One or more labile proteins regulate the stability of chimeric mRNA containing the 3 '-untranslated region of cholesterol-7 alpha-hydroxylase mRNA

Multiple AUUUA elements similar to those that regulate the degradation of s everal different mRNAs are conserved in the 3'-untranslated region (3'-UTR) of cholesterol-7 alpha-hydroxylase (CYP7A1) mRNAs from several species. We examined if stabilization of mRNA decay could account for the >20-fold inc rease in the expression of CYP7A1 mRNA without a detectable change in trans cription following dexamethasone treatment of rat hepatoma cells (L35 cells ). Following RNA polymerase II-dependent transcription block or protein syn thesis block, the decay of CYP7A1 mRNA displayed a short half-life (similar to 30 min). Control experiments showed that in cells pre-treated with a RN A polymerase II inhibitor, dexamethasone had no detectable effect on CYP7A1 mRNA decay, Stable expression of luciferase reporter mRNAs in L35 cells sh owed that the CYP7A1 3'-UTR was required to observe a dexamethasone inducti on. To examine the hypothesis that a labile protein is required for dexamet hasone-induced mRNA stabilization, cells were stably transfected with a tet racycline-repressible promoter that drives the expression of a green fluore scent protein analogue (ECFP) with or without the 3'-UTR of CYP7A1, Cells e xpressing ECFP with the 5'-UTR of CYP7A1 displayed a 3-fold dexamethasone i nduction 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 de xamethasone-induced stabilization of mRNA decay, These data provide compell ing evidence that a putative labile protein and the 3'-UTR of CYP7A1 act to gether to decrease the rate of CYP7A1 mRNA degradation.