title: Posttranscriptional regulation of endothelial nitric oxide synthase during cell growth.
abstract: The expression of the endothelial NO synthase (eNOS) is dramatically influenced by the state of cell growth. In proliferating cells, mRNA levels are increased 4-fold compared with postconfluent, nonproliferating cells. Nuclear run-on analysis indicated that there is no difference in the transcriptional rate of eNOS in proliferating versus postconfluent cells. The half-life of eNOS mRNA, measured after actinomycin D transcriptional arrest, was 3-fold greater in preconfluent compared with confluent endothelial cells. Using UV-cross-linking analysis, a cytoplasmic protein with an apparent molecular mass of 51 kDa was found to bind to terminal 545-nt eNOS mRNA 3-fold more in confluent cells than in proliferating cells. Further characterization of the eNOS mRNA indicated that a 43-nt sequence at the origin of the 3'-untranslated region (UTR) is critical in binding of this protein. Endothelial cells were stably transfected with a chimeric cDNA plasmid containing chloramphenicol acetyl transferase (CAT) ligated to the eNOS coding region and either the wild-type 3'-UTR (pcDNACAT/eNOS((wtUTR))) or a mutant 3'-UTR lacking the 43 nt found to bind the 51-kDa protein (pcDNACAT/eNOS((DeltaUTR))). The CAT/eNOS mRNA half-life was dramatically stabilized in these latter cells as compared with cells transfected with pcDNACAT/eNOS((wtUTR))). Thus, this 43-nt region plays a critical role in destabilizing eNOS mRNA. These studies demonstrate a mechanism for modulation of eNOS expression during cell growth via interactions between the proximal 3'-UTR and a novel approximately 51-kDa cytosolic protein.
authors.affiliation: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: C D
authors.initials: CD
authors.lastname: Searles
authors.forename: Y
authors.initials: Y
authors.lastname: Miwa
authors.forename: D G
authors.initials: DG
authors.lastname: Harrison
authors.forename: S
authors.initials: S
authors.lastname: Ramasamy
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002455:Cell Division; D002478:Cells, Cultured; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D006207:Half-Life; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D006706:Homeostasis; D008970:Molecular Weight; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000502:physiology; D014158:Transcription, Genetic
chemical_list: D020413:3' Untranslated Regions; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D015500:Chloramphenicol O-Acetyltransferase