One or more labile proteins regulate the stability of chimeric mRNA containing the 3′-untranslated region of cholesterol-7α-hydroxylase mRNA
One or more labile proteins regulate the stability of chimeric mRNA containing the 3′-untranslated region of cholesterol-7α-hydroxylase mRNA
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DOI：
10.1074/jbc.m002351200
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发表时间：
2000-06-30
影响因子：
4.8
通讯作者：
Davis, RA
中科院分区：
文献类型：
--
作者：
Baker, DM;Wang, SL;Davis, RA
来自多个物种的胆固醇 7 α-羟化酶 (CYP7A1) mRNA 的 3'-非翻译区 (3'-UTR) 中保守有多个 AUUUA 元件，这些元件与调节几种不同 mRNA 降解的元件相似。我们检查了 mRNA 衰减的稳定是否可以解释在地塞米松处理大鼠肝癌细胞（L35 细胞）后 CYP7A1 mRNA 的表达增加超过 20 倍而转录没有可检测到的变化。在RNA聚合酶II依赖性转录阻断或蛋白质合成阻断之后，CYP7A1 mRNA的衰变显示出较短的半衰期（类似于30分钟）。对照实验表明，在用 RNA 聚合酶 II 抑制剂预处理的细胞中，地塞米松对 CYP7A1 mRNA 衰减没有可检测到的影响。L35 细胞中荧光素酶报告基因 mRNA 的稳定表达表明，观察地塞米松诱导需要 CYP7A1 3'-UTR。为了检验地塞米松诱导的 mRNA 稳定需要不稳定蛋白的假设，用四环素抑制型启动子稳定转染细胞，该启动子驱动绿色荧光蛋白类似物 (ECFP) 的表达，无论有或没有 CYP7A1 的 3'-UTR，表达带有 CYP7A1 5'-UTR 的 ECFP 的细胞显示出 3 倍的地塞米松诱导 ECFP mRNA，而表达 ECFP 的细胞显示出 3 倍的地塞米松诱导 ECFP mRNA没有 3'-UTR 的 ECFP 则不会。此外，通过添加四环素类似物多西环素对包含 3'-UTR 的 ECFP 转录进行特异性阻断，清楚地显示了地塞米松诱导的 mRNA 降解稳定。这些数据提供了令人信服的证据，证明假定的不稳定蛋白和 CYP7A1 的 3'-UTR 共同作用，降低了 CYP7A1 mRNA 降解的速率。
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-7 alpha-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 (similar to 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 5'-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.