Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase.

Abstract

The structure of the intervening sequence (IVS) of the Tetahymena rRNA precursor mediates cleavageligation reactions that result in pre-rRNA splicing and IVS cyclization. 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 oligodeoxynucleotide primers was found to stop (or pause) one nucleotide prior to 1-methyladenosine, 3-methylcytidine, and 7-ethoxycarbonyladenosine residues. The detection of 1-methyladenosine 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 comparison. Thus, IVS RNA autocyclization does not involve major 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.Many eukaryotic genes are interrupted by intervening sequences (introns, IVSs), which are removed from transcripts of these genes by the process of RNA splicing (1, 2). We have been studying one example of such an event, the splic- MATERIALS AND METHODSPreparation of RNA. RNA was synthesized by transcription of pIVS11 DNA with purified Escherichia coli RNA polymerase (3) in a solution containing ATP, CTP, and GTP, each at 200 ,uM; 100 ,uM [3H]UTP (New England Nuclear); 250 mM NaCl; 5 mM MgCl2; 1 mM CaCl2; 50 mM Tris Cl, pH 8.0; and 0.07% 2-mercaptoethanol. During transcription at 30°C for 2 hr, RNA splicing and a limited amount of IVS RNA cyclization also occurred. The resultant IVS RNAs were purified by gel electrophoresis (12) followed by gel filtration on Sephadex G-50 and were then ethanol-precipitated in the absence of carrier. For cyclization, L IVS RNA was incubated in 150 mM NaCl/50 mM Na cacodylate, pH 7.5/10 mM MgCl2 at 30°C for 1 hr. The resulting RNA mixture, containing >80% C IVS, was used directly for chemical or enzymatic treatment without further purification.Chemical Modification. The general procedures and precautions described by Peattie (12) were followed. For modification of native C IVS RNA, 0.5 1,u of dimethyl sulfate (Me2SO4) (Aldrich) or 10 1.l of diethyl pyrocarbonate (DEP) (Aldrich) was added to 200 ul of solution containing 0.5-4 pmol of freshly cyclized IVS RNA and incubated at 30°C for 30 min (Me2SO4) or 60 min (DEP). For the "high Mg2+" and "low Mg2+" analyses, the concentrations of NaCl and MgCl2 were adjusted by dilution immediately after the 1-hr cyclization, and the above modification procedures then were ca...

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