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Nonsense codon activation of endonuclease-mediated mRNA decay

核酸内切酶介导的 mRNA 衰变的无义密码子激活

基本信息

批准号：
8208188
负责人：
DANIEL R. SCHOENBERG
金额：
$ 30.14万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-01-01 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8208188
关键词：
Abbreviations Active Sites Acute Erythroblastic Leukemia Alleles Alternative Splicing Anemia Behavior Biochemical Biological Assay Cell Line Cells Child Code Complex Cooley&apos s anemia Cycloheximide Dactinomycin Detection Development Disease Dominant-Negative Mutation Doxycycline Enzymes Erythroid Erythroid Cells Exons Family Fluorescence Resonance Energy Transfer Genes Globin Goals Hemoglobin Histidine Human Indium Inherited Knowledge Life Mediating Messenger RNA Modeling Molecular Mus Mutate Mutation Names Nonsense Codon Orthologous Gene Patients Process Production Protein Tyrosine Kinase Proteins RNA Interference RNA Splicing Research Ribonucleases Role Sampling Signal Transduction Site Terminator Codon Tetanus Helper Peptide Tetracyclines Thalassemia Transgenic Mice Triose-Phosphate Isomerase Untranslated Regions Work Xenopus Xenopus Proteins abstracting base beta Chain Antigen T Cell Receptor beta Globin beta Thalassemia endonuclease mRNA Decay mRNA Surveillance mouse model novel novel strategies premature

项目摘要

Project summary/abstract In erythroid cells a premature termination codon (PTC) in exons 1 or 2 of the beta-globin gene activates a cytoplasmic endonuclease that degrades beta-globin mRNA. The resulting loss of beta-globin expression is the mechanism underlying Cooley's anemia (beta-thalassemia), an often fatal disorder of hemoglobin production. Using cultured erythroid cells we showed that a PTC in exon 2 activates endonuclease cleavage by mPMR1, the mammalian ortholog of the Xenopus mRNA endonuclease PMR1. Biochemical differences in the decay of the PTC-containing beta-globin mRNA in erythroid versus non-erythorid cells indicate that a distinct process targets the decay of this mRNA in its native cell context. Aim 1 will characterize the details of the PTC- stimulated endonuclease decay in erythroid cells and determine the role of 3'-UTR elements in stabililzing the decay intermediates. A sensitive FRET-based assay developed to study the polarity of mRNA decay will be used to quantify the selective loss of exon 1 by endonuclease cleavage. Aim 2 will examine the relationship between the PTC-stimulated degradation of beta-globin mRNA and key proteins involved in mRNA surveillance (NMD) using RNAi and dominant negative proteins to interfere with key steps in the detection of a PTC and subsequent activation of endonuclease decay. Aim 3 will characterize the role of mPMR1 in the degradation of PTC-containing beta-globin mRNA and characterize tyrosine kinase activation of this process. Aim 4 will determine how the signal for recognition of a PTC is transduced to activate endonuclease cleavage using directed protein interaction analysis, tethering and RNAi to examine interactions between mPMR1 and key proteins involved in mRNA surveillance. The long-term goal of this research is to develop new treatments for beta-thalassemia by understanding the novel mechanisms involved in beta-globin mRNA decay in erythroid cells.

项目概要/摘要在红系细胞中，β-珠蛋白基因外显子1或2中的提前终止密码子（PTC）激活了一个转录因子。降解β-珠蛋白mRNA的细胞质核酸内切酶。导致的β-珠蛋白表达的丧失是库利氏贫血（β-地中海贫血）是一种致命的血红蛋白生成障碍。使用培养的红系细胞，我们发现外显子2中的PTC激活mPMR 1的核酸内切酶切割，非洲爪蟾mRNA核酸内切酶PMR 1的哺乳动物直系同源物。生物化学的差异，在红系细胞和非红系细胞中含有PTC β-珠蛋白mRNA表明，靶向这种mRNA在其天然细胞环境中的衰变。目标1将描述PTC的细节- 刺激的核酸内切酶在红系细胞中的衰变，并确定3 '-UTR元件在稳定红系细胞中的作用。衰变中间体一个敏感的FRET为基础的分析开发研究极性的mRNA衰减将是用于定量核酸内切酶切割导致的外显子1的选择性丢失。目标2将研究 PTC刺激的β-珠蛋白mRNA降解和mRNA中涉及的关键蛋白之间的关系使用RNAi和显性阴性蛋白干扰检测中的关键步骤， PTC和随后的核酸内切酶衰变的激活。目的3将描述mPMR 1在以下过程中的作用：含PTC的β-珠蛋白mRNA的降解，并表征该过程的酪氨酸激酶活化。目的4将确定识别PTC的信号如何被转导以激活核酸内切酶切割使用定向蛋白质相互作用分析、系链和RNAi来检查mPMR 1和参与mRNA监视的关键蛋白质。这项研究的长期目标是开发新的治疗方法通过了解红细胞中β-珠蛋白mRNA衰变的新机制，细胞