Trans-Splicing Reactions by Ribozymes

  • Joshua T. Jones
  • Seong-Wook Lee
  • Bruce A. Sullenger
Part of the Methods in Molecular Biology™ book series (MIMB, volume 74)

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).
Fig. 1.

Ribozyme-mediated destruction of RNA vs ribozyme-mediated revision of RNA.

Fig. 2.

Self-splicing and targeted trans-splicing.

References

  1. 1.
    Sullenger, B. A. (1995) Revising messages traveling along the cellular information superhighway. Chem. Biol. 2, 249–253.PubMedCrossRefGoogle Scholar
  2. 2.
    Cech, T. R (1990) Self splicing of group I introns. Annu. Rev. Biochem. 59, 543–568.PubMedCrossRefGoogle Scholar
  3. 3.
    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.PubMedCrossRefGoogle Scholar
  4. 4.
    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.PubMedCrossRefGoogle Scholar
  5. 5.
    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.PubMedCrossRefGoogle Scholar
  6. 6.
    Sullenger, B. A. and Cech, T. R. (1994) Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature 371, 619–622.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1997

Authors and Affiliations

  • Joshua T. Jones
    • 1
    • 2
  • Seong-Wook Lee
    • 1
    • 2
  • Bruce A. Sullenger
    • 1
    • 2
  1. 1.Program in Molecular Therapeutics, Department of Experimental SurgeryDuke University Medical CenterDurham
  2. 2.Program in Molecular Therapeutics, Department of GeneticsDuke University Medical CenterDurham

Personalised recommendations