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: 7751927
• 负责人: DANIEL R. SCHOENBERG
• 金额: $ 30.44万
• 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/7751927
• 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; 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; 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; public health relevance

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，在β-珠蛋白基因的外显子1或2中的过早终止密码子（PTC）激活了降解β-蛋白mRNA的细胞质内核酸酶。 β-珠蛋白表达的丧失是Cooley贫血（β-地中海贫血）的机制，这是一种通常是致命的血红蛋白产生疾病。使用培养的红细胞细胞，我们表明外显子2中的PTC激活了Xenopus mRNA核酸内切酶PMR1的哺乳动物直系同源物mpmr1激活内切酶的裂解。在红细胞中与非胸细胞中含PTC的β-珠蛋白mRNA衰减的生化差异表明，一个独特的过程靶向该mRNA在其天然细胞环境中的衰减。 AIM 1将表征红斑细胞中PTC刺激的内切酶衰减的细节，并确定3'-UTR元素在稳定衰减中间体中的作用。开发了一种基于敏感的基于FRET的测定方法来研究mRNA衰变的极性，以量化外核酸酶裂解的选择性损失。 AIM 2将检查使用RNAI和显性负蛋白参与MRNA监视（NMD）的PTC刺激的β-珠蛋白mRNA的降解与关键蛋白之间的关系，从。 AIM 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。 AIM 4将确定如何使用定向的蛋白质相互作用分析，绑定和RNAI识别PTC的信号来激活内切酶裂解，以检查MPMR1与MRNA监测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β-珠蛋白mRNA衰变的新型机制来开发β-杜比杜比杜比杜比中硫酸氢蛋白酶的新疗法。 公共卫生相关性Cooley的贫血是一种常见的遗传疾病，会产生弥补生命的贫血，尤其是在幼儿中。它通常是由血红蛋白β链突变引起的，它激活了由缺陷基因产生的mRNA的破坏。这项研究试图了解这些缺陷的基因产品如何被破坏，以及如何使用此信息来开发这种使人衰弱的疾病的新疗法。