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Inhibition of splicing of wild-type and mutated luciferase-adenovirus pre-mRNAs by antisense oligonucleotides

D Hodges and ST Crooke

Department of Anatomy and Neurobiology, University of California, Irvine 92717, USA.

We report the construction, characterization, and use of luciferase reporters to test the ability of antisense oligonucleotides to inhibit RNA splicing. beta-Globin and adenovirus introns were inserted into a luciferase cDNA, and luciferase expression was analyzed in transiently transfected cells. The adenovirus reporter expressed large amounts of luciferase, but two beta-globin constructs were inactive. RNA analyses determined that the beta-globin pre-mRNAs were not spliced. Mutagenesis of the beta-globin 5' splice site, branchpoint, and 3' splice site sequences to the adenovirus intron sequences promoted maximal splicing and luciferase activity; reciprocal changes in all three elements of the adenovirus intron eliminated luciferase activity. Wild-type and 3' splice site mutated adenovirus reporters were used to determine the ability of phosphorothioate deoxy and 2' methoxy oligonucleotides to inhibit splicing. RNase H activating oligodeoxynucleotides were better inhibitors of wild-type adenovirus expression than were 2' methoxy analogues. However, 2' methoxy oligonucleotides specific for the branchpoint were more effective inhibitors of splicing of adenovirus transcript containing the beta-globin branchpoint and 3' splice site. We suggest that pre-mRNAs with weak splice sites are potential targets for oligonucleotides that inhibit splicing by occupancy rather than cleavage of the transcripts.

Volume 48, Issue 5, pp. 905-918, 11/01/1995
Copyright © 1995 by American Society for Pharmacology and Experimental Therapeutics




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Copyright © 1995 by the American Society for Pharmacology and Experimental Therapeutics