Gayen A K, Peffley D M
Department of Pharmacology and Molecular Biology, University of Health Sciences, Chicago Medical School, Illinois 60064, USA.
Arch Biochem Biophys. 1995 Oct 1;322(2):475-85. doi: 10.1006/abbi.1995.1491.
Transcripts for hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase are heterogeneous in length. This heterogeneity is due to variations in the length of 5'-untranslated leader (UTL) sequences, which are generated by both alternate splicing within the first exon as well as alternate transcription start sites. Because mRNA 5'-UTL sequences have a role in regulating translational efficiency, the level and distribution of HMG-CoA reductase transcripts were measured in both total cellular RNA and polysomes from the Syrian hamster cell line C100. Cells were treated with either lovastatin alone, lovastatin and 25-hydroxycholesterol (25-OH C), or lovastatin, 25-OH C, and mevalonate, three treatment regimens used in an earlier study to demonstrate nonsterol-mediated translational control of HMG-CoA reductase synthesis [D. M. Peffley (1992) Somat. Cell Mol. Genet. 18, 19-32]. When reductase mRNA was measured by 5'-extension analysis under the same conditions, levels of transcripts with 5'-UTL regions ranging from 41 to 81 bases were reduced approximately four- to eightfold. In contrast, transcripts with 5'-UTL regions 93 to 100 bases in length were not reduced, and transcripts with 5'-UTL regions approximately 300-400 bases in length increased twofold. The addition of 25-OH C alone or both 25-OH C and mevalonate to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels fivefold in total cellular RNA as determined by RNase protection assay. No comparable change was observed with control ribosomal protein S17 mRNA. Postmitochondrial supernatants representing both translationally inactive monosomes and translationally active polysomes were prepared by sucrose gradient fractionation from cells incubated with the standard three treatments. Because 5'-UTL sequences of many mRNAs have a role in regulating translational efficiency we isolated RNA from each fraction and measured levels of reductase transcripts by 5'-extension analysis. Under all three conditions, transcripts with 5'-UTL sequences 41-103 bases in length were primarily associated with dense sucrose fractions that contain polysomes. In contrast, reductase transcripts with leader sequences 300 to 400 bases were almost exclusively associated with the less dense sucrose fractions containing monosomes. These results indicate that both the level and polysome distribution of individual reductase transcripts are influenced by the length of 5'-UTL sequences.
仓鼠3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶的转录本长度各异。这种异质性是由于5'-非翻译前导（UTL）序列长度的变化所致，这些变化是由第一个外显子内的可变剪接以及可变转录起始位点产生的。由于mRNA的5'-UTL序列在调节翻译效率方面发挥作用，因此在叙利亚仓鼠细胞系C100的总细胞RNA和多核糖体中测量了HMG-CoA还原酶转录本的水平和分布。细胞分别用单独的洛伐他汀、洛伐他汀和25-羟基胆固醇（25-OH C）或洛伐他汀、25-OH C和甲羟戊酸处理，这三种处理方案在早期研究中用于证明HMG-CoA还原酶合成的非甾醇介导的翻译控制[D.M.佩夫利（1992年）《体细胞与分子遗传学》18，19 - 32]。当在相同条件下通过5'-延伸分析测量还原酶mRNA时，5'-UTL区域长度在41至81个碱基之间的转录本水平降低了约四至八倍。相比之下，5'-UTL区域长度为93至100个碱基的转录本没有减少，而5'-UTL区域长度约为300 - 400个碱基的转录本增加了两倍。通过核糖核酸酶保护试验测定，单独添加25-OH C或同时添加25-OH C和甲羟戊酸至洛伐他汀处理的细胞中，总细胞RNA中的HMG-CoA还原酶mRNA水平降低了五倍。对照核糖体蛋白S17 mRNA未观察到类似变化。通过蔗糖梯度分级分离法，从用标准三种处理孵育的细胞中制备了代表翻译无活性单体和翻译活性多核糖体的线粒体后上清液。由于许多mRNA的5'-UTL序列在调节翻译效率方面发挥作用，我们从每个级分中分离RNA，并通过5'-延伸分析测量还原酶转录本的水平。在所有三种条件下，5'-UTL序列长度为41 - 103个碱基的转录本主要与含有多核糖体的高密度蔗糖级分相关。相比之下，前导序列长度为300至400个碱基的还原酶转录本几乎完全与含有单体的低密度蔗糖级分相关。这些结果表明，单个还原酶转录本的水平和多核糖体分布均受5'-UTL序列长度的影响。
