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Cited by 57RNA Research and SplicingNitric Oxide and Endothelin EffectsRNA and protein synthesis mechanisms

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

Lai, P·2003·Cardiovascular Research
DOI 10.1016/s0008-6363(03)00296-7
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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. 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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  • TNFα downregulates NOS3 expression and abundance by decreasing the stability of NOS3 mRNA, effectively shortening its half-life.

    “was shown to downregulate NOS3 expression and abundance [123, 131-134] by decreasing the stability of NOS3 mRNA [135, 136], effectively shortening its half-life [137].”

    From Regulation of obesity and insulin resistance by nitric oxide (2014) · cites this paper as [132]

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Authors
Lai, P.
Affiliations
St. Michael's Hospital (CA)
Corresponding author
Lai, P
ORCID
Lai, P
Venue
Cardiovascular Research
Publisher
Oxford University Press (OUP)
Published
2003-07-01
Volume
59
Issue
1
Pages
160-168
ISSN
0008-6363 (ISSN-L)
DOI
10.1016/s0008-6363(03)00296-7
OpenAlex
W2041378565
Cited by
57
References
26
Citations by year
2026: 1 · 2022: 4 · 2021: 4 · 2019: 1 · 2018: 2 · 2016: 2 · 2015: 6 · 2014: 3 · 2013: 1 · 2012: 7
FWCI
1.15
Type
journal article
Language
en
Open access
closed
Locations
  • Cardiovascular Research (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
Keywords
endothelial nitric oxide synthase, TNF-α, 3' untranslated region, RNA-protein interactions, mRNA stability, AU-rich elements, ribonucleoprotein complexes, endothelial cells, gene silencing, adenoviral gene transfer, translational regulation
MeSH terms
Animals, Base Sequence, Cattle, Cells, Cultured, Cloning, Molecular, Cytoplasm, Endothelium, Vascular, Gene Expression Regulation, Humans, Molecular Sequence Data, Protein Binding, RNA, Messenger, Tumor Necrosis Factor-alpha, Sequence Homology, Nitric Oxide Synthase, Reverse Transcriptase Polymerase Chain Reaction, 3' Untranslated Regions, Nitric Oxide Synthase Type III

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