The N-glycosidase activity of the ribosome-inactivating protein ME 1 targets single-stranded regions of nucleic acids independent of sequence or structural motifs

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 loop of large eukaryotic and prokaryotic rRNAs. We have investigated the effect of modifying the structure of non-ribosomal 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).