title: Active stabilization of human endothelial nitric oxide synthase mRNA by hnRNP E1 protects against antisense RNA and microRNAs.
abstract: Human endothelial nitric oxide synthase (eNOS) mRNA is highly stable in endothelial cells (ECs). Posttranscriptional regulation of eNOS mRNA stability is an important component of eNOS regulation, especially under hypoxic conditions. Here, we show that the human eNOS 3' untranslated region (3' UTR) contains multiple, evolutionarily conserved pyrimidine (C and CU)-rich sequence elements that are both necessary and sufficient for mRNA stabilization. Importantly, RNA immunoprecipitations and RNA electrophoretic mobility shift assays (EMSAs) revealed the formation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1)-containing RNP complexes at these 3'-UTR elements. Knockdown of hnRNP E1 decreased eNOS mRNA half-life, mRNA levels, and protein expression. Significantly, these stabilizing RNP complexes protect eNOS mRNA from the inhibitory effects of its antisense transcript sONE and 3'-UTR-targeting small interfering RNAs (siRNAs), as well as microRNAs, specifically, hsa-miR-765, which targets eNOS mRNA stability determinants. Hypoxia disrupts hnRNP E1/eNOS 3'-UTR interactions via increased Akt-mediated serine phosphorylation (including serine 43) and increased nuclear localization of hnRNP E1. These mechanisms account, at least in part, for the decrease in eNOS mRNA stability under hypoxic conditions. Thus, the stabilization of human eNOS mRNA by hnRNP E1-containing RNP complexes serves as a key protective mechanism against the posttranscriptional inhibitory effects of antisense RNA and microRNAs under basal conditions but is disrupted under hypoxic conditions.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: J J David
authors.initials: JJ
authors.lastname: Ho
authors.forename: G Brett
authors.initials: GB
authors.lastname: Robb
authors.forename: Sharon C
authors.initials: SC
authors.lastname: Tai
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Turgeon
authors.forename: Imtiaz A
authors.initials: IA
authors.lastname: Mawji
authors.forename: H S Jeffrey
authors.initials: HS
authors.lastname: Man
authors.forename: Philip A
authors.initials: PA
authors.lastname: Marsden
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D061745:Antioxidant Response Elements; D001483:Base Sequence; D001665:Binding Sites; D015687:Cell Hypoxia; D002478:Cells, Cultured; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins; D006207:Half-Life; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D052250:Nitric Oxide Synthase Type III / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins; D011817:Rabbits; D043244:Ribonuclease III / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D035683:MicroRNAs; C094604:PCBP1 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases