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Post‐transcriptional regulation of cGMP‐dependent protein kinase mRNA in vascular smooth muscle cells.
Author(s) -
Sellak Hassan,
Lincoln Thomas M.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a540
Subject(s) - messenger rna , untranslated region , vascular smooth muscle , microbiology and biotechnology , cgmp dependent protein kinase , transcription (linguistics) , complementary dna , three prime untranslated region , transcriptional regulation , protein kinase a , biology , post transcriptional regulation , gene expression , chemistry , kinase , gene , cyclin dependent kinase 2 , biochemistry , endocrinology , smooth muscle , linguistics , philosophy
It is well established that type‐I cGMP‐dependent protein kinase (PKG‐I) mediates many functions in vascular smooth muscle cells (VSMC). Despite its important role in the vasculature, PKG expression regulation is not yet completely understood. We previously reported that PKG gene transcription is under the control of both Sp1/3 transcription factors and upstream stimulatory factors (USF1/2). Further investigations reveal that PKG transcript half‐life in actinomycin D‐treated VSMC was approximately10h. In agreement with this observation, the half‐life of PKG protein was between 72h and 92h, suggesting a plausible involvement of post‐transcriptional regulation in the stability of PKG mRNA. In the current study, we examined the role of the 3′untranslated region (UTR)‐PKG‐I mRNA in the control of PKG expression and stability in VSMC. Using a 3′ RACE system for the amplification of cDNA ends, we generated and cloned a 1.1kb 3′UTR‐PKG mRNA in pGL3 control vector down stream of the luciferase reporter gene. The analysis of the cloned 3′UTR‐PKG mRNA revealed the existence of 4 AU‐rich regions (AU1, AU2, AU3, and AU4). Different riboprobes were generated either by 5′labeling of designed ribonucleotides containing individual AU‐rich regions, or by in vitro transcription from the 1.1kb cDNA cloned in pGEM7 vector. RNA‐EMSA and UV‐cross linking assay showed that AU1 and AU3 as well as the 1.1 kb probe were able to retard two cytosolic proteins of about 30 and 50 kD. Serial deletions and functional studies revealed that among the deleted constructs, only the 1.1Kb 3′UTR PKG mRNA increased luciferase activity in transfected VSMC. This 3′UTR PKG mRNA construct contains a functional polyA site. Taken together, these data suggest that PKG expression is subjected to post‐transcriptional regulation in VSMC through its 3′UTR mRNA.