Part of the Methods in Molecular Biology™ book series (MIMB, volume 74)
Trans-Splicing Reactions by Ribozymes
Trans-cleaving ribozymes can be targeted to cut specific RNAs in vitro, which has led to much interest in their potential to destroy specific messages inside cells. Here, we will describe a different reaction catalyzed by ribozymes that may allow them to be employed to “revise” genetic instructions, not just destroy them (see Fig. 1) (1). The Tetrahymena thermophila self-splicing group I intron naturally excises itself from pre-rRNAs by performing two successive transesterification reactions (see Fig. 2A) (2). First, the phosphodiester bond that attaches it to a 5′-exon is broken. Then, holding onto the 5′-exon via base pairing with its 5′-exon binding site, the ribozyme catalyzes the ligation of the 5′-exon onto the 3′-exon to liberate itself. Careful characterization of this reaction has illustrated that the vast majority of sequence requirements for such splicing are contained within the intron. The sequence of the 3′-exon can be made of virtually any sequence, and the 5′-exon must only have a uridine at the splice site and maintain base pairing between the end of the exon and the intron’s 5′-exon binding site (see Fig. 2A).
© Humana Press Inc. 1997