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Unformatted text preview: Characterization of the RNA motif responsible for the specific interaction of potato spindle tuber viroid RNA (PSTVd) and the tomato protein Virp1 Mariyana Gozmanova 1 , 2 , 3 , Michela Alessandra Denti 1 , Ivan Nikiforov Minkov 3 , Mina Tsagris 1 , 2 and Martin Tabler 1 , * 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 Received July 3, 2003; Revised and Accepted August 14, 2003 ABSTRACT 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 inter- action. 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 alter- ations 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. INTRODUCTION Viroids are subviral RNA pathogens that are able to infect some higher plants. They are unique for their small size and their propagation strategy [for review and general statements on viroids see (1–4)]. Their genome consists of a single- stranded, covalently closed RNA molecule, which adopts in most cases an unbranched rod-like secondary structure. Despite their simplicity, viroids are able to replicate auto- nomously. This capability is remarkable since there is no indication that the viroid RNA has the capacity to encode a peptide. Nevertheless, the non-coding viroid RNA supplies genetic information such as replicapability, pathogenicity, mobility and host-specificity. It is assumed that the RNA sequence folds in a specific secondary structure that provides binding signals for host (protein) factors. It is further assumed that these factors exert in combination with the viroid RNA the...
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