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.
Highlights
Endothelial nitric-oxide synthase mRNA levels are abnormal in diseases of the cardiovascular system, but changes in gene expression cannot be accounted for by transcription alone
We found that the major 2.9-kb sONE mRNA species was encoded by a 12-exon gene (NOS3AS) spanning 6.5 kb of genomic DNA (Fig. 1a, Tables III–VI)
We noted that the human sONE mRNA contains four regions that were perfectly complementary to the Endothelial nitric-oxide synthase (eNOS) mRNA (Fig. 1, a and b) and that sONE is complementary to regions spanning portions of exons 23–26 and representing 182 amino acids of human eNOS
Summary
CDNA and Genomic Characterization—An oligo(dT)-primed gt human testis cDNA library (Clontech) was screened under conditions of high stringency by using a full-length human eNOS cDNA (25). Quantitation of sONE mRNA levels was performed by using primers spanning NOS3AS exons 7/8 5Ј-CGC CTG ATG AGG AGA AGC C-3Ј, 5Ј-TCT GTG GTC ACC TGA AAC CCT-3Ј, and 5Ј-VICTM TGC CTC TAG CCC CAG ACA ACA GTG G TAMRATM-3Ј (123-bp amplicon) (VICTM, Applied Biosystems). Northern Blot Analysis and in Situ cRNA Hybridization—Multiple tissue Northern blots containing ϳ2 g of poly(A)ϩ RNA per lane (Clontech) were hybridized with human cDNA probes corresponding to exons 8 –16 of NOS3 or 4 –9 of NOS3AS. These probes are located in nonoverlapping genomic regions.
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