A modified group I intron can function as both a ribozyme and a 5' exon in a trans-exon ligation reaction.
Here, we show that a single RNA molecule derived from a group-I intron can provide the catalytic activity, the substrate recognition domain and the attacking nucleophile in a reaction that mimics the exon ligation step of splicing. To accomplish this reaction, we have linked a 5' exon sequence to the 3' end of an attenuated form of the self-splicing Tetrahymena rRNA intron. The ribozyme (I-E1) attacks an oligoribonucleotide analog of the 3' splice site (I'-E2) to generate a product containing ligated exons (I-E1-E2) and a small intron fragment (I'). Two modified introns were constructed and tested for activity. A construct designed to interact with the 3' splice site through intermolecular P9.0 and P10 helices was found to be inactive due to failure to form a stable ribozyme-substrate complex. A second modified intron and substrate combination was engineered, in which the complex was further stabilized by an intermolecular P9.2 helix. In this case, stable complexes and reaction products were identified. The reaction efficiency was low compared to splicing of the unmodified intron-containing precursor, and will be optimized in future experiments. Following optimization, we believe that this system may be exploited to examine the functional consequences of a wide variety of 3' splice-site modifications, and may provide the basis for development of highly selective trans-acting ribozymes.
Markey Center for Molecular Genetics, Department of Microbiology and Molecular Genetics, University of Vermont, Burlington 05405.
K I
KI
Tasiouka
J M
JM
Burke
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D005091:Exons; D006868:Hydrolysis; D007438:Introns; D008969:Molecular Sequence Data; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000378:metabolism*; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D013768:Tetrahymena / Q000235:genetics; D014158:Transcription, Genetic
D016337:RNA, Catalytic; D016053:RNA, Protozoan; D012335:RNA, Ribosomal