Regulation of endothelial nitric oxide synthase by small RNA

Proc Natl Acad Sci U S A. 2005 Nov 22;102(47):16967-72. doi: 10.1073/pnas.0503853102. Epub 2005 Nov 11.

Abstract

Repeats (27-nt) in intron 4 have been shown to play a cis-acting role in endothelial nitric oxide synthase (eNOS) promoter activity. We hypothesize that the 27-nt repeats could be the source of small nuclear RNA specifically regulating eNOS expression. In this study, we used synthesized 27-nt RNA duplex and found that the eNOS gene transcriptional efficiency was reduced 63% (0.047 +/- 0.009 vs. 0.126 +/- 0.015, P < 0.01) by nuclear run-on assay. In endothelial cells transfected with the 27-nt small RNA duplex, we found that the eNOS mRNA and protein levels were decreased by >64% (P < 0.01). Conversely, a randomly selected 27-nt from luciferase gene had no effect on the eNOS expression. Furthermore, this eNOS silencing effect appeared to be reversible under the stimulation of vascular endothelial growth factor (10 ng/ml), which is known to up-regulate eNOS expression. Using in situ hybridization and Northern blotting, we observed the presence of endogenous eNOS intron 4-derived 27-nt small RNA, which was confined to the nucleus. In summary, we demonstrated that intron-based microRNAs in eNOS can induce significant gene specific transcriptional suppression, which could be an effective negative feedback regulator for gene expression.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / enzymology
  • Gene Expression / physiology
  • Humans
  • Introns
  • Nitric Oxide Synthase Type III / genetics
  • Nitric Oxide Synthase Type III / metabolism*
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / physiology*
  • Transfection
  • Vascular Endothelial Growth Factor A / physiology

Substances

  • RNA, Small Interfering
  • Vascular Endothelial Growth Factor A
  • Nitric Oxide Synthase Type III