Reverse self-splicing of group II intron RNAs in vitro

Abstract

GROUP II introns, which are classed together on the basis of a conserved secondary structure¹, are found in organellar genes of lower eukaryotes and plants. Like introns in nuclear pre-messenger RNA, they are excised by a two-step splicing reaction to generate branched circular RNAs, the so-called lariats². A remarkable feature of group II introns is their self-splicing activity in vitro^3–6. In the absence of a nucleotide cofactor, the intron RNAs catalyse two successive transesterification reactions which lead to autocatalytic excision of the lariat IVS from pre-mRNA and concomitantly to exon ligation. By virtue of its ability to specifically bind the 5' exon⁷, the intron can also catalyse such reactions on exogenous RNA substrates⁸. This sequence-specific attachment could enable group II introns to integrate into unreá-lated RNAs by reverse splicing, in a process similar to that described for the self-splicing Tetrahymena group I intron⁹. Here we report that group II lariat IVS can indeed reintegrate itself into an RNA composed of the ligated exons in vitro. This occurs by a process of self-splicing that completely reverses both transesterification steps of the forward reaction: it involves a transition of the 2'–5' phosphodiester bond of the lariat RNA into the 3'–5' bond of the reconstituted 5' splice junction.