Ing Nancy H, Massuto Dana A, Jaeger Laurie A
Department of Animal Science, Texas A & M University, College Station, Texas 77843; Veterinary Integrative Biosciences, Texas A & M University, College Station, Texas 77843.
Veterinary Integrative Biosciences, Texas A & M University, College Station, Texas 77843.
J Biol Chem. 2008 Jan 18;283(3):1764-1772. doi: 10.1074/jbc.M704745200. Epub 2007 Nov 19.
Estradiol up-regulates expression of the estrogen receptor alpha gene in the uterus by stabilizing estrogen receptor alpha mRNA. Previously, we defined two discrete minimal estradiol-modulated stability sequences (MEMSS) within the extensive 3'-untranslated region of estrogen receptor alpha mRNA with an in vitro stability assay using cytosolic extracts from sheep uterus. We report here that excess MEMSS RNA inhibited the enhanced stability of estrogen receptor alpha mRNA in extracts from estradiol-treated ewes compared with those from control ewes. Several estradiol-induced MEMSS-binding proteins were characterized by UV cross-linking in uterine extracts from ewes in a time course study (0, 8, 16, and 24 h after estradiol injection). The pattern of binding proteins changed at 16 h post-injection, concurrent with enhanced estrogen receptor alpha mRNA stability and the highest rate of accumulation of estrogen receptor alpha mRNA. The predominant MEMSS-binding protein induced by estradiol treatment was identified as AUF1 (A + U-rich RNA-binding factor 1) protein isoform p45 (a product of the heterogeneous nuclear ribonucleoprotein D gene). Immunoblot analysis indicated that only two of four AUF1 protein isoforms were present in the uterine cytosolic extracts and that estradiol treatment strongly increased the ratio of AUF1 isoforms p45 to p37. Nonphosphorylated recombinant AUF1p45 protected estrogen receptor alpha mRNA in vitro in a dose-dependent manner. These studies describe estrogenic induction of AUF1p45 binding to the estrogen receptor alpha mRNA as a molecular mechanism for post-transcriptional up-regulation of gene expression.
雌二醇通过稳定雌激素受体α mRNA上调子宫中雌激素受体α基因的表达。此前，我们利用绵羊子宫的胞质提取物进行体外稳定性分析，在雌激素受体α mRNA广泛的3'-非翻译区内定义了两个离散的最小雌二醇调节稳定性序列（MEMSS）。我们在此报告，与对照母羊的提取物相比，过量的MEMSS RNA抑制了来自经雌二醇处理的母羊提取物中雌激素受体α mRNA的增强稳定性。在一项时间进程研究（注射雌二醇后0、8、16和24小时）中，通过紫外线交联对来自母羊子宫提取物中的几种雌二醇诱导的MEMSS结合蛋白进行了表征。注射后16小时，结合蛋白的模式发生变化，同时雌激素受体α mRNA稳定性增强，雌激素受体α mRNA积累速率最高。经雌二醇处理诱导的主要MEMSS结合蛋白被鉴定为富含AU元件的RNA结合因子1（AUF1）蛋白异构体p45（异质性核糖核蛋白D基因的产物）。免疫印迹分析表明，子宫胞质提取物中仅存在四种AUF1蛋白异构体中的两种，并且雌二醇处理强烈增加了AUF1异构体p45与p37的比例。非磷酸化的重组AUF1p45在体外以剂量依赖的方式保护雌激素受体α mRNA。这些研究描述了雌激素诱导AUF1p45与雌激素受体α mRNA结合是基因表达转录后上调的分子机制。
