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: 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
• 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' 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

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.

项目摘要/摘要 在红细胞中，在β-珠蛋白基因1或2中的外显子1或2中的过早终止密码子（PTC）激活A 降解β-珠蛋白mRNA的细胞质核酸内切酶。 β-珠蛋白表达的导致丧失是 Cooley贫血（β-甲性疾病）的机制，这是一种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MPMR1的内切酶裂解， 爪蟾mRNA核酸内切酶PMR1的哺乳动物直系同源物。衰变的生化差异 含PTC的β-珠蛋白mRNA在红斑中与非甲状腺细胞中的细胞表明一个独特的过程 靶向该mRNA在其本地细胞环境中的衰变。 AIM 1将表征PTC-的细节 刺激红细胞细胞中的核酸内切酶衰减，并确定3'-UTR元素在稳定稳定中的作用 衰减中间体。为研究mRNA衰变的极性而开发的一种敏感的基于FRET的测定将是 用于定量通过核酸内切酶裂解的外显子1的选择性损失。 AIM 2将检查关系 在PTC刺激的β-珠蛋白mRNA的降解与参与mRNA的关键蛋白之间 使用RNAi和显性负蛋白的监视（NMD）干扰检测A的关键步骤 PTC和后核酸酶衰减的随后激活。 AIM 3将表征MPMR1在 含PTC的β-珠蛋白mRNA的降解并表征了该过程的酪氨酸激酶激活。 AIM 4将确定如何转导PTC的信号以激活内核酸酶裂解 使用定向的蛋白质相互作用分析，绑扎和RNAi检查mpmr1和 与mRNA监测有关的关键蛋白质。这项研究的长期目标是开发新的治疗方法 通过了解β-珠蛋白mRNA衰变的新型机制，用于β-甲硫0thalassyria。 细胞。

项目成果

Quantitative analysis of deadenylation-independent mRNA decay by a modified MBRACE assay.

通过改进的 MBRACE 测定对不依赖于去腺苷化的 mRNA 衰减进行定量分析。

• DOI: 10.1007/978-1-62703-971-0_28
• 发表时间: 2014
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Dougherty,JulieA;Mascarenhas,Roshan;Schoenberg,DanielR
• 通讯作者: Schoenberg,DanielR