Nonsense codon activation of endonuclease-mediated mRNA decay

项目摘要/摘要 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 降解β-珠蛋白信使核糖核酸的胞质内切酶。由此导致的β-珠蛋白表达的丧失是 库利氏贫血(β-地中海贫血)的发病机制，这是一种通常致命的血红蛋白生成障碍。 利用培养的红系细胞，我们发现外显子2的PTC激活了mPMR1对核酸内切酶的切割， 非洲爪猴基因内切酶PMR1的哺乳动物同源基因。细胞腐烂过程中的生化差异 红系细胞和非红系细胞中含有PTC的β-珠蛋白mRNA提示了一个不同的过程 靶标是在其自然细胞环境中这种mRNA的衰变。目标1将描述PTC的细节- 刺激红系细胞核酸内切酶降解并确定3‘-非编码区元件在稳定红系细胞中的作用 腐烂中间体。一种灵敏的基于FRET的分析方法将被开发用于研究mRNA衰退的极性 用于通过内切酶切割来量化外显子1的选择性丢失。目标2将考察两者之间的关系 PTC诱导的β-珠蛋白mRNA降解与相关关键蛋白之间的关系 使用RNAi和显性负蛋白干扰检测的关键步骤的监视(NMD) PTC和随后激活的核酸内切酶衰变。目标3将描述mPMR1在 含有PTC的β-珠蛋白mRNA的降解和这一过程中酪氨酸激酶的激活特征。 AIM 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 利用定向蛋白质相互作用分析、Tethering和RNAi检测mPMR1和mPMR1之间的相互作用 参与mRNA监测的关键蛋白质。这项研究的长期目标是开发新的治疗方法 通过了解红系β-珠蛋白mRNA衰变的新机制来治疗β-地中海贫血 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.