SYNTHESIS OF CIRCULAR RNA IN BACTERIA AND YEAST USING RNA CYCLASE RIBOZYMES DERIVED FROM A GROUP-I INTRON OF PHAGE-T4

Studies on the function of circular RNA and RNA topology in vivo have been limited by the difficulty in expressing circular RNA of desired s equence. To overcome this, the group I intron from the phage T4 td gen e was split in a peripheral loop (L6a) and rearranged so that the 3' h alf intron and 3' splice site are upstream and a 5' splice site and 5' half intron are downstream of a single exon. The group I splicing rea ctions excise the internal exon RNA as a circle (RNA cyclase ribozyme activity). We show that foreign sequences can be placed in the exon an d made circular in vitro. Expression of such constructs (RNA cyclase r ibozymes) in Escherichia coli and yeast results in the accumulation of circular RNA in these organisms. In yeast, RNA cyclase ribozymes can be expressed from a regulated promoter like an mRNA, containing 5' lea der and 3' trailer regions, and a nuclear pre-mRNA intron. RNA cyclase ribozymes have broad application to questions of RNA structure and fu nction including end requirements for RNA transport or function, RNA t opology, efficacy of antisense or ribozyme gene control elements, and the biosynthesis of extremely long polypeptides.