ESTROGEN AND VIGILIN CONTROL OF MRNA STABILITY

以卵黄蛋白原mRNA稳定性为模型，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Viglin，它是一种雌激素诱导蛋白，与卵黄蛋白原基因3‘端非翻译区结合，该区域在雌激素介导的稳定中起重要作用。这种耐人寻味但大部分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。Viglin通过与特定的mRNAs、tRNA和延伸因子lpha(EF Lpha)结合，形成一个多组分的雌激素调节复合体。其具体目的是：(1)验证Viglin通过作为整合蛋白发挥作用来控制mRNA降解的假设，形成一个多组分的复合体，将雌激素和其他特定激素信号的影响与细胞蛋白质合成的总体速度的感觉联系起来。为了确定更多的Viglin相互作用伙伴，我们将使用酵母双杂交分析和RNA凝胶位移分析。我们将在DT40细胞中制备Viglin基因的靶向干扰，并使用这些细胞、下拉实验、RNA凝胶位移分析和哺乳动物2-杂交研究对Viglin-mRNA-tRNA-EF lpha复合体进行物理和功能定位。我们将确定Viglin S与tRNA和EFα的相互作用是否会改变其对特定mRNA的亲和力。为了分析Viglin-mRNA-tRNA-EFα相互作用在控制mRNA稳定性中的功能重要性，我们在Viglin细胞系中进行了转基因，并分析了不同水平的Viglin和不同的mRNA转译率对mRNA降解的影响。我们将调节网织红细胞裂解蛋白合成系统中的翻译率和Viglin水平，并检测多聚体mRNA内切酶PMR-1对mRNA靶标的降解。(2)克隆和分析Viglin启动子，分析雌激素和生长因子对Viglin转录的调控，以探讨雌激素以及细胞生长和蛋白质合成的变化是如何诱导Viglin的。我们将评估由游离Viglin进行的自我调节在控制Viglin水平方面的潜在作用。(3)为了探讨PMR-1mRNase在卵黄蛋白原mRNA降解中的作用，我们确定了卵黄蛋白原的起始裂解位点，并分析了PMR-1在无细胞体系和Viglin细胞系中对Viglin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.