ESTROGEN ACTION IN XENOPUS LIVER

改变RNA的稳定性是调节转录因子表达的主要机制。 编码癌基因、同源框蛋白、淋巴因子、细胞因子的基因， 细胞骨架蛋白和生长因子受体等。 转录后过程也控制着许多转录因子的表达。 基因调控（Genes Regulated）生长激素、酪蛋白、载脂蛋白]。 的 可利用的一个受调节的RNA是特别有用的研究， RNA稳定性的基本机制，因为添加或去除 刺激提供了明确的分子开关。 迄今 赋予许多mRNA稳定性或不稳定性的序列具有 被描述。 然而，没有真核核糖核酸酶催化RNA 代谢已被确认。 本提案的主题是 雌激素对爪蟾血清白蛋白mRNA稳定性的调节 左肝 E在体内或肝脏外植体培养物中的给药 导致编码主要血清蛋白的mRNA从细胞中消失， 细胞质 不稳定的RNA是独特的，因为它们具有非常短的 (17残基）、离散的聚（A）尾。 Poly（A）长度不受E. 在前一个授予期间，E诱导的核酸酶[称为xln， 非洲爪蟾肝核酸酶）被鉴定为具有预期的性质， 一种催化调节RNA降解的酶。 具体目标为 本研究的主要内容是：1）分离克隆xln，制备抗体， 表达载体，并使用这些工具来研究RNA的性质 RNA切割位点的选择性和结构要求; 2）映射 在体内引入的白蛋白RNA切割位点在E之后通过Sl 保护和引物延伸，并确定其结构背景; 3） 利用Aim 1中开发的抗体和cDNA克隆研究其调控机制， 通过E的核酸酶; 4）转染将产生杂交体的构建体 将球蛋白-白蛋白mRNA导入原代肝细胞培养物中以绘制功能图 不稳定性的决定因素，并检查多聚（A）长度的作用， 调节mRNA的不稳定性;和5）重建白蛋白RNA降解 体外 本研究项目的长期目标是确定和 表征参与调节mRNA的所有分子组分 不稳定性[ie.核酸酶，RNP蛋白，RNA靶向和切割位点， 其他相互作用的RNA]以便重建受调节的过程 体外RNA降解。

Altering RNA stability is the major mechanism regulating the expression of genes encoding oncogenes, homeobox proteins, lymphokines, cytokines, cytoskeletal proteins, and growth factor receptors to name a few. Posttranscriptional processes also control the expression of a number of hormone-regulated genes [eg. growth hormone, casein, apolipoproteins]. The availability of a hormone-regulated RNA is particularly useful for study of fundamental mechanisms of RNA stability, since the addition or removal of the stimulus provides a well-defined molecular switch. To date the sequences that impart stability or instability to a number of mRNAs have been described. However no eucaryotic ribonuclease that catalyzes RNA metabolism has been identified. The subject of the present proposal is the regulation of serum albumin mRNA stability by estrogen (E) in Xenopus laevis liver. Administration of E in vivo or to liver explant cultures causes the mRNAs encoding the major serum proteins to disappear from the cytoplasm. The destabilized RNAs are unique in that they have very short (17 residue), discrete poly(A) tails. Poly(A) length is unaffected by E. During the preceding grant period an E-induced nuclease [termed xln for Xenopus liver nuclease) was identified that has the expected properties for an enzyme that catalyzes regulated RNA degradation. The Specific Aims for this proposal are: 1) To isolate and clone xln, prepare antibodies and expression vectors, and use these tools to study the nature of RNA selectivity and structural requirements of RNA cleavage sites; 2) To map the sites of albumin RNA cleavage introduced in vivo following E by Sl protection and primer extension, and determine their structural context; 3) To use antibody and cDNA clones developed in Aim 1 to study the regulation of the nuclease by E; 4) To transfect constructs that will produce hybrid globin-albumin mRNA into primary hepatocyte cultures to map functional instability determinants and examine the role of poly(A) length in regulated mRNA instability; and 5) To reconstitute albumin RNA degradation in vitro. The long term goals of this research project are to identify and characterize all of the molecular components involved in regulated mRNA instability [ie. nuclease, RNP proteins, RNA targeting and cleavage sites, other interacting RNAs] in order to reconstitute the process of regulated RNA degradation in vitro.