The N-glycosidase activity of the ribosome-inactivating protein ME1 targets single-stranded regions of nucleic acids independent of sequence or structural motifs
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 75oC rather than 60oC 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.
Direct action of ME1 on a single-stranded plant pathogenic RNA. A. Thermal denaturation of linear PSTVd under low ionic strength conditions. TGGE analysis of linear 32P-labeled PSTVd RNA was carried out at temperatures ranging from 30 to 60oC. B. Depurination of PSTVd by ME1. RNA transcripts synthesized in vitro were partially denatured, incubated with ME1, treated with aniline, and fractionated on a 7 M urea/ 6% [w/v] polyacrylamide gel. C. & D. Infectivity of ME1-treated PSTVd RNAs. Cotyledons of week-old tomato seedlings were dusted with carborundum and inoculated with aliquots (10 ul) of either untreated or ME1-treated PSTVd RNA transcripts serially diluted in 20 mM Na phosphate, pH 7.0. Inoculated seedlings were maintained in a greenhouse and photographed 5 wks post inoculation.
Sang-Wook Park, Ramarao Vepachedu, Robert A Owens and Jorge M. Vivanco. The N-glycosidase activity of the ribosome-inactivating protein ME1 targets single-stranded regions of nucleic acids independent of sequence or structural motifs. J.Biol.Chem.279(in press).