Alternative hammerhead structures in the self-cleavage of avocado sunblotch viroid RNAs

The plus and minus RNAs of the 247 nt avocado sunblotch viroid (ASBV) undergo site specific RNA self-cleavage reactions in vitro. As with several other self-cleaving RNAs, we proposed hammerhead secondary structures for the sequence around the site of self-cleavage of both RNAs. We have shown previously that, during transcription of a dimeric plus ASBV RNA, a double-hammerhead structure formed and was necessary for self-cleavage. Here, we show that the purified full-length dimeric plus RNA, when incubated under our standard self-cleavage conditions, also self-cleaved by a double-hammerhead structure. In contrast, a dimeric minus ASBV RNA self-cleaved by a double-hammerhead structure during transcription, but by a single-hammerhead structure after purification. This illustrates the importance of the pathway of folding of the RNA in determining which active self-cleaving structure is formed.