Beta-globin mRNA decay in erythroid cells

描述(申请人提供)：贝塔-地中海贫血是一种常见的遗传性疾病，由贝塔珠蛋白基因突变引起。这些突变中的许多都引入了提前终止密码子(PTC)，导致受影响的等位基因编码的mRNA降解。携带这两种β-珠蛋白等位基因突变的个体有严重的贫血、溶血和因骨髓扩张而导致的继发性病理。大多数含有PTC的mRNAs是通过一种与核相关的监视途径降解的，这种途径被称为无义介导的mRNA衰退(NMD)。以前的工作表明，人β-珠蛋白基因外显子2上的PTC激活了β-珠蛋白mRNA的细胞质降解，并产生了亚稳态衰变中间产物。我们在使用小鼠红白血病细胞的模型系统中复制了这一结果，并确定了mRNA的衰退是内核溶解切割的结果。正常和含PTC的β-珠蛋白mRNA的降解都是由一种多聚体相关的核糖核酸酶催化的，该酶的性质类似于非洲爪哇中鉴定的一种多聚体相关的内切酶xPMR1。我们认为，内切酶催化的含有PTC的β-珠蛋白mRNA在红系细胞中的降解是NMD的一种特殊形式。目标1将使用转录脉冲追逐和一种新的、灵敏的基于FRET的方法来研究mRNA衰退过程的细节以及它们与β-珠蛋白mRNA的结构特征之间的关系。目的2研究PTC诱导的β-珠蛋白mRNA的降解与UPF1的RNAi、优势负性蛋白的表达之间的关系，以及这一过程与下游内含子剪接的关系，以及它与第一轮翻译的关系。目标3将描述β-珠蛋白信使核糖核酸，重点是最近发现的被认为编码这种酶的独特基因。这将需要表达重组蛋白，鉴定其生化性质，并实验研究通过RNA干扰使mPMR1失活对PTC刺激的β-珠蛋白mRNA降解的影响。长期目标是通过更好地了解导致含有PTC的mRNAs降解的酶机制，确定治疗由PTC引起的β-地中海贫血和其他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.