Nucleic Acids Research, 2003, Vol. 31, No. 19 5534-5543
© 2003 Oxford University Press
Characterization of the RNA motif responsible for the specific interaction of potato spindle tuber viroid RNA (PSTVd) and the tomato protein Virp1
1 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, PO Box 1527, GR-71110 Heraklion/Crete, Greece, 2 Department of Biology, University of Crete, GR-71110 Heraklion/Crete, Greece and 3 Department of Plant Physiology and Molecular Biology, University of Plovdiv, 24, Tsar Assen Street, 4000 Plovdiv, Bulgaria
*To whom correspondence should be addressed. Tel: +30 2810 394365; Fax: +30 2810 394408; Email: tabler{at}imbb.forth.gr
Present address:
Michela Alessandra Denti, Dipartimento di Genetica e Biologia Molecolare, Universita’ La Sapienza, P.le Aldo Moro 5, 00185 Roma, Italia
Viroids are small non-coding parasitic RNAs that are able to infect their host plants systemically. This circular naked RNA makes use of host proteins to accomplish its proliferation. Here we analyze the specific binding of the tomato protein Virp1 to the terminal right domain of potato spindle tuber viroid RNA (PSTVd). We find that two asymmetric internal loops within the PSTVd (+) RNA, each composed of the sequence elements 5'-ACAGG and CUCUUCC-5', are responsible for the specific RNA–protein interaction. In view of the nucleotide composition we call this structural element an ‘RY motif’. The RY motif located close to the terminal right hairpin loop of the PSTVd secondary structure has an 
5-fold stronger binding affinity than the more centrally located RY motif. Simultaneous sequence alterations in both RY motifs abolished the specific binding to Virp1. Mutations in any of the two RY motifs resulted in non-infectious viroid RNA, with the exception of one case, where reversion to sequence wild type took place. In contrast, the simultaneous exchange of two nucleotides within the terminal right hairpin loop of PSTVd had only moderate influence on the binding to Virp1. This variant was infectious and sequence changes were maintained in the progeny. The relevance of the phylogenetic conservation of the RY motif, and sequence elements therein, amongst various genera of the family Pospiviroidae is discussed.
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