Choi J W, Peffley D M
Department of Pharmacology and Molecular Biology, University of Health Sciences, Chicago Medical School, IL 60064, USA.
Biochem J. 1995 Apr 1;307 ( Pt 1)(Pt 1):233-8. doi: 10.1042/bj3070233.
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.
在一项早期研究中[崔、伦德奎斯特和佩夫利（1993年），《生物化学杂志》296卷，859 - 866页]，我们确定25 - 羟基胆固醇通过一种需要蛋白质合成的转录后机制来调节3 - 羟基 - 3 - 甲基戊二酰辅酶A（HMG - CoA）还原酶mRNA。为了研究3' - 非翻译序列在25 - 羟基胆固醇介导的转录后调控中是否起作用，我们将约1400 bp的HMG - CoA还原酶cDNA的3' - 非翻译区连接到人类β - 珠蛋白DNA的编码区。然后，β - 珠蛋白 - 3' - 非翻译还原酶融合构建体在已知可调节还原酶mRNA的条件下在中国仓鼠卵巢成纤维细胞中瞬时表达。在用还原酶的竞争性抑制剂洛伐他汀孵育的转染细胞中，无论有无洛伐他汀，β - 珠蛋白RNA水平均无差异。然而，在存在洛伐他汀和一种氧化甾醇25 - 羟基胆固醇的情况下，β - 珠蛋白RNA水平大约降低了2倍。用放线菌酮抑制蛋白质合成可阻断25 - 羟基胆固醇对β - 珠蛋白RNA的影响。此外，用1367 bp的猿猴病毒40增强子区域替换3' - 非翻译序列消除了25 - 羟基胆固醇的调控作用。由于融合构建体没有转录所需的甾醇调节元件，我们的结果表明β - 珠蛋白RNA的变化发生在转录后水平。此外，我们已经表明，HMG - CoA还原酶cDNA的3' - 非翻译区赋予β - 珠蛋白RNA氧化甾醇介导的转录后调控，这种效应需要蛋白质合成。
