A shortened form of the Tetrahymena thermophila group I intron can catalyze the complete splicing reaction in trans

Abstract

The group I intron from Tetrahymena thermophila is able to catalyze its own excision from a precursor RNA. The intron recognizes the splice sites through an intron-encoded sequence called the internal guide sequence, or IGS. The 5' and 3' exons are thought to align on the IGS and form a pseudoknot structure consisting of two stems (P1 and P10). We created a shortened form of the intron that lacks the exon sequences and the entire IGS. This RNA is unable to react upon itself. It can catalyze a sequential two-step transesterification reaction on a P1P10 substrate added in trans that completely mimics splicing. The reaction works for different substrates that contain a U.G base-pair preceding the 5' cleavage site and a guanosine base preceding the 3' cleavage site, but that are otherwise unrelated in sequence. The ribozyme uses primarily the correct 5' and 3' splice sites even in the presence of potential cryptic splice sites, and therefore it must rely on the structure of the substrate (formation of the P1 and P10 helices) for correct splice site recognition. A C-G base-pair after the 5' splice site in P1 decreases activity while a U.G or G.U base-pair enhances activity. The relative position in P1 of the U.G base-pair preceding the 5' splice site is an important determinant. The ability of the intron to recognize primarily a specific structure, rather than a sequence, has ramifications for splice-site selection, for molecular modeling of the group I intron, and for ribozyme-based gene targeting.