Identification of a non-junction phosphodiester that influences an autolytic processing reaction of RNA

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Abstract
Oligoribonucleotides with specific sequences derived from the satellite RNA of tobacco ringspot virus undergo autolytic cleavage at the CpA phosphodiester that is the junction between unit sequences of multimeric satellite RNA. Buzayan et al. (Nucleic Acids Res., 16, 4009–4023 (1988)) showed that an oligoribonucleotide with 97 satellite RNA-derived nucleotide residues self-cleaved with greatly reduced efficiency when it was synthesized in vitro from adenosine-5'-O-(1-thiotriphosphate) (abbreviated rATPαS) and three rNTPs. No other substitution of one rNTP by the corresponding rNTPαS had this effect, suggesting that a phosphorothioate CpA junction inhibits self-cleavage. Here, we replaced the usual CpA junction of a small self-cleaving oligoribonucleotide with a CpU junction. Self-cleavage of this molecule was reduced not only by rUTPαS-substitution, as expected, but also by partial and complete rATPαS-substitution. By analysis of the locations of rAMPS residues in cleavage products derived from partially rATPaSsubstituted oligoribonucleotides, we identified A26 as the residue contributing the non-junction phosphorothioate diester that most strongly inhibited self-cleavage. Manganese ions strongly stimulated the self-cleavage of the rATPαS-substituted, CpU-junction oligoribonucleotide but was less effective when the junction was CpA.