Structural requirements for selection of 5′- and 3′ splice sites of group II introns

The group II intron bl1 in the gene for apocytochrome b in yeast mitochondrial DNA (COB) is self-splicing in vitro. It could recently be shown that self-splicing of this intron is fully reversible in vitro. In addition, intron integration is not restricted to parental exons, since the intron can also integrate into a foreign RNA. The position of insertion seems to be immediately 3' to a cryptic intron binding site 1 (IBS1). We confirmed and extended these results by sequencing 26 individual RNAs with transposed introns after reverse transcription and PCR amplification. Results show that intron integration into authentic exons is generally correct, but that integration into a foreign RNA is often inaccurate, i.e. insertion is one nt downstream or upstream of the 3' end of IBS1. This leads to the generation of 5' splice junctions of the new intron-harbouring 'preRNAs' with addition (or deletion) of a single A residue at the 3' end of IBS1. To investigate which structures help to define the position of 5'- and 3' cleavage, preRNAs of i) these clones with aberrant 5' splice junctions and ii) preRNAs with artificial hairpins between domains 5 and 6 of the intron were spliced under different reaction conditions. Results obtained let us conclude that i) branchpoint dependent 5' cleavage is directed by the 5' terminal G residue of the intron and, ii) the first nucleotide(s) of the 3' exon play an important role in defining the 3' splice site.