Structural Analyses of Avocado sunblotch viroid Reveal Differences in the Folding of Plus and Minus RNA Strands

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.

The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins

“…2b). As indicated above, such an observation is consistent with others made previously in gels of similar porosity, but using ml ASBVd (+) and (À) RNAs [23], and with the more stable conformation predicted for the mc ASBVd (+) RNA by the three software programs (see above). However, we found that while the ml ASBVd (À) RNA resulting from self-cleavage migrated in 8 % gels as a sharp band, the ml ASBVd (+) RNA also from selfcleavage migrated as a blurred band (data not shown), thus hinting at the co-existence of different conformations in the latter.…”

“…In vitro SHAPE of mc ASBVd (+) and (À) RNAs generated well-resolved structures that were almost identical with the two acylating reagents used (NMIA and NAI), in line with the adoption of predominant conformations by both RNAs in vitro as inferred from their migration as single bands in non-denaturing PAGE. These structures also agree with: (i) those predicted in silico, particularly with the RNAfold software program, and (ii) those derived previously from in vitro SHAPE [24], while differing somewhat from others obtained with the same in vitro approach [23]. Furthermore, the rod-like conformations proposed in vitro for the mc ASBVd (+) and (À) RNAs are also consistent with the solubility in 2 M LiCl of the mc ASBVd RNAs extracted from infected tissue, mainly formed by the (+) strand [45], and of the ml ELVd (+) and (À) RNAs resulting from in vitro self-cleavage that adopt quasi-rod-like secondary structures [36].…”

“…On the other hand, the SHAPE reactivity of some nucleotides forming the lower strand of the left terminal loop (positions 123-128) did not support their involvement in a kissing-loop interaction with the lower strand of a right terminal loop (positions 231-236) (Fig. 4a, b), as proposed previously for a ml ASBVd (À) form [23].…”

“…Although the ml (+) and (À) RNAs of eggplant latent viroid (ELVd, 333 nt) [35] are clearly discriminated by electrophoresis in gels of this porosity [36], they might not have sufficient power to resolve RNAs of smaller size like those of ASBVd. Therefore, we performed our experiments in 8 % gels in which, according to earlier data, ml ASBVd (+) and (À) RNAs migrate differentially [23]. Considering that the in vitro SHAPE analysis to be carried out subsequently entails a thermal denaturation/renaturation, we also tested whether such a treatment had any effect on the electrophoretic mobilities of mc ASBVd (+) and (À) RNAs with the same approach as used before with the two other viroids [26,36].…”

“…While this thermodynamics-based methodology predicts rod-like or quasi-rod-like conformations for both strands, in vitro examination of the ml ASBVd (+) and (À) RNAs by selective 2¢-hydroxyl acylation analysed by primer extension (SHAPE), complemented by gel electrophoresis and other physical approaches [23][24][25], has led to secondary structures exhibiting significant differences. Prominent among them is the presence [23] or absence [24] of a kissing-loop interaction stabilizing the ml (À) RNA.…”