HORMONAL REGULATION OF MESSENGER RNA STABILITY

信使 RNA 稳定性的激素调节

• 批准号: 2396907
• 负责人: DANIEL R. SCHOENBERG
• 金额: $ 24.71万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2396907
• 关键词: Xenopus; Xenopus oocyte; chemical stability; electrospray ionization mass spectrometry; enzyme induction /repression; enzyme mechanism; enzyme structure; estrogens; hormone regulation /control mechanism; liver cells; liver metabolism; messenger RNA; molecular cloning; nucleic acid sequence; pancreatic ribonuclease; posttranscriptional RNA processing; posttranslational modifications; protein purification; transfection; western blottings; yeast two hybrid system

Steroid and peptide hormones, growth factors and neurotransmitters can effect changes in the stability of specific mRNAs in target cells. A major focus of research in this laboratory is on the mechanism by which estrogen (E) elicits such changes. The model system under study is the liver of the South African frog Xenopus laevis. The translational profile of this tissue is reprogrammed by E from one in which serum protein synthesis predominates to one in which translation is directed toward production of the yolk protein precursor vitellogenin (VTG). This is accomplished by the induction and stabilization of VTG mRNA, and the destabilization of mRNAs for the serum proteins. In the past funding period we purified an cloned a unique ribonuclease identified on liver polysomes of E-treated frogs, and demonstrated that this enzyme is involved in the in vivo degradation of albumin mRNA. Since this is the first polysomal messenger ribonuclease so identified we have named this enzyme PMR-1. PMR defines a new class of RNase as it has no homology to known vertebrate members of the RNASE superfamily. Biochemical and cDNA sequence data suggest PMR is phosphorylated in the N-terminal Half, and expression experiments locate catalytic activity to the C-terminal half. Aim 1 will examine the structure and post-translational modifications found on PMR extracted from liver polysomes using electrospray mass spectrometry. In addition, this Aim will develop baculoviurs vectors to express recombinant protein, use expression in E. Coli to map residues involved in catalysis, and clone its human homolog. Aim 2 will use Xenopus oocyte injection to examine the relationship between PMR and the destabilization of albumin mRNA and determine their relationship to PMR cleavage sites. Aim 3 will use expression screening of a phage library, the yeast 2-hybrid system, and conventional chromatography coupled to surface plasmon resonance (BIAcore) to identify and clone PMR-binding protein(s) (PMR-BP). These will be evaluated by co-immunoprecipitation with PMR and for their ability to influence the degradation of albumin mRNA in the oocyte injection system developed in Aim 2. E has no effect on the amount of PMR within the cell, suggesting that increased PMR activity on polysomes after E is caused by post- translational activation of pre-existing enzyme. The experiments in Aim 4 will use both primary hepatocyte cultures and transfected HepG2 cells to examine the hypothesis that PMR is activated by $E-induced changes in its phosphorylation. The presence of multiple phosphorylation sites on PMR raises the possibility that this enzyme may integrate signaling by various intracellular pathways to effect selective mRNA destabilization in response to different extracellular stimuli.

类固醇和肽类激素、生长因子和神经递质 可以影响靶细胞中特定mRNA的稳定性的变化。 该实验室的一个主要研究重点是 哪种雌激素（E）会引起这种变化。 下的模型系统 研究对象是南非非洲爪蟾的肝脏。 的 该组织的翻译谱由E从一个重新编程 其中血清蛋白质合成占主导地位， 翻译是针对蛋黄蛋白的生产 前体卵黄蛋白原（VTG）。 这是由 VTG mRNA的诱导和稳定，以及 血清蛋白的mRNA。 在过去的一段时间里，我们 纯化并克隆了在肝脏上鉴定的独特核糖核酸酶 多核糖体的E处理青蛙，并证明这种酶是 参与白蛋白mRNA的体内降解。 由于这 是第一个多核糖体信使核糖核酸酶， 命名为PMR-1。 PMR定义了一类新的RNase， 它与已知的脊椎动物RNASE成员没有同源性 超家族 生化和cDNA序列数据表明PMR 在N-末端的一半被磷酸化， 实验将催化活性定位于C-末端的一半。 要求1 将检查发现的结构和翻译后修饰 使用电喷雾质量从肝多聚体中提取的PMR 光谱法 此外，该Aim还将开发杆状病毒 表达重组蛋白的载体，在E.杆菌以 绘制参与催化作用的残基，并克隆其人类同源物。 目的2将用非洲爪蟾卵母细胞注射液检测 PMR和白蛋白mRNA的不稳定性之间的关系， 确定它们与PMR切割位点的关系。 目标3将使用 噬菌体文库的表达筛选，酵母双杂交系统， 和与表面等离子体激元偶联的常规色谱法 共振（BIAcore）以鉴定和克隆PMR结合蛋白 （PMR-BP）。 这些将通过免疫共沉淀进行评价 与PMR和他们的能力，以影响降解的 Aim 2中开发的卵母细胞注射系统中的白蛋白mRNA。 E对细胞内PMR的量没有影响，表明 E后多核糖体上PMR活性增加是由后 预先存在的酶的翻译激活。 中的实验 目的4将使用原代肝细胞培养物和转染的 HepG2细胞，以检验PMR被激活的假设。 $E诱导其磷酸化的变化。 的存在 PMR上的多个磷酸化位点提出了这样的可能性， 这种酶可以整合各种细胞内信号传导， 影响选择性mRNA不稳定的途径 不同的细胞外刺激