The RNA of both polarities of the peach latent mosaic viroid self-cleaves in vitro solely 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 self-cleavage 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.