Abstract 256: Post-transcriptional Regulation of Endothelial Nitric Oxide Synthase Expression by Translation Elongation Factor 1-alpha
BACKGROUND: Nitric oxide (NO) is a potent cell-signaling molecule that plays important and diverse roles in biological processes. NO produced by endothelial nitric oxide synthesis (eNOS) is critical to the maintenance of vascular homeostasis. Because of its important biological effects, NO production by eNOS is under complex and tight control. Accumulating evidence indicates that the binding of cytosolic protein(s) to eNOS 3′ untranslated region (3′-UTR) plays a critical role in the regulation of eNOS mRNA stability. However, the identity of these proteins has not been elucidated so far.
METHODS AND RESULTS: eNOS 3′-UTR binding proteins were purified by RNA affinity chromatography from a cytosolic fraction of TNF-α stimulated bovine aortic endothelial cells (BAECs). We found that two bands at 60 and 52 kDa were uniquely present in eNOS 3′-UTR ribonucleoprotein complexes. LC-MS/MS analysis of the 52 kDa band identified three peptides (YYVTIIDAPGHR, EHALLAYTLGVK, VETGVLKPGMVVTFAPVNVTTEVK) that correspond to peptides deduced from translation elongation factor 1-alpha (eEF1A). Immuno-precipitation and reverse transcription-PCR assays further confirmed the specific interaction of eEF1A and eNOS mRNA 3′-UTR in TNF-α stimulated BAECs. In addition, RNA gel mobility shift and UV crosslinking assays suggested that recombinant GST-eEF1A fusion protein specifically binds to an UC-rich sequence in the 3′-UTR of eNOS mRNA. Treatment of BAECs with TNF-α (20 ng/ml) rapidly increased eEF1A expression by 12-fold, reaching a maximum after 1hr of stimulation. TNF-α incubation did not change the cellular localization of eEF1A, but markedly enhanced the binding of eEF1A to eNOS mRNA in BAECs, as demonstrated by RNA affinity pull-down assays. In addition, overexpression of eEF1A inhibited the expression of luciferase gene fused with eNOS 3′-UTR in both COS-7 cells and BAECs. Coexpression of eEF1A and eNOS in COS-7 cells substantially inhibited eNOS expression by 80%. Furthermore, eNOS mRNA stability was markedly decreased in BAECs transduced with recombinant adenovirus harboring full length eEF1A gene.
CONCLUSIONS: these results demonstrate that eEF1A is a novel eNOS 3′-UTR binding protein that plays a critical role in the regulation of eNOS mRNA stability.