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.
Jones
J T
JT
Department of Experimental Surgery, Duke University Medical Center, Durham, North Carolina 27710, USA.
Lee
S W
SW
Sullenger
B A
BA
D016475:3T3 Cells / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D003593:Cytoplasm / Q000502:physiology; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D016808:Tetrahymena thermophila / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins
D012329:RNA, Bacterial; D016337:RNA, Catalytic; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases