Identification of functional domains in theself-cleaving Neurospora VS ribozyme using damage selection

Abstract

Varkud Satellite (VS) RNA contains a small self-cleaving RNA motif that is distinct in its sequence and secondary structure from the hammerhead, hairpin, and hepatitis delta virus ribozymes, which are found in other natural RNAs. We have used a base specific chemical damage selection (modification interference) assay to identify functionally important nucleotides and structural elements in VS RNA. Many modified bases interfered with self-cleavage and most of these clustered at helix junctions, certain internal loops, and in a long-range pseudoknot; these correspond to previously determined sites of magnesium-dependent protection from chemical modification. The clustering suggests that these bases are important not only for a large number of individual interactions, but because they form a smaller number of structural elements that are important for activity. Modification of bases in other single-stranded regions, which did not exhibit magnesium-dependent protection, generally did not interfere with activity, suggesting that some of these regions might be dispensable for function. Surprisingly, we found a separate cluster of bases whose modification significantly enhanced cleavage. These bases appear to form a structural element that naturally attenuates the self-cleavage reaction. In natural VS RNA this attenuator structure may affect the cleavage/ligation equilibrium by inhibiting circle re-opening, thereby helping to maintain the RNA in a circular form, which is the predominant form of VS RNA in vivo. Taken together, the results of the damage selection experiments localize the catalytic core of VS RNA to a small subset of the previously determined minimal contiguous sequence.