3'-untranslated sequences mediate post-transcriptional regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA by 25-hydroxycholesterol.
In an earlier study [Choi, Lundquist and Peffley (1993) Biochem. J. 296, 859-866], we determined that 25-hydroxycholesterol regulates 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA through a post-transcriptional mechanism that requires protein synthesis. To investigate whether 3'-untranslated sequences play a role in 25-hydroxycholesterol-mediated post-transcriptional control, we ligated approx. 1400 bp of the 3'-untranslated region of HMG-CoA reductase cDNA to the coding region of human beta-globin DNA. beta-Globin-3'-untranslated reductase fusion constructs were then transiently expressed in Chinese hamster ovary fibroblasts under conditions known to regulate reductase mRNA. There were no differences in beta-globin RNA levels in transfected cells incubated with or without lovastatin, a competitive inhibitor of reductase. However, in the presence of lovastatin and an oxysterol, 25-hydroxycholesterol, beta-globin RNA levels were decreased approx. 2-fold. Inhibition of protein synthesis with cycloheximide blocked the effects of 25-hydroxycholesterol on beta-globin RNA. Moreover, replacing the 3'-untranslated sequences with 1367 bp of the simian virus 40 enhancer region eliminated the regulatory effect of 25-hydroxycholesterol. Because the fusion construct has no sterol regulatory elements necessary for transcription, our results indicate that the change in beta-globin RNA occurred at a post-transcriptional level. In addition, we have shown that the 3'-untranslated region of HMG-CoA reductase cDNA imparted oxysterol-mediated post-transcriptional regulation to beta-globin RNA, an effect that required protein synthesis.
Choi
J W
JW
Department of Pharmacology and Molecular Biology, University of Health Sciences, Chicago Medical School, IL 60064, USA.
Peffley
D M
DM
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D003513:Cycloheximide / Q000494:pharmacology; D003864:Depression, Chemical; D004790:Enzyme Induction / Q000187:drug effects; D017930:Genes, Reporter; D005914:Globins / Q000096:biosynthesis / Q000235:genetics; D006888:Hydroxycholesterols / Q000494:pharmacology*; D006903:Hydroxymethylglutaryl CoA Reductases / Q000096:biosynthesis / Q000235:genetics*; D008148:Lovastatin / Q000494:pharmacology; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid
D003470:Culture Media; D006888:Hydroxycholesterols; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C007997:25-hydroxycholesterol; D005914:Globins; D003513:Cycloheximide; D008148:Lovastatin; D006903:Hydroxymethylglutaryl CoA Reductases