The RNA of both polarities of the peach latent mosaic viroid self-cleavesin vitrosolely by single hammerhead structures

Abstract

Hammerhead self-cleavage of dimeric, monomeric, truncated and mutated transcripts derived from both polarities of the peach latent mosaic viroid (PLMVd) were characterized. In contrast to some results previously published for a very close sequence variant (see ref. 1), these RNAs exhibit a virtually identical selfcleavage during transcription and after purification. By self-cleavage of dimeric transcripts with normal and mutated hammerhead domains and by complementation experiments, we show that the cleavage reactions involve only single hammerhead structures. This observation contrasts with the case of avocado sunblotch viroid (ASBVd), the other self-cleaving viroid, whose mechanism involves mostly double hammerhead structures, whereas single hammerhead cleavage is associated with viroid-like plant satellite RNAs. The difference in stability between the native secondary structures adopted by viroids and the autocatalytic structures, including the hammerhead motif, governs the efficiency of the self-cleavage reaction. The transition between these conformers is the limiting step in catalysis and is related exclusively to the left arm region of PLMVd secondary structure, which includes the hammerhead sequences. Most of the mutations between the variant we used and the sequence variant previously published are located in this left arm region, which may explain to a great extent the differences in their cleavage efficiency. No interactions with long-range sequences contributing to the autocatalytic tertiary structure were revealed in these experiments.

A Short Double-Stranded RNA Motif of Peach Latent Mosaic Viroid Contains the Initiation and the Self-Cleavage Sites of Both Polarity Strands

“…In some instances, a second (nested) PCR amplification was performed. 4, respectively), thus showing that the experimental approach developed was sensitive and specific.…”

“…Finally, longer-than-unit viroid strands accumulate in ASBVd-infected avocado (11,28), but they are absent in PLMVd-infected peach (Fig. 1) or accumulate at very low concentrations (8); this difference may result from the higher catalytic efficiency of PLMVd ribozymes, which are stable and operate as single hammerhead structures (4,27), compared to those of ASBVd, which are unstable and operate as doublehammerhead structures (23). Overall, these significant divergences between ASBVd and PLMVd made unlikely the existence of similar transcription initiation sites and created the stimulus to determine those of the latter.…”

Mapping in Solution Shows the Peach Latent Mosaic Viroid To Possess a New Pseudoknot in a Complex, Branched Secondary Structure

“…DNA templates for in vitro transcription of the three different RNA molecules used in this study were synthesized from the pPD1 vector (3). Briefly, this construct possesses two tandemly repeated PLMVd sequences (from the Armking peach cultivar [9]) cloned into the PstI restriction site of pBluescript II KS.…”

Identification of a Peach Latent Mosaic Viroid Hairpin Able To Act as a Dicer-Like Substrate

“…P-radiolabeled PLMVd transcripts were synthesized in vitro in the presence of 50 Ci of [␣-32 P]GTP (3,000 Ci/mmol) from the recombinant plasmid pPD1 that possesses two tandemly repeated PLMVd sequences (2). During transcription, RNAs of both polarities possessing hammerhead sequences were produced and self-cleaved efficiently, yielding 338-nt monomeric transcripts.…”