Key result
The 3'UTR of PKG-I mRNA stabilizes its expression in vascular smooth muscle cells through interactions with cytosolic proteins at specific AU-rich regions.
PKG-I expression in vascular smooth muscle cells is subject to post-transcriptional regulation through the 3'UTR of its mRNA.
No change to vascular therapy; leaves open whether 3'UTR-PKG-I interactions translate to human disease.
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.
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Sellak et al. (2011) studied Vascular smooth muscle cell regulation (in vitro). 3'UTR of PKG-I mRNA vs. Control vector was evaluated on Luciferase reporter activity and mRNA stability. The 3'UTR of PKG-I mRNA stabilizes its expression in vascular smooth muscle cells through interactions with cytosolic proteins at specific AU-rich regions.
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