title: Downregulation of eNOS mRNA expression by TNFalpha: identification and functional characterization of RNA-protein interactions in the 3'UTR.
abstract: OBJECTIVE
  We have previously shown that downregulation of endothelial nitric oxide synthase (eNOS) expression by tumour necrosis factor-alpha (TNF alpha) resulted entirely from the marked destabilization of the eNOS mRNA. As the 3'-untranslated region (3'UTR) in many eukaryotic mRNA has been well documented to bind regulatory trans-factors in the control of transcript stability, we have examined protein binding to this region of the eNOS mRNA. A high degree of homology amongst human and bovine 3'UTR also suggests that important functional features that are conserved through evolution are present within this region.
  
  
  METHODS
  RNA-protein interactions were studied in cross-linking assays, in which radiolabelled RNA encoding the human eNOS 3'UTR or selected sequences was incubated with cytoplasmic extracts of cultured human umbilical vein endothelial cells (HUVECs). Serial 5'- and 3'-truncated deletional mutations of the eNOS 3'UTR were generated to identify the specific binding sequences. eNOS mRNA expression in HUVECs was assessed by RT-PCR analysis.
  
  
  RESULTS
  Using radiolabelled RNA encoding the entire 418-nucleotide 3'UTR, we have identified ribonucleoprotein complexes (RNPs) of approximate molecular weights of 53, 56 and 66 kDa in the endothelial extracts. The formation of the 53- and 56-kDa RNPs was upregulated by TNF alpha, while the formation of the 66-kDa RNP was downregulated. Formation of the 53-kDa RNP was favoured by RNA fragments that contained sequences from the proximal and distal portions of the 3'UTR, whereas the formation of the 66-kDa RNP was favoured by RNA fragments with the AU-rich distal end. RNA fragments containing a CU-rich 158-nucleotide sequence from the medial portion of the eNOS 3'UTR (designated M158) favoured the formation of the 56-kDa RNP. Adenoviral gene transfer and overexpression of M158 RNA, as a protein-binding decoy to prevent the formation of the 56-kDa RNP on the endogenous transcripts, attenuated the TNF alpha-induced downregulation of eNOS mRNA in cultured endothelial cells.
  
  
  CONCLUSIONS
  Our results demonstrate that the regulation of eNOS expression involves the specific binding of cytoplasmic proteins to highly conserved elements along the 3'UTR, and the 56-kDa RNP represents a novel regulatory trans-factor in the destabilization of eNOS transcripts.
authors.affiliation: The Terrence Donnelly Research Laboratories, Division of Cardiology, St. Michael's Hospital, and Institute of Medical Science, University of Toronto, Ontario, Canada.
authors.forename: Patrick F H
authors.initials: PF
authors.lastname: Lai
authors.forename: Farida
authors.initials: F
authors.lastname: Mohamed
authors.forename: Juan-Carlos
authors.initials: JC
authors.lastname: Monge
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Stewart
mesh_terms: D020413:3' Untranslated Regions / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017385:Sequence Homology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D020413:3' Untranslated Regions; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III