Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells

Abstract

A nuclear extract for the processing of oligomeric viroid RNA in vitro has been prepared from nuclei isolated from healthy potato cells grown in suspension culture. Linear RNA molecules containing concatameric units of (+) or (-) strands, respectively, of the potato spindle tuber viroid (PSTV) were synthesized in vitro with the aid of the SP6 RNA polymerase and used as substrates for processing. When oligomeric linear PSTV (+)RNAs are incubated with the nuclear extract, monomeric linear molecules are accurately excised from them, and ligated to monomeric PSTV (+)RNA circles representing the viroid proper. Oligomeric PSTV (-)RNAs are likewise processed but with a much lower efficiency. Viroid-processing operates although other nucleolytic activities are still present in the extract. These results substantiate our previous finding that oligomeric PSTV does not process autocatalytically under in vitro conditions where certain introns and other RNAs do. This is the first report of an in vitro RNA processing system derived from higher plants.