Altered mRNA Splicing and Inhibition of Human E-selectin Expression by an Antisense Oligonucleotide in Human Umbilical Vein Endothelial Cells

Thomas P Condon,C Frank Bennett

doi:10.1074/jbc.271.48.30398

Abstract

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.

Highlights

The field of ASO1 research has grown rapidly over the past few years (1–3)
If ISIS 2679 worked by an RNase H cleavage mechanism, a cleavage product would be difficult to resolve by Northern blot because ISIS 2679 hybridizes to bases 40 –59
ISIS 4730 was designed to hybridize at position 2006 –2025 of the E-selectin mRNA, and the expected RNase H cleavage products would both be approximately 1900 bases in length

Summary

Introduction

The field of ASO1 research has grown rapidly over the past few years (1–3). In cell culture-based experiments, most of the work reported has utilized either phosphodiester or phosphorothioate ASO. Potential mechanisms whereby an ASO may inhibit expression of its targeted mRNA include inhibition at the transcriptional or translational levels, as well as specific RNA processing steps (e.g. 5Ј capping, polyadenylation, splicing, nuclear export, and degradation of the target RNA by RNases). E-selectin, known as endothelial leukocyte adhesion molecule-1 (originally ELAM-1), is transiently expressed on endothelial cells by induction with inflammatory mediators such as interleukin-1, TNF-␣, or bacterial lipopolysaccharide (7). We have shown that ASO are specific and potent inhibitors of ICAM-1, vascular cell adhesion molecule-1, and E-selectin expression in HUVEC. One ASO, ISIS 4730, designed to be complementary to an area in the 3Ј-UTR of the human E-selectin mRNA, displayed a marked reduction in mRNA and protein expression levels. We have characterized this lower molecular weight transcript and demonstrate that ISIS 4730 targets the E-selectin pre-mRNA, promoting cleavage of the pre-mRNA at the hybridization site. We demonstrate that ISIS 4730 prevents splicing of the last intron (intron 13) of E-selectin, resulting in an additional 1300 bases of sequence associated with the stable cleavage product

Methods

Results

Conclusion