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

• 批准号: 8004999
• 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
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8004999
• 关键词: Abbreviations; Active Sites; Acute Erythroblastic Leukemia; Alleles; Alternative Splicing; Anemia; Behavior; Biochemical; Biological Assay; Cell Line; Cells; Child; Code; Complex; Cooley' s anemia; Cycloheximide; Dactinomycin; Detection; Development; Disease; Dominant-Negative Mutation; Doxycycline; Enzymes; Erythroid; Erythroid Cells; Exons; Family; Fluorescence Resonance Energy Transfer; Genes; Globin; Goals; Health; 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; base; beta Chain Antigen T Cell Receptor; beta Globin; beta Thalassemia; endonuclease; mRNA Decay; mRNA Surveillance; mouse model; novel; novel strategies; premature

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE Cooley's anemia is a common inherited disease that produces life-theatening anemia, particularly in young children. It is often caused by mutations in the hemoglobin beta- chain that activate the destruction of the mRNA made from the defective gene. This research seeks to understand how these defective gene products are destroyed and how this information might be used to develop new treatments for this debilitating disease.

描述（由申请人提供）：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）激活降解β-珠蛋白mRNA的细胞质核酸内切酶。β-珠蛋白表达的丧失是库利氏贫血（β-地中海贫血）的潜在机制，这是一种经常致命的血红蛋白产生障碍。使用培养的红系细胞，我们表明，PTC在外显子2激活内切酶切割mPMR 1，哺乳动物的直系同源的非洲爪蟾mRNA内切酶PMR 1。在红系细胞与非红系细胞中含PTC的β-珠蛋白mRNA衰变的生物化学差异表明，一个独特的过程靶向该mRNA在其天然细胞环境中的衰变。目的1将描述红系细胞中PTC刺激的核酸内切酶衰变的细节，并确定3 '-UTR元件在稳定衰变中间体中的作用。开发用于研究mRNA衰减极性的灵敏的基于FRET的测定将用于定量核酸内切酶切割导致的外显子1的选择性丢失。目的2将检查PTC刺激的β-珠蛋白mRNA降解与mRNA监视（NMD）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PTC检测中的关键步骤和随后的内切核酸酶衰变激活。目的3将表征mPMR 1在降解含PTC的β-珠蛋白mRNA中的作用，并表征该过程中酪氨酸激酶的活化。目的4将确定如何识别PTC的信号转导激活核酸内切酶切割使用定向蛋白质相互作用分析，拴系和RNAi检查mPMR 1和mRNA监视中涉及的关键蛋白之间的相互作用。这项研究的长期目标是通过了解红系细胞中β-珠蛋白mRNA衰变的新机制来开发β-地中海贫血的新治疗方法。 库利氏贫血症是一种常见的遗传性疾病，可引起危及生命的贫血，特别是在幼儿中。它通常是由血红蛋白β链的突变引起的，这种突变激活了由缺陷基因制成的mRNA的破坏。这项研究旨在了解这些有缺陷的基因产物是如何被破坏的，以及这些信息如何用于开发这种使人衰弱的疾病的新疗法。