Abstract
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).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sullenger, B. A. (1995) Revising messages traveling along the cellular information superhighway. Chem. Biol. 2, 249–253.
Cech, T. R (1990) Self splicing of group I introns. Annu. Rev. Biochem. 59, 543–568.
Zaug, A. J., Grosshans, C. A., and Cech, T. A. (1988) Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes. Biochemistry 27, 8924–8931.
Inoue, T., Sullivan, F. X., and Cech, T. R. (1985) Intermolecular exon ligation of the rRNA precursor of Tetrahymena: oligonucleotides can function as 5′ exons. Cell 43, 431–437.
Been, M. D. and Cech, T. R. (1986) One binding site determines sequence specificity of Tetrahymena pre-rRNA self splicing, trans-splicing, and RNA enzyme activity. Cell 47, 207–216.
Sullenger, B. A. and Cech, T. R. (1994) Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature 371, 619–622.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1997 Humana Press Inc.
About this protocol
Cite this protocol
Jones, J.T., Lee, SW., Sullenger, B.A. (1997). Trans-Splicing Reactions by Ribozymes. In: Turner, P.C. (eds) Ribozyme Protocols. Methods in Molecular Biology™, vol 74. Humana Press. https://doi.org/10.1385/0-89603-389-9:341
Download citation
DOI: https://doi.org/10.1385/0-89603-389-9:341
Publisher Name: Humana Press
Print ISBN: 978-0-89603-389-4
Online ISBN: 978-1-59259-560-0
eBook Packages: Springer Protocols