Secondary Structure Probing of Potato Spindle Tuber Viroid (PSTVd) and Sequence Comparison with Other Small Pathogenic RNA Replicons Provides Evidence for Central Non-canonical Base-pairs, Large A-rich Loops, and a Terminal Branch

Abstract Using PCR and in vitro transcription, linear (non-circular) unit-length (+)strand RNA molecules of a lethal PSTVd variant were produced which are able to initiate typical disease symptoms when inoculated into tomato. Non-denaturing gel electrophoresis shows that these transcripts can adopt the same two conformations as circular PSTVd molecules, namely a fast migrating rod-like and a slowly migrating cruciform structure. The rod-like conformer of two end-labelled transcripts was probed with nucleases and dimethyl sulphate, revealing that in solution its right part is identical to computer prediction. In the left part, however, three unique features could be substantiated. (1) In the central region a UV-cross-link able loop is closed and thus contains non-canonical base-pairs (“loop E structure”). (2) Three large “pre-melting loops” are present at 25° to 37°C. The structure of the leftmost one, which is A-rich and conserved in most viroids, correlates with pathogenicity. (3) Two small stem-loops instead of an unbranched structure are found at the left terminus. These hairpins can form in all “large” viroids (≈300 nucleotides or longer), thus placing the dodecamer conserved among these viroids, GGUUCCUGUGGU, within the upper helix and the branch junction. A large viroid from Iresine lacks one of these hairpins, whereas all “small” viroids (≈300 nucleotides or smaller) lack both. In several plant virus satellite RNAs and the newt satellite RNA, the motif GAUUU(U) and dodecamer remnants appear in an equivalent structure comprising two or three hairpins. Using lead- and terbium-induced cleavage of the RNA, metal binding sites were found, mostly in loops. Thus, probing of PSTVd RNA and comparison with other viroids, virusoids, and satellite RNAs contributes to our understanding of the secondary structure of these small pathogenic RNAs.