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）：在红细胞中，β-珠蛋白基因的外显子 1 或 2 中的提前终止密码子 (PTC) 会激活降解 β-珠蛋白 mRNA 的细胞质核酸内切酶。由此产生的β-珠蛋白表达丧失是库利氏贫血（β-地中海贫血）的潜在机制，库利氏贫血是一种通常致命的血红蛋白产生疾病。使用培养的红系细胞，我们发现外显子 2 中的 PTC 可激活 mPMR1 的核酸内切酶裂解，mPMR1 是非洲爪蟾 mRNA 核酸内切酶 PMR1 的哺乳动物直系同源物。红系细胞与非红系细胞中含 PTC 的 β-珠蛋白 mRNA 衰变的生化差异表明，有一个独特的过程针对该 mRNA 在其天然细胞环境中的衰变。目标 1 将表征红细胞中 PTC 刺激的核酸内切酶衰变的细节，并确定 3'-UTR 元素在稳定衰变中间体中的作用。为研究 mRNA 衰变的极性而开发的基于 FRET 的灵敏测定将用于量化核酸内切酶切割导致的外显子 1 的选择性丢失。目标 2 将检查 PTC 刺激的 β-珠蛋白 mRNA 降解与参与 mRNA 监视 (NMD) 的关键蛋白之间的关系，使用 RNAi 和显性失活蛋白干扰 PTC 检测和随后核酸内切酶衰变激活的关键步骤。目标 3 将表征 mPMR1 在含 PTC 的 β-珠蛋白 mRNA 降解中的作用，并表征该过程的酪氨酸激酶激活。目标 4 将确定如何转导识别 PTC 的信号以激活核酸内切酶裂解，使用定向蛋白质相互作用分析、束缚和 RNAi 来检查 mPMR1 和参与 mRNA 监视的关键蛋白质之间的相互作用。这项研究的长期目标是通过了解红细胞中 β 珠蛋白 mRNA 衰变的新机制来开发治疗 β 地中海贫血的新疗法。 公众健康相关性 库利氏贫血是一种常见的遗传性疾病，会导致危及生命的贫血，特别是在幼儿中。它通常是由血红蛋白β链突变引起的，该突变激活了由缺陷基因产生的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，以及如何利用这些信息来开发针对这种使人衰弱的疾病的新疗法。