Abstract
The mRNA encoding the human low density lipoprotein (LDL) receptor is transiently stabilized after phorbol ester treatment of HepG2 cells and has been shown to associate with components of the cytoskeleton in this cell line (G. M. Wilson, E. A. Roberts, and R. G. Deeley, J. Lipid Res. 1997. 38: 437-446). Using an episomal expression system, fragments of the 3' untranslated region (3'UTR) of LDL receptor mRNA were transcribed in fusion with the coding region of beta-globin mRNA in HepG2 cells. Analyses of the decay kinetics of these beta-globin-LDL receptor fusion mRNA deletion mutants showed that sequences in the proximal 3'UTR of LDL receptor mRNA including several AU-rich elements (AREs) were sufficient to confer short constitutive mRNA half-life in the heterologous system. Stabilization of LDL receptor mRNA in the presence of PMA required sequences in the distal 3'UTR, at or near three Alu-like repetitive elements. Furthermore, the 3'UTR of LDL receptor mRNA conferred cytoskeletal association on the otherwise unassociated beta-globin mRNA, by a mechanism involving at least two distinct RNA elements. Comparisons of decay kinetics and subcellular localization of endogenous LDL receptor mRNA and beta-globin-LDL receptor mRNA fusions in HepG2 cells have demonstrated that several cis-acting elements in the receptor 3'UTR contribute to post-transcriptional regulation of receptor expression, and provide further support for involvement of the cytoskeleton in the regulation of LDL receptor mRNA turnover.
Highlights
The mRNA encoding the human low density lipoprotein (LDL) receptor is transiently stabilized after phorbol ester treatment of HepG2 cells and has been shown to associate with components of the cytoskeleton in this cell line
Maintenance of the open reading frame of globin ensured that translation of the G-LDLR3ЈUTR fusion mRNA would be terminated at the endogenous STOP codon of LDL receptor mRNA
These data indicate that elements within the 3Ј untranslated region (3ЈUTR) of receptor mRNA contribute to both the short constitutive half-life and stabilization in response to PMA treatment associated with endogenous LDL receptor mRNA in HepG2 cells
Summary
The mRNA encoding the human low density lipoprotein (LDL) receptor is transiently stabilized after phorbol ester treatment of HepG2 cells and has been shown to associate with components of the cytoskeleton in this cell line Several signal transduction pathways, including the cyclic AMP, inositol 1,4,5-trisphosphate-Ca2ϩ, and diacylglycerol-protein kinase C (PKC) systems [16, 17], have been shown to mediate receptor mRNA induction in HepG2 cells, and activation of PKC by treatment with phorbol esters results in increased LDL-binding and catabolism [18]. Activation of these signal transduction pathways is known to promote intracellular events required for cell proliferation or differentiation in many systems (reviewed in 19–21).
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