RNA transfection is a versatile tool to investigate endothelin-1 posttranscriptional regulation.
Endothelin-1 (ET-1) is a potent endothelial-derived vasoconstrictor and cellular mitogen. Perturbations in ET-1 levels have been observed in a number of cardiovascular and renal disorders. Steady-state ET-1 mRNA expression is regulated in the vascular endothelium by an inducible promoter and a constitutively short mRNA half-life. Recent studies have identified mRNA stabilization as a pathophysiologically relevant mechanism of ET-1 induction in vascular endothelial cells. However, mechanistic studies on posttranscriptional pathways in physiologically relevant postconfluent primary endothelial cell monolayers have remained challenging because of endothelial resistance to DNA-based gene transfer and expression. To overcome these challenges, we developed an RNA transfection method to study ET-1 posttranscriptional regulation. Reporter transcripts transfected into either preconfluent or postconfluent primary endothelial cells were rapidly and robustly expressed. RNA transfection reconstituted poly(A)-tail-dependent protein expression and ET-1 3'-UTR-dependent mRNA destabilization, suggesting that the transfected RNA accessed endothelial cell posttranscriptional pathways. Because RNA transfection uncouples transcription from expression, the influence of the ET-1 3'-UTR on posttranscriptional expression kinetics could also be monitored. Taken together, our results suggest that RNA transfection is a versatile tool to investigate ET-1 posttranscriptional regulation in endothelial cell culture models.
Mawji
Imtiaz A
IA
Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, University Health Network, University of Toronto, Medical Sciences Building, Room 7358, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
Marsden
Philip A
PA
D020413:3' Untranslated Regions; D000818:Animals; D001011:Aorta / Q000166:cytology; D002417:Cattle; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D020413:3' Untranslated Regions; D019332:Endothelin-1; D012313:RNA; D008156:Luciferases