Role of the 3'-untranslated region of human endothelin-1 in vascular endothelial cells. Contribution to transcript lability and the cellular heat shock response.
Endothelin-1 (ET-1) is a potent vasoconstrictor peptide expressed in the vascular endothelium. Stringent control over ET-1 expression is achieved through a highly regulated promoter and rapid mRNA turnover. Since little is known about mechanisms governing ET-1 post-transcriptional regulation, and changes in ET-1 mRNA stability are implicated in disease processes, we characterized these pathways using a variety of functional approaches. We expressed human ET-1 and luciferase transcripts with or without a wild type ET-1 3'-untranslated region (3'-UTR) and found that the 3'-UTR had potent mRNA destabilizing activity. Deletion analysis localized this activity to two domains of the 3'-UTR we have termed destabilizing elements 1 and 2 (DE1 and DE2). Mutational studies revealed that DE1 functions as an AU-rich element (ARE) dependent on a 100-nucleotide region. This activity was further localized to a 10-nucleotide region at position 978-987 of the 3'-UTR. Depletion of AUF1 by RNA interference up-regulated ET-1 in endothelial cells suggesting AUF1-dependent regulation. Since AUF1 functions through the ubiquitin-proteasome pathway, we disrupted this pathway with heat shock and proteasome inhibitor in endothelial cells and observed stabilization of endogenous ET-1 mRNA. Chimeric transcripts bearing wild type ET-1 3'-UTRs were also stabilized in response to proteasome inhibition whereas DE1 mutants failed to respond. Taken together, these findings suggest a complex model of ARE-mediated mRNA turnover dependent on two 3'-UTR domains, DE1 and DE2. Furthermore, DE1 functions as an ARE directing mRNA half-life through the proteasome. Finally, this data provides evidence for a novel pathway of ET-1 mRNA stabilization by heat shock.
Mawji
Imtiaz A
IA
Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada.
Robb
G Brett
GB
Tai
Sharon C
SC
Marsden
Philip A
PA
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000378:metabolism; D019332:Endothelin-1 / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D018869:Heat-Shock Response / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction
D020413:3' Untranslated Regions; D019332:Endothelin-1; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex