Elucidation of the structures of all members of the Avsunviroidae family

Abstract

Viroids are small single-stranded RNA pathogens which cause significant damage to plants. As their nucleic acids do not encode for any proteins, they are dependant solely on their structure for their propagation. The elucidation of the secondary structures of viroids has been limited because of the exhaustive and time consuming nature of classic approaches. Here, the method of high-throughput selective 2'-hydroxyl acylation analysed by primer extension (hSHAPE) has been adapted to probe the viroid structure. The data obtained using this method were then used as input for computer-assisted structure prediction using RNA structure software in order to determine the secondary structures of the RNA strands of both (+) and (–) polarities of all Avsunviroidae members, one of the two families of viroids. The resolution of the structures of all of the members of the family provides a global view of the complexity of these RNAs. The structural differences between the two polarities, and any plausible tertiary interactions, were also analysed. Interestingly, the structures of the (+) and (–) strands were found to be different for each viroid species. The structures of the recently isolated grapevine hammerhead viroid-like RNA strands were also solved. This species shares several structural features with the Avsunviroidae family, although its infectious potential remains to be determined.To our knowledge, this article represents the first report of the structural elucidation of a complete family of viroids.

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

“…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].…”

“…Conversely, PLMVd and CChMVd RNAs, which fold into complex multi-branched conformations stabilized by a kissing-loop interaction, are insoluble under the same saline conditions [45]. In this same context, our in vitro SHAPE results do not support the existence in the mc ASBVd (-) RNA of the kissing-loop interaction proposed to stabilize a ml ASBVd (À) form, which has neither been inferred by in vitro SHAPE for another ml ASBVd (À) form [23,24]. Moreover, we have not detected by native PAGE any Mg 2+ -induced difference in the mobility of the mc ASBVd (À) RNA, while we have observed for it a slower mobility than for its (+) counterpart that neither is influenced by Mg 2+ .…”

“…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.…”

Chrysanthemum Chlorotic Mottle Viroid-Mediated Trafficking of Foreign mRNA into Chloroplasts

“…1E) (Giguère et al, 2014), was able to facilitate RNA trafficking into chloroplasts, although to different extents, with the latter being less efficient. In the case of ELVd, the region that was able to traffic fused mRNA for GFP into the chloroplasts was the left one-third of the RNA structure (nucleotides 52-150, containing two stem-loop structures of the left three-way junction), without the rest of the molecule, although not as efficiently as the full-length molecule Pallas, 2010b, 2012).…”

“…In the case of ELVd, the region that was able to traffic fused mRNA for GFP into the chloroplasts was the left one-third of the RNA structure (nucleotides 52-150, containing two stem-loop structures of the left three-way junction), without the rest of the molecule, although not as efficiently as the full-length molecule Pallas, 2010b, 2012). Since ELVd and CChMVd do not contain any sequence similarity, beyond the limited sequences (11 of 13 nt split into four parts) involved in hammerhead ribozymes (Giguère et al, 2014), then the ability of both RNAs to traffic RNAs into chloroplasts most likely resides in their structures. Since CChMVd, ELVd and PLMVd all contain multiple branch junctions, but ASBVd does not, according to the current structural models (Giguère et al, 2014), then either these branching-structure aspects are not necessary for trafficking into chloroplasts, or they are and ASBVd adopts a different structure (with or without the help of binding proteins) to facilitate its movement into chloroplasts.…”