INTERLOCKED CIRCLE FORMATION BY GROUP-I INTRONS - STRUCTURAL REQUIREMENTS AND MECHANISM

Precursor RNA transcribed from the yeast mitochondrial gene coding for the large ribosomal RNA contains a group I intron that can excise its elf in vitro. Apart from group I specific sequence elements the intron also contains a gene encoding a DNA endonuclease involved in intron d ispersal. A precursor RNA derivative from which this gene has been rem oved self-splices efficiently, but due to activation of cryptic openin g sites located in the 5' exon, the 3' part of this exon is sometimes co-excised with the intron. Upon further reaction, this enlarged intro n molecules give rise to interlocked circles, comprising small circles derived from 5' exon parts and large circles of the intron. Sequence comparison between cryptic opening sites and authentic splice sites re veals in most cases homology with the 3' exon part that is capable of interacting with the Internal Guide Sequence. The role of the IGS was further substantiated by replacing the cryptic opening sites with well defined sequences of authentic splice sites: one corresponding to the 3' splice site and its mutant derivatives, the other to a fragment co ntaining the natural 5'-3' exon junction. Precursor RNAs derived from these constructs give rise to interlocked circles, and mutation studie s confirm that the 3' exon nucleotides flanking a 3' splice site are e ssential for their formation. The results underline the crucial role o f the IGS in interlocked circle formation which behaves similarly as i n the normal self-splicing reactions. It has been proposed that the tw o short helices formed by basepairing of the IGS with the 5' and 3' ex on can co-axially stack on top of each other forming a quasi continuou s RNA double helix or pseudoknot. We present a model explaining how tr ansesterification reactions of a mutant precursor RNA in such a pseudo knot can lead to interlocked circles. The experiments support the noti on that a similar structure is also operative in splicing of wild type precursor RNA.