US EPA

3231490 

Journal Article 

Downregulation of eNOS mRNA expression by TNF alpha: identification and functional characterization of RNA-protein interactions in the 3 ' UTR 

Lai, PFH; Mohamed, F; Monge, JC; Stewart, DJ 

2003 

Yes 

Cardiovascular Research
ISSN: 0008-6363
EISSN: 1755-3245 

59 

1 

160-168 

10.1016/S0008-6363(03)00296-7 

WOS:000184221300019 

Objective: We have previously shown that downregulation of
endothelial nitric oxide synthase (eNOS) expression by tumour necrosis factor-alpha (TNFalpha)
resulted entirely from the marked destabilization of the cNOS 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 YUTR were generated to identify the specific binding sequences.
eNOS rnRNA 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 TNFalpha,. 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 YUTR (designated M158)
favoured the formation of the 56-kDa RNP. Adenoviral gene transfer and overexpression of M 158
RNA, as a protein-binding decoy to prevent the formation of the 56-kDa RNP on the endogenous
transcripts, attenuated the TNFalpha-induced downregulation of eNOS mRNA in Cultured endothelial
cells. Conclusion: Our results demonstrate that the regulation of eNOS expression involves the
specific binding of cytoplasmic proteins to highly conserved elements along the YUTR, and the 56
-kDa RNP represents a novel regulatory trans-factor in the destabilization of eNOS transcripts.
(C) 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved. 

gene expression; endothelial factors; nitric oxide synthase; tumour necrosis factor alpha; RNA-binding factors