HORMONAL REGULATION OF MESSENGER RNA STABILITY

信使 RNA 稳定性的激素调节

• 批准号: 2734567
• 负责人: DANIEL R. SCHOENBERG
• 金额: $ 30.61万
• 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/2734567
• 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.

类固醇和肽激素，生长因子和神经递质