Nonsense codon activation of endonuclease-mediated mRNA decay

项目概要/摘要 在红系细胞中，β-珠蛋白基因外显子1或2中的提前终止密码子（PTC）激活了一个转录因子。 降解β-珠蛋白mRNA的细胞质核酸内切酶。导致的β-珠蛋白表达的丧失是 库利氏贫血（β-地中海贫血）是一种致命的血红蛋白生成障碍。 使用培养的红系细胞，我们发现外显子2中的PTC激活mPMR 1的核酸内切酶切割， 非洲爪蟾mRNA核酸内切酶PMR 1的哺乳动物直系同源物。生物化学的差异， 在红系细胞和非红系细胞中含有PTC β-珠蛋白mRNA表明， 靶向这种mRNA在其天然细胞环境中的衰变。目标1将描述PTC的细节- 刺激的核酸内切酶在红系细胞中的衰变，并确定3 '-UTR元件在稳定红系细胞中的作用。 衰变中间体一个敏感的FRET为基础的分析开发研究极性的mRNA衰减将是 用于定量核酸内切酶切割导致的外显子1的选择性丢失。目标2将研究 PTC刺激的β-珠蛋白mRNA降解和mRNA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检测中的关键步骤， PTC和随后的核酸内切酶衰变的激活。目的3将描述mPMR 1在以下过程中的作用： 含PTC的β-珠蛋白mRNA的降解，并表征该过程的酪氨酸激酶活化。 目的4将确定识别PTC的信号如何被转导以激活核酸内切酶切割 使用定向蛋白质相互作用分析、系链和RNAi来检查mPMR 1和 参与mRNA监视的关键蛋白质。这项研究的长期目标是开发新的治疗方法 通过了解红细胞中β-珠蛋白mRNA衰变的新机制， 细胞

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.