Abstract
Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado sunblotch viroid (ASBVd), a member of the family Avsunviroidae, replicates via a symmetric RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo viroid life cycle.
Highlights
Viroids, a class of plant pathogens, are single-stranded, covalently closed circular RNA molecules with a chain length between 246 and 401 nt [1]
To evaluate the catalytic activity of monomeric Avocado sunblotch viroid (ASBVd) (+) and (−) encompassing the full hammerhead ribozyme (HHR), self-cleavage time course experiments of mASBVd-HHR (+) and mASBVd-HHR (−) RNAs were performed as indicated in the Experimental Section
Similar self-cleavage time course experiments were observed for the mASBVd-HHR (−) at 50 mM and 100 mM MgCl2 at each temperature, with the fraction of uncleaved molecules at the end-point of the reaction being less than 20% in all cases (Figure 1B)
Summary
A class of plant pathogens, are single-stranded, covalently closed circular RNA molecules with a chain length between 246 and 401 nt [1]. Since viroid RNAs do not code for any protein, the different phases of their life cycle, such as cellular transport, replication, and induction of pathogenicity, depend entirely on the capability of the RNA molecule to interact with cellular host factors. The determination of viroid secondary and tertiary structures of both strand polarities is crucial to better understanding how these small RNAs are able to use host machineries. In contrast to Pospiviroidae, the four members of Avsunviroidae including Avocado sunblotch viroid (ASBVd), Peach latent mosaic viroid (PLMVd), Chrysanthemum chlorotic mottle viroid (CChMVd) and Eggplant latent viroid (ELVd), are able to self-cleave, in both polarity strands, through a hammerhead ribozyme and replicate via a symmetric rolling circle pathway [10,11]
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