Self-splicing of the Tetrahymena intron from mRNA in mammalian cells.
The Tetrahymena pre-rRNA self-splicing intron is shown to function in the unnatural context of an mRNA transcribed by RNA polymerase II in mammalian cells. Mutational analysis supports the conclusion that splicing in cells occurs by the same RNA-catalyzed mechanism established for splicing in vitro. Insertion of the intron at five positions spanning the luciferase open reading frame revealed 10-fold differences in accumulation of ligated exons and in luciferase activity; thus, the intron self-splices in many exon contexts, but the context can have a significant effect on activity. In addition, even the best self-splicing constructs, which produced half as much mRNA as did an uninterrupted luciferase gene, gave approximately 100-fold less luciferase enzyme activity, revealing an unexpected discontinuity between mRNA production and translation in cells. The finding that production of accurately spliced mRNA in cells does not guarantee a corresponding level of protein production is surprising, and may have implications for the development of trans-splicing ribozymes as therapeutics.
Hagen
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Department of Chemistry and Biochemistry and Howard Hughes Medical Institute, University of Colorado, Boulder, CO 80309-0215, USA.
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D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D001517:Coleoptera / Q000235:genetics; D005091:Exons; D017930:Genes, Reporter; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000235:genetics; D008322:Mammals; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics*; D013768:Tetrahymena / Q000235:genetics*; D014162:Transfection
D012322:RNA Precursors; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D008156:Luciferases