Beta-globin mRNA decay in erythroid cells

描述（由申请人提供）：β-地中海贫血是由β珠蛋白基因突变引起的常见遗传性疾病。这些突变中的许多引入导致受影响等位基因编码的mRNA降解的提前终止密码子（PTC）。在两个β-珠蛋白等位基因中遗传突变的个体具有严重的贫血、溶血和由骨髓扩张引起的继发性病理。大多数含有PTC的mRNA通过称为无义介导的mRNA衰变（NMD）的核相关监视途径降解。先前的工作表明，在人类β-珠蛋白基因的外显子2中的PTC激活β-珠蛋白mRNA的细胞质降解，产生亚稳态衰变中间体。我们在一个模型系统中使用小鼠红白血病细胞复制了这一点，并确定mRNA衰变是由核酸内切裂解引起的。正常和含PTC的β-珠蛋白mRNA的降解都是由多核糖体相关的核糖核酸酶催化的，该核糖核酸酶的性质类似于在非洲爪蟾中鉴定的多核糖体相关的内切核酸酶，称为xPMR 1。我们认为，内切核酸酶催化降解的PTC-含有β-珠蛋白mRNA在红系细胞是一个专门的形式NMD。目的1将使用转录脉冲追踪和一种新的，灵敏的FRET为基础的分析，以检查mRNA衰变过程的细节，以及它们如何与β-珠蛋白mRNA的结构特征。目的2将通过Upf 1的RNAi、显性负性蛋白的表达、检测该过程与下游内含子剪接的关系以及通过检测其与翻译的先锋轮的关系来检测PTC刺激的β-珠蛋白mRNA降解与NMD之间的关系。目标3将描述β-珠蛋白mRNA的特征，重点是最近发现的一个独特的基因，据信编码这种酶。这将需要重组蛋白的表达，其生化特性的表征，以及研究通过RNA干扰灭活mPMR 1对PTC刺激的β-珠蛋白mRNA降解的影响的实验。长期目标是通过更好地了解负责降解含PTC mRNA的酶机制，确定治疗β-地中海贫血和其他由PTC引起的疾病的新治疗靶点。

DESCRIPTION (provided by applicant): The beta-thalassemias are common genetic disorders that result from mutations in the beta globin gene. Many of these mutations introduce a premature termination codon (PTC) that results in the degradation of mRNA encoded by the affected allele. Individuals inheriting mutations in both beta-globin alleles have severe anemia, hemolysis, and secondary pathology resulting from expansion of the bone marrow. Most PTC containing mRNAs are degraded through a nucleus-associated surveillance pathway termed nonsense mediated mRNA decay (NMD). Previous work demonstrated that a PTC in exon 2 of the human beta-globin gene activated the cytoplasmic degradation of beta-globin mRNA, with the production of metastable decay intermediates. We replicated this in a model system using murine erythroleukemia cells, and determined that mRNA decay results from endonucleolytic cleavage. The degradation of both normal and PTC-containing beta-globin mRNA is catalyzed by a polysome-associated ribonuclease whose properties are similar to a polysome-associated endonuclease identified in Xenopus termed xPMR1. We propose that the endonuclease-catalyzed degradation of PTC-containing beta-globin mRNA in erythroid cells is a specialized form of NMD. Aim 1 will use transcription pulse-chase and a new, sensitive FRET-based assay to examine details of the mRNA decay process and how they relate to structural features of beta-globin mRNA. Aim 2 will examine the relationship between the PTC-stimulated degradation of beta-globin mRNA and NMD by RNAi of Upf1, expression of dominant negative proteins, examining the relationship of this process to downstream intron splicing, and by examining its relationship to the pioneer round of translation. Aim 3 will characterize the beta-globin mRNA, focusing on a recently identified unique gene believed to encode this enzyme. This will entail expression of recombinant protein, characterization of its biochemical properties, and experiments studying the impact of inactivating mPMR1 by RNA interference on the PTC-stimulated degradation of beta-globin mRNA. The long-term goal is to identify new therapeutic targets for treating beta-thalassemia and other diseases caused by PTCs by better understanding the enzymatic mechanisms responsible for the degradation of PTC-containing mRNAs.