ESTROGEN AND VIGILIN CONTROL OF MRNA STABILITY

以卵黄原蛋白mRNA稳定性为模型，我们正在研究雌激素对mRNA稳定性的转录后调控。本研究的重点是鉴定和克隆雌激素诱导蛋白vigilin，该蛋白与卵黄原蛋白mRNA 3'-非翻译区结合，在雌激素介导的稳定中起重要作用。这个有趣的，但在很大程度上未被研究的蛋白质在所有真核细胞中都是保守的，包含14个不同的RNA结合域。Vigilin通过与特定mrna、tRNA和延伸因子（EF α）结合形成多组分雌激素调节复合物。具体目的是：(1)验证vigilin通过作为整合蛋白来控制mRNA降解的假设，形成一个多组分复合物，将雌激素和其他特定激素信号的作用与细胞蛋白质合成的总体速率联系起来。为了确定额外的维吉林相互作用伙伴，我们将使用酵母2杂交分析和RNA凝胶转移分析。我们将准备在DT40细胞中靶向破坏vigilin基因，并利用这些细胞，下拉实验，RNA凝胶转移试验和哺乳动物2-杂交研究进行vigilin- mrna - trna - ef α复合物的物理和功能定位。我们将确定vigilin与tRNA和EF α的相互作用是否会改变其对特定mrna的亲和力。为了分析vigilin-mRNA- trna - ef α -相互作用在控制mRNA稳定性中的功能重要性，我们将在vigilin细胞系中进行转染，分析不同vigilin水平和不同mRNA翻译速率对mRNA降解的影响。我们将调节网织细胞裂解蛋白合成系统的翻译率和维林水平，并通过多体mRNA内切酶PMR-1检测mRNA靶标的降解。(2)为了研究雌激素以及细胞生长和蛋白产生的变化如何诱导vigilin，我们将克隆和分析vigilin启动子，分析雌激素和生长因子对vigilin转录的调控。我们将评估游离维林在控制维林水平中的自动调节的潜在作用。(3)为了评估PMR-1 mRNA酶在卵黄原蛋白mRNA降解中的作用，我们将确定卵黄原蛋白mRNA的初始裂解位点，并分析PMR-1在无细胞系统和vigilin细胞系中对警戒蛋白控制的mRNA降解的影响。这些研究应该为雌激素受体作用、蛋白质合成速率和mRNA降解调节之间的新联系提供新的见解。

Using vitellogenin mRNA stabilization as a model, we are investigating the posttranscriptional regulation of mRNA stability by estrogen. This proposal focuses on our identification and cloning of vigilin as the estrogen-inducible protein which binds to a segment of the vitellogenin mRNA 3'- untranslated region important in estrogen-mediated stabilization. This intriguing, but largely unstudied, protein is conserved in all eukaryotic cells, and contains 14 distinct RNA binding domains. Vigilin forms a multi-component estrogen-regulated complex by binding to specific mRNAs, tRNA and elongation factor lalpha(EF lalpha). The Specific Aims are: (1) To test the hypothesis that vigilin controls mRNA degradation by functioning as an integrator protein, forming a multi-component complex which links the effects of estrogen and other specific hormone signals with sensing of the overall rate of cell protein synthesis. To identify additional vigilin interaction partners we will use yeast 2-hybrid analysis, and RNA gel shifts assays. We will prepare targeted disruptions of the vigilin gene in DT40 cells and use these cells, pull-down experiments RNA gel shift assays and mammalian 2-hybrid studies carry out physical and functional mapping of the vigilin-mRNA-tRNA-EF lalpha complex. We will determine whether vigilin s interactions with tRNA and EF lalpha alter its affinity for specific mRNAs. To analyze the functional importance of vigilin-mRNA-tRNA-EF lalpha-interactions in the control of mRNA stability, we will carry out transfections in the vigilin cell line and analyze the effects of different vigilin levels and of different mRNA translation rates on mRNA degradation. We will regulate translation rates and vigilin levels in the reticulocyte lysate protein-synthesizing system and examine the degradation of mRNA targets by the polysomal mRNA endonuclease, PMR-1. (2) To investigate how estrogen and changes in cell growth and protein production induce vigilin, we will clone and analyze the vigilin promoter and analyze the regulation of vigilin transcription by estrogen and by growth factors. We will evaluate the potential role of autoregulation by free vigilin in control of vigilin levels. (3) To evaluate the role of the PMR-1 mRNase in vitellogenin mRNA degradation, we will identify the initial vitellogenin mRNA cleavage site and analyze the effect of PMR-1 on vigilin-controlled mRNA degradation in cell-free systems and in the vigilin cell line. These studies should provide new insights into a novel link between estrogen receptor action, protein synthesis rates, and the regulation of mRNA degradation.