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，β -珠蛋白基因外显子1或2的过早终止密码子（PTC）激活胞质内切酶，降解β -珠蛋白mRNA。导致的-珠蛋白表达缺失是库利贫血（-地中海贫血）的潜在机制，这是一种经常致命的血红蛋白生成紊乱。利用培养的红细胞，我们发现外显子2的PTC激活了爪蟾mRNA内切酶PMR1的哺乳动物同源物mPMR1的内切酶切割。红细胞与非红细胞中含有ptc的β -珠蛋白mRNA衰变的生化差异表明，在其原生细胞环境中，有一个不同的过程针对该mRNA的衰变。目的1将描述PTC刺激的红细胞内切酶衰变的细节，并确定3'- utr元件在稳定衰变中间体中的作用。一种用于研究mRNA衰减极性的敏感的基于fret的检测将用于量化内切酶切割导致的外显子1的选择性损失。目的2将研究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检测和随后激活内切酶衰变的关键步骤。目的3将描述mPMR1在含ptc的β -珠蛋白mRNA降解中的作用，并描述这一过程的酪氨酸激酶激活。目的4将确定识别PTC的信号如何被转导为激活内切酶切割，使用定向蛋白质相互作用分析，系带和RNAi来检查mPMR1与mRNA监视中涉及的关键蛋白质之间的相互作用。本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，开发新的治疗-地中海贫血的方法。库利贫血是一种常见的遗传性疾病，可导致贫血，尤其是在幼儿中。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这种使人衰弱的疾病的新疗法。