Secondary structure of the circular intervening sequence: A technique using chemical probes and reverse (RNA splicing/RNA catalysis/base modification/base methylation/R)

The structure of the intervening sequence (IVS) of the Tetrahymena rRNA precursor mediates cleavage- ligation reactions that result in pre-rRNA splicing and IVS cy- clization. We have developed a method for RNA structure analysis and applied it to the circular form of the IVS RNA. The native RNA was treated with dimethyl sulfate or diethyl pyrocarbonate to modify bases not involved in secondary or tertiary interactions. The RNA was then used as a template for reverse transcription. Elongation of synthetic oligodeoxynu- cleotide primers was found to stop (or pause) one nucleotide prior to 1-methyladenosine, 3-methylcytidine, and 7-ethoxy- carbonyladenosine residues. The detection of l-methyladeno- sine is particularly useful for locating single-stranded regions. After chemical cleavage of the RNA, 7-methylguanosine also could be detected. In general, the sites of modification were consistent with a previous model of the secondary structure of the linear form of the IVS RNA, a model based on enzymatic cleavage data, free energy calculations, and phylogenetic com- parison. Thus, IVS RNA autocyclization does not involve ma- jor rearrangements of the secondary structure, although there is evidence for a conformational change in one region of the molecule. The methods described here should be of general use for obtaining information about structure far from the ends of RNA molecules.