Tagging ribozyme reaction sites to follow trans-splicing in mammalian cells.
In mammalian cells, genetic instructions are usually revised by RNA splicing before they are translated to proteins. Here we demonstrate that a trans-splicing group I ribozyme can be employed to intentionally modify the sequence of targeted transcripts in tissue culture cells. By analyzing the ribozyme reaction products, we demonstrate that targeted trans-splicing can proceed in murine fibroblasts with high fidelity, providing direct evidence that ribozymes function as anticipated in a therapeutically relevant setting. Trans-splicing is not very specific however, and the ribozyme reacted with and tagged a variety of cellular transcripts with its 3' exon sequence. RNA tagging provides a unique approach to study RNA catalysis in mammalian cells. Such analysis should facilitate the logical development of safe, therapeutic ribozymes that can repair mutant RNAs associated with a variety of inherited diseases.
Duke Scholars
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Related Subject Headings
- Viral Proteins
- Transfection
- Transcription, Genetic
- Tetrahymena thermophila
- Substrate Specificity
- RNA, Catalytic
- RNA, Bacterial
- RNA Splicing
- RNA
- Polymerase Chain Reaction
Citation
Published In
DOI
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Viral Proteins
- Transfection
- Transcription, Genetic
- Tetrahymena thermophila
- Substrate Specificity
- RNA, Catalytic
- RNA, Bacterial
- RNA Splicing
- RNA
- Polymerase Chain Reaction