ESTROGEN REGULATED MRNA BINDING PROTEIN IN RNA STABILITY

我们已经鉴定出一种雌激素诱导蛋白，它能与高密度脂蛋白结合。 对雌激素3‘-非翻译区的一段专一性- 稳定的编码卵黄前体蛋白卵黄蛋白原的mRNA。 由于该蛋白广泛分布于非洲爪哇的不同组织中 性激素同时受雌激素和睾丸素的调节，我们有 鉴定出一种人类同源物，它可能在类固醇中起着重要作用 激素对mRNA降解的控制。我们的方法是克隆、表达 并对蛋白质进行表征，以研究雌激素对其调节作用 和其他激素，决定了基因的精确序列和结构 信使核糖核酸结合位点，确定额外的与蛋白质结合的信使核糖核酸 研究其在调节mRNA稳定性中的作用。 蛋白质与其识别结合的高度特异性 序列，以及我们建立的硝化纤维滤膜结合试验 直接筛选cdna表达文库是首选方法 用于克隆和初始表达。如果这被证明是不可能的，我们会 使用包括RNA亲和层析在内的生化技术来纯化 蛋白质，获得部分氨基酸序列，并使用简并 寡核苷酸筛选cDNA文库。非洲爪哇的cDNA克隆将是 用于分离人结合蛋白的cDNA克隆。 将使用核酸探针和针对表达蛋白的抗体 为了确定雌激素是否诱导了mRNA和蛋白质，还是增加了 预先存在的蛋白质对其信使核糖核酸结合部位的亲和力。我们会 然后对监管机制进行了较为详细的分析。 为了进一步表征mRNA结合位点，我们将使用迭代中的 体外筛选方法、核糖核酸酶保护和诱变。试管苗 选择提供了一种鉴定共识信使核糖核酸的不寻常的方法 结合部位，并产生结合部位的上结合突变体，以及 将有助于对结合部位的二级结构进行建模。我们 将使用共识结合位点的序列和结构来 识别其他稳定性受调控的含有潜在的mRNAs 蛋白质的结合部位。 我们将确定雌激素诱导、结合 卵黄蛋白原基因的蛋白质识别序列及其调控 稳定性。我们还将研究结合蛋白在 控制一个人的mRNA的稳定性，我们在其中识别一个结合 地点。我们将确定初始RNase切割位点是否为 位于蛋白质结合部位附近。 这些研究应该为类固醇激素提供重要的见解。 控制信使核糖核酸稳定性。

We have identified an estrogen-inducible protein which binds with high specificity to a segment of the 3'-untranslated region of the estrogen- stabilized mRNA encoding the egg yolk precursor protein, vitellogenin. Since this protein is widely distributed in different tissues of Xenopus laevis, is regulated by both estrogen and testosterone, and we have identified a human homologue, it likely plays an important role in steroid hormone control of mRNA degradation. Our approach is to clone, express and characterize the protein, to investigate its regulation by estrogen and other hormones, determine the precise sequence and structure of the mRNA binding site, identify additional mRNAs which bind the protein and investigate its role in the regulation of mRNA stability. The high specificity with which the protein binds to its recognition sequence, and our development of a nitrocellulose filter binding assay make direct screening of a cDNA expression library our preferred method for cloning and initial expression. If this proves impossible, we will use biochemical techniques including RNA affinity chromatography to purify the protein, obtain partial amino acid sequence, and use degenerate oligonucleotides to screen a cDNA library. The Xenopus cDNA clone will be used to isolate a human binding protein cDNA clone. Nucleic acid probes and antibodies to the expressed protein will be used to determine whether estrogen induces the mRNA and protein, or increases the affinity of pre-existing protein for its mRNA binding site. We will then carry out a more detailed analysis of the regulatory mechanism. To further characterize the mRNA binding site we will use an iterative in vitro selection method, RNase protection and mutagenesis. The in vitro selection provides an unusual approach to identifying a consensus mRNA binding site, and generating up-binding mutants of the binding site, and will facilitate modeling the secondary structure of the binding site. We will use the sequence and structure of the consensus binding site to identify other mRNAs whose stability is regulated which contain potential binding sites for the protein. We will determine the relationship between estrogen induction, binding of the protein to its recognition sequence and control of vitellogenin mRNA stability. We will also examine the role of the binding protein in controlling the stability of one human mRNA in which we identify a binding site. We will determine whether the initial RNase cleavage site is located near the protein binding site. These studies should provide important insights into steroid hormone control of mRNA stability.