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。许多这些突变引入了一个过早终止密码子（PTC），导致受影响等位基因编码的mRNA降解。遗传两个-珠蛋白等位基因突变的个体有严重的贫血、溶血和由骨髓扩张引起的继发性病理。大多数含有mRNA的PTC通过称为无义介导的mRNA衰变（NMD）的核相关监视途径降解。先前的研究表明，人类β -珠蛋白基因外显子2中的PTC激活了β -珠蛋白mRNA的胞质降解，并产生亚稳态衰变中间体。我们在使用小鼠红白血病细胞的模型系统中复制了这一过程，并确定mRNA衰变是由核内溶分裂引起的。正常和含ptc的β -珠蛋白mRNA的降解都是由一种多聚体相关核糖核酸酶催化的，其性质与在爪蟾中发现的一种多聚体相关内切酶xPMR1相似。我们认为，内切酶催化的红细胞中ptc -球蛋白mRNA的降解是NMD的一种特殊形式。目的1将使用转录脉冲追踪和一种新的、灵敏的基于fret的检测来检查mRNA衰变过程的细节，以及它们与β -珠蛋白mRNA结构特征的关系。目的2将研究ptc刺激的β -珠蛋白mRNA降解和NMD之间的关系，通过Upf1的RNAi，显性负蛋白的表达，研究这一过程与下游内含子剪接的关系，并通过研究其与翻译的先驱轮的关系。目的3将描述-珠蛋白mRNA，重点关注最近发现的被认为编码这种酶的独特基因。这将需要重组蛋白的表达，其生化特性的表征，以及通过RNA干扰灭活mPMR1对ptc刺激的β -珠蛋白mRNA降解的影响的实验研究。长期目标是通过更好地了解ptc - mrna降解的酶机制，确定治疗β -地中海贫血和其他由ptc引起的疾病的新的治疗靶点。

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.