Altered mRNA splicing and inhibition of human E-selectin expression by an antisense oligonucleotide in human umbilical vein endothelial cells.
We have characterized the mechanism of action of an antisense oligodeoxynucleotide (ASO) targeting human endothelial leukocyte adhesion molecule, E-selectin. ISIS 4730, a 20-base ASO designed to be complementary to a region in the 3'-untranslated region (3'-UTR) of human E-selectin, is a potent and specific inhibitor of both mRNA and protein expression in human umbilical vein endothelial cells. Following treatment with ISIS 4730, a lower molecular weight mRNA (3300 bases) species was detected by Northern blot analysis with a corresponding decrease in the mature E-selectin transcript (3875 bases). The ASO-induced low molecular weight mRNA is stable and remains in the nucleus. We demonstrate that ISIS 4730 targets E-selectin pre-mRNA in the nucleus and promotes cleavage of the pre-mRNA at the hybridization site, resulting in prevention of splicing of the last intron. The change in molecular weight of the E-selectin transcript is the result of loss of the 3'-UTR due to ASO-mediated RNA cleavage and retention of the last intron. Cleavage of the E-selectin pre-mRNA appears to be due to endogenous RNase H or a related enzyme activity.
Condon
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ISIS Pharmaceuticals, Department of Molecular Pharmacology, Carlsbad, California 92008, USA.
Bennett
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D017398:Alternative Splicing / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D019040:E-Selectin / Q000235:genetics*; D004730:Endothelium, Vascular / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D013329:Structure-Activity Relationship; D014471:Umbilical Veins
D019040:E-Selectin; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger