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

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Abstract
Objective: We have previously shown that downregulation of endothelial nitric oxide synthase (eNOS) expression by tumour necrosis factor-α (TNFα) 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 TNFα, 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 TNFα-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 3′UTR, and the 56-kDa RNP represents a novel regulatory trans-factor in the destabilization of eNOS transcripts.

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