title: Post-transcriptional regulation of endothelial nitric-oxide synthase by an overlapping antisense mRNA transcript.
abstract: 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.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
authors.forename: G Brett
authors.initials: GB
authors.lastname: Robb
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Carson
authors.forename: Sharon C
authors.initials: SC
authors.lastname: Tai
authors.forename: Jason E
authors.initials: JE
authors.lastname: Fish
authors.forename: Sundeep
authors.initials: S
authors.lastname: Singh
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yamada
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Scherer
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Nakabayashi
authors.forename: Philip A
authors.initials: PA
authors.lastname: Marsden
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011233:Precipitin Tests; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects*; D016372:RNA, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D016372:RNA, Antisense; D012333:RNA, Messenger; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III