Nonsense codon activation of endonuclease-mediated mRNA decay

描述(申请人提供)：在红系细胞中，β-珠蛋白基因外显子1或2上的提前终止密码子(PTC)激活了一种细胞质核酸内切酶，从而降解了β-珠蛋白mRNA。由此导致的β-珠蛋白表达的丧失是导致库利氏贫血(β-地中海贫血)的机制，这种贫血通常是一种致命的血红蛋白生成障碍。在培养的红系细胞中，我们发现外显子2上的PTC激活了mPMR1对核酸内切酶的切割，mPMR1是非洲爪哇mRNA内切酶PMR1的哺乳动物同源基因。在红系和非红系细胞中，含有PTC的β-珠蛋白信使核糖核酸衰变的生化差异表明，在其天然细胞环境中，有一个不同的过程针对该信使RNA的衰变。目的1研究PTC刺激的红系细胞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。一种灵敏的基于FRET的分析方法被用来研究mRNA衰变的极性，它将被用来定量核酸内切酶切割外显子1的选择性丢失。目的2利用RNAi技术研究PTC刺激的β-珠蛋白mRNA降解与参与mRNA监测(NMD)的关键蛋白之间的关系，以及干扰PTC检测和随后激活核酸内切酶衰退的关键步骤的显性负性蛋白。目的3研究mPMR1在含PTC的β-珠蛋白mRNA降解中的作用及其酪氨酸激酶的激活过程。AIM 4将使用直接蛋白质相互作用分析、拴系和RNAi来确定识别PTC的信号是如何被转导以激活核酸内切酶的，以检测mPMR1与参与mRNA监测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系细胞中β-珠蛋白mRNA衰退的新机制来开发治疗β-地中海贫血的新方法。与公共卫生相关库利氏贫血是一种常见的遗传性疾病，会导致严重的贫血，尤其是在幼儿中。它通常是由血红蛋白β链突变引起的，这种突变激活了由缺陷基因产生的信使核糖核酸的破坏。这项研究试图了解这些有缺陷的基因产物是如何被破坏的，以及这些信息可能如何被用来开发这种令人衰弱的疾病的新疗法。

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.