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BASIC BIOLOGY

RNA Transfection Is a Versatile Tool to Investigate Endothelin-1 Posttranscriptional Regulation

Imtiaz A. Mawji^* and Philip A. Marsden^,1

^* Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, University Health Network, University of Toronto, Toronto, Canada; and Renal Division and Department of Medicine, St. Michael’s Hospital and University of Toronto, Toronto, Canada

To whom requests for reprints should be addressed at ¹ University of Toronto, Medical Sciences Building, Room 7358, 1 King’s College Circle, Toronto, ON M5S 1A8. E-mail: p.marsden{at}utoronto.ca

Abstract

Endothelin-1 (ET-1) is a potent endothelial-derived vasocon-strictor 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.

Key Words: 3'-untranslated region • endothelin • endothelium • gene regulation • mRNA stability • RNA transfection