HORMONAL REGULATION OF MESSENGER RNA STABILITY

类固醇和多肽激素、生长因子和神经递质 可以影响靶细胞中特定mRNAs稳定性的变化。 这个实验室的一个主要研究重点是通过 哪种雌激素(E)会引起这种变化。模式下的系统 研究是南非非洲爪蛙的肝脏。这个 这个组织的翻译图谱被E从一重新编程 其中血清蛋白质合成占主导地位，而不是 翻译是针对蛋黄蛋白的生产。 前体卵黄蛋白原(VTG)。这是由 VTG基因的诱导和稳定及其失稳 血清蛋白的mRNAs。在过去的资助期，我们 纯化和克隆了一种在肝脏中鉴定的独特的核糖核酸酶 E处理的青蛙的多聚体，并证明该酶是 参与白蛋白mRNA在体内的降解。既然是这样 我们鉴定出的第一个多体信使核糖核酸酶 将这种酶命名为PMR-1。PMR将新的RNase类定义为 它与已知的脊椎动物核糖核酸酶成员没有同源性 超级大家庭。生化和cDNA序列数据显示PMR 在N-末端的一半被磷酸化，并表达 实验确定了C-末端的催化活性。目标1 将检查发现的结构和翻译后修改 电喷雾质谱法提取肝多聚体中的PMR 光谱分析。此外，这一目标将发展杆状病毒 载体表达重组蛋白，在大肠杆菌中表达 绘制与催化有关的残基，并克隆其人类同源物。 目标2将使用非洲爪哇卵母细胞注射来检查两者之间的关系 PMR与白蛋白mRNA失稳之间的关系 确定它们与PMR裂解位点的关系。AIM 3将使用 噬菌体文库、酵母双杂交系统的表达筛选 与表面等离子激元偶联的常规层析 共振(Biacore)鉴定和克隆PMR结合蛋白(S) (PMR-BP)。这些将通过免疫共沉淀法进行评估。 与PMR和它们影响降解的能力 在目标2中开发了卵母细胞注射系统中的白蛋白信使核糖核酸。 E对细胞内PMR的量没有影响，提示 E后多聚体上PMR活性增加是由后 预先存在的酶的翻译激活。在中国的实验 Aim 4将使用原代肝细胞培养和转染法 以检验PMR被激活的假设 $E可引起其磷酸化的改变。.的存在 PMR上的多个磷酸化位点增加了一种可能性 这种酶可以整合不同细胞内的信号 影响选择性信使核糖核酸失稳的途径 不同的细胞外刺激。

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.