susceptible host plants. Others have shown that RNA sequence changes involving only a single nucleotide converts Potato spindle tuber viroid (PSTVd) into a form that is highly infectious for tobacco. Many plants, including tobacco, contain so-called "ribosome inactivating proteins", enzymes that inactivate RNA molecules by removing a single adenosine residue from a common structural feature known as "loop E". Because PSTVd and related viroids are known to form loop E-like structures, it is possible that their host range is controlled by the presence or absence of ribosome inactivating proteins. This manuscript describes our first attempts to prove/disprove this hypothesis. Understanding the factor(s) controlling host range is key to our efforts to render plants resistant to viroid infection. This report will be of greatest interest to researchers interested in the molecular interactions between viroids or viruses and their plant hosts.

Technical Abstract: 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 (S/R) loop of the large eukaryotic and prokaryotic rRNAs. We have investigated the effect of partial denaturation on the interaction of ME1 and other RIPs with non-ribosomal RNA substrates following partial denaturation. 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 depend on the physical availability of nucleotides, and denaturation of nucleic acid substrates increased their interaction with ME1. Pretreatment of pap-h mRNA at 75 C rather than 60 C lowered the apparent KD from 87.1 nm to 73.9 nM. Exposure to ME1 completely abolished the infectivity partially-denatured RNA transcripts of potato spindle tuber viroid, suggesting that RIPs may target invading nucleic acids before they reach the host ribosomes. The extensive folding of many potential substrates, however, interferes with their ability to interact with RIPs, thereby blocking their inactivation by ME1 or other RIPs.