title: Stabilization of cGMP-dependent protein kinase G (PKG) expression in vascular smooth muscle cells: contribution of 3'UTR of its mRNA.
abstract: The type-I cGMP-dependent protein kinase (PKG-I) expression regulation is not yet completely understood. In this study, we examined the role of 3'-untranslated region (3'UTR)-PKG-I messenger RNA (mRNA) in the control of PKG-I expression in vascular smooth muscle cells (VSMCs). Using a 3'-rapid amplification of cDNA ends (RACE) for the amplification of complementary DNA (cDNA) ends, we generated and cloned a 1.2-kb-3'UTR mRNA PKG-I in pGL3 control vector downstream of the luciferase reporter gene. Serial deletions and functional studies revealed that among the deleted constructs, only the 1.2-kb-3'UTR PKG-I mRNA possesses the highest activity in transfected VSMC. Kinetic luciferase assays in the presence of actinomycin D showed that this construct stabilizes luciferase activity compared to the control vector. Sequence analysis of 3'UTR-PKG-I mRNA revealed the existence of four AU-rich regions (AU1 through AU4) in addition to a potential poly(A) site. Different riboprobes were generated either by 5'-end-labelling of designed ribonucleotides, containing individual AU-rich regions or by in vitro transcription assay using cloned 1.2-kb cDNA as a template. RNA-electrophoretic mobility shift assay (EMSA) and ultra-violet cross-linking (UV-CL) assays showed that AU1, AU3, AU4 and 1.2-kb probes were able to retard cytosolic and nuclear proteins. Taken together, these data suggest that PKG-I expression is subjected to post-transcriptional regulation in VSMC through the 3'UTR of its mRNA.
authors.affiliation: University of South Alabama, College of Medicine, Department of Physiology, Mobile, AL 36688, USA. hsellak@usouthal.edu
authors.forename: Hassan
authors.initials: H
authors.lastname: Sellak
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Lincoln
authors.forename: Chung-Sik
authors.initials: CS
authors.lastname: Choi
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D012333:RNA, Messenger; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases