The strands of both polarities of a small circular RNA from carnation self-cleavein vitrothrough alternative double- and single-hammerhead structures

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Abstract
The sequence of a circular RNA from carnation has been determined and found to consist of 275 nucleotide residues adopting a branched secondary structure of minimum free energy. Both plus and minus strands of this RNA can form the hammerhead structures proposed to mediate the in vitro self-cleavage of a number of small infectious plant RNAs and the transcript of satellite 2 DNA from the newt. Minus full- and partial-length transcripts of the carnation circular RNA including the hammerhead structure showed self-cleavage during transcription and after purification, indicating the involvement of a single-hammerhead structure in the self-cleavage reaction. In the case of the plus transcripts only a dimeric RNA, but not a monomeric one, self-cleaved efficiently during transcription and after purification, strongly supporting the implication in this process of a double-hammerhead structure theoretically more stable than the corresponding single cleavage domain. However, a plus monomeric transcript self-cleaved after purification at a slow rate in a concentration-independent reaction which most probably occurs through an intramolecular mechanism. Comparative sequence analysis has revealed that the circular RNA from carnation shares similarities with some representative members of the viroid and viroid-like satellites RNAs from plants, suggesting that it is a new member of either these two groups of small pathogenic RNAs.