ESTROGEN ACTION IN XENOPUS LIVER

改变RNA的稳定性是调节其表达的主要机制 编码癌基因、同源盒蛋白、淋巴因子、细胞因子、 细胞骨架蛋白和生长因子受体等等。 转录后过程也控制着许多基因的表达 激素调节基因[例如。生长激素、酪蛋白、载脂蛋白]。这个 荷尔蒙调节的RNA的可用性对于研究 RNA稳定性的基本机制，因为添加或删除了 刺激提供了一个定义明确的分子开关。到目前为止 赋予许多mRNA稳定性或不稳定性的序列具有 都被描述过。然而，没有催化RNA的真核核糖核酸酶 新陈代谢已被确定。本提案的主题是 雌激素(E)对非洲爪哇血清白蛋白基因稳定性的调节 莱维斯肝。E在体内或肝脏外植体培养中的应用 导致编码主要血清蛋白的mRNAs从 细胞质。不稳定的RNA是独特的，因为它们具有非常短的 (17个残基)，离散的聚(A)尾巴。Poly(A)长度不受E. 在之前的授权期内，E诱导的核酸酶[称为XLN for 非洲爪哇肝脏核酸酶)被鉴定为具有预期的性质 核糖核酸降解酶一种催化调节的核糖核酸降解的酶具体目标是 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裂解位点的选择性和结构要求；2)作图 白蛋白RNA在体内被S1切割后的切割部位 保护和引物延伸，并确定其结构背景；3) 利用目标1中开发的抗体和cDNA克隆来研究其调控 用E；4)导入将产生杂交的构建体 珠蛋白-白蛋白基因导入原代培养肝细胞进行功能定位 不稳定的决定因素和检验聚(A)长度在 调节mRNA的不稳定性；5)重建白蛋白RNA降解 在试管中。这项研究项目的长期目标是确定和 鉴定参与调控的mRNA的所有分子成分 不稳定[即核酸酶、RNP蛋白、RNA靶向和裂解位点， 其他相互作用的RNA]，以重建受调控的过程 RNA在体外的降解。

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.