One or more labile proteins regulate the stability of chimeric mRNAs containing the 3'-untranslated region of cholesterol-7alpha -hydroxylase mRNA.
Multiple AUUUA elements similar to those that regulate the degradation of several different mRNAs are conserved in the 3'-untranslated region (3'-UTR) of cholesterol-7alpha-hydroxylase (CYP7A1) mRNAs from several species. We examined if stabilization of mRNA decay could account for the >20-fold increase in the expression of CYP7A1 mRNA without a detectable change in transcription following dexamethasone treatment of rat hepatoma cells (L35 cells). Following RNA polymerase II-dependent transcription block or protein synthesis block, the decay of CYP7A1 mRNA displayed a short half-life ( approximately 30 min). Control experiments showed that in cells pre-treated with a RNA polymerase II inhibitor, dexamethasone had no detectable effect on CYP7A1 mRNA decay. Stable expression of luciferase reporter mRNAs in L35 cells showed that the CYP7A1 3'-UTR was required to observe a dexamethasone induction. To examine the hypothesis that a labile protein is required for dexamethasone-induced mRNA stabilization, cells were stably transfected with a tetracycline-repressible promoter that drives the expression of a green fluorescent protein analogue (ECFP) with or without the 3'-UTR of CYP7A1. Cells expressing ECFP with the 3'-UTR of CYP7A1 displayed a 3-fold dexamethasone induction of ECFP mRNA, whereas cells expressing ECFP without the 3'-UTR did not. Moreover, specific block of the transcription of ECFP containing the 3'-UTR by adding the tetracycline analogue doxycycline clearly displayed dexamethasone-induced stabilization of mRNA decay. These data provide compelling evidence that a putative labile protein and the 3'-UTR of CYP7A1 act together to decrease the rate of CYP7A1 mRNA degradation.
Baker
D M
DM
Mammalian Cell and Molecular Biology Laboratory, San Diego State University, San Diego, California 92182-4614, USA.
Wang
S L
SL
Bell
D J
DJ
Drevon
C A
CA
Davis
R A
RA
D020413:3' Untranslated Regions; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002790:Cholesterol 7-alpha-Hydroxylase / Q000235:genetics* / Q000378:metabolism*; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004318:Doxycycline / Q000494:pharmacology; D017930:Genes, Reporter; D005938:Glucocorticoids / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010957:Plasmids; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D020413:3' Untranslated Regions; D000900:Anti-Bacterial Agents; D005938:Glucocorticoids; D008164:Luminescent Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D004004:Dichlororibofuranosylbenzimidazole; D003907:Dexamethasone; D003513:Cycloheximide; D008156:Luciferases; D002790:Cholesterol 7-alpha-Hydroxylase; D004318:Doxycycline