Downregulation of eNOS mRNA expression by TNFα:: identification and functional characterization of RNA-protein interactions in the 3′UTR

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.