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J. Biol. Chem., Vol. 279, Issue 36, 37982-37996, September 3, 2004

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Post-transcriptional Regulation of Endothelial Nitric-oxide Synthase by an Overlapping Antisense mRNA Transcript^*

G. Brett Robb, Andrew R. Carson, Sharon C. Tai¶, Jason E. Fish, Sundeep Singh¶, Takahiro Yamada, Stephen W. Scherer||**, Kazuhiko Nakabayashi, and Philip A. Marsden

From the Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, the Department of Genetics and Genomic Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, the ^||Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, and ^¶Renal Division and Department of Medicine, St. Michael's Hospital and University of Toronto, Toronto, Ontario M5S 1A8, Canada

Endothelial nitric-oxide synthase (eNOS) mRNA levels are abnormal in diseases of the cardiovascular system, but changes in gene expression cannot be accounted for by transcription alone. We found evidence for the existence of an antisense mRNA (sONE) that is derived from a transcription unit (NOS3AS) on the opposite DNA strand from which the human eNOS (NOS3) mRNA is transcribed at human chromosome 7q36. The genes are oriented in a tail-to-tail configuration, and the mRNAs encoding sONE and eNOS are complementary for 662 nucleotides. The mRNA for sONE could be detected in a variety of cell types, both in vivo and in vitro, but not vascular endothelial cells. In contrast, expression of eNOS is highly restricted to vascular endothelium. Most surprisingly, interrogation of transcriptional events across NOS3/NOS3AS genomic regions, using single- and double-stranded probes for nuclear run-off analyses and chromatin immunoprecipitation-based assessments of RNA polymerase II distribution, indicated that NOS3 and NOS3AS gene transcription did not correlate with steady-state mRNA levels. We found strong evidence supporting a role for NOS3AS in the post-transcriptional regulation of NOS3 expression. RNA interference-mediated inhibition of sONE expression in vascular smooth muscle cells increased eNOS expression. Overexpression of sONE in endothelial cells blunted eNOS expression. Finally, the histone deacetylase inhibitor trichostatin A is known to regulate the expression of eNOS via a post-transcriptional mechanism. We found that trichostatin A treatment of vascular endothelial cells increased expression of sONE mRNA levels prior to the observed decrease in eNOS mRNA expression. Taken together, these results indicate that an antisense mRNA (sONE) participates in the post-transcriptional regulation of eNOS and provide a newer model for endothelial cell-specific gene expression.

Received for publication, January 12, 2004 , and in revised form, May 25, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY316116, AY515311, AY515312, and AY515313.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** Investigator of the Canadian Institutes of Health Research and International Scholar of the Howard Hughes Medical Institute with operating support from Genome Canada.

Recipient of a Heart and Stroke Foundation of Canada Career Investigator Award and supported by Operating Grant T-5003 from the Heart and Stroke Foundation of Canada. To whom correspondence should be addressed: Rm. 7358, Medical Sciences Bldg., University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-2441; Fax: 416-978-8765; E-mail: p.marsden{at}utoronto.ca.

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