Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A naked rod-like conformation similar but not identical to that observed in vitro

With a minimal (250-400nt), non-protein-coding, circular RNA genome, viroids rely on sequence/structural motifs for replication and colonization of their host plants. These motifs are embedded in a compact secondary structure whose elucidation is crucial to understand how they function. Viroid RNA structure has been tackled in silico with algorithms searching for the conformation of minimal free energy, and in vitro by probing in solution with RNases, dimethyl sulphate and bisulphite, and with selective 2-hydroxyl acylation analyzed by primer extension (SHAPE), which interrogates the RNA backbone at single-nucleotide resolution. However, in vivo approaches at that resolution have not been assayed. Here, after confirming by 3 termodynamics-based predictions and by in vitro SHAPE that the secondary structure adopted by the infectious monomeric circular (+) RNA of potato spindle tuber viroid (PSTVd) is a rod-like conformation with double-stranded segments flanked by loops, we have probed it in vivo with a SHAPE modification. We provide direct evidence that a similar, but not identical, rod-like conformation exists in PSTVd-infected leaves of Nicotiana benthamiana, verifying the long-standing view that this RNA accumulates in planta as a naked form rather than tightly associated with protecting host proteins. However, certain nucleotides of the central conserved region, including some of the loop E involved in key functions such as replication, are more SHAPE-reactive in vitro than in vivo. This difference is most likely due to interactions with proteins mediating some of these functions, or to structural changes promoted by other factors of the in vivo habitat.