Beta-globin mRNA decay in erythroid cells

红细胞中的 β-珠蛋白 mRNA 衰减

• 批准号: 6752342
• 负责人: DANIEL R. SCHOENBERG
• 金额: $ 14.95万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6752342
• 关键词: RNA interference; chemical stability; endonuclease; erythrocytes; fluorescence resonance energy transfer; gene mutation; globin; messenger RNA; nucleic acid structure; polysomes; recombinant proteins; thalassemia; transcription termination

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.

描述（由申请人提供）：β-地中海贫血是由β球蛋白基因突变引起的常见遗传疾病。这些突变中的许多突变引入了过早的终止密码子（PTC），导致受影响等位基因编码的mRNA降解。 β-珠蛋白等位基因中遗传突变的个体均具有严重的贫血，溶血和继发性病理，这是由于骨髓的扩张而导致的。大多数含有mRNA的PTC通过核相关的监视途径降解，称为废话介导的mRNA衰变（NMD）。先前的工作表明，人β-珠蛋白基因的外显子2中的PTC激活了β-珠蛋白mRNA的细胞质降解，并产生了可稳态的衰减中间体。我们在使用鼠红血症细胞的模型系统中复制了这一点，并确定mRNA衰减是由核解核液裂解引起的。正常和含PTC的β-珠蛋白mRNA的降解是由多聚糖相关的核糖核酸酶催化的，其性质与称为XPMR1的Xenopus中鉴定出的多核体相关的内核酸酶相似。我们提出，含PTC的β-珠蛋白mRNA在红细胞细胞中的核酸酶催化降解是NMD的一种专门形式。 AIM 1将使用转录脉冲追踪和一种新的，基于敏感的FRET测定方法来检查mRNA衰变过程的细节，以及它们与β-珠蛋白mRNA的结构特征如何相关。 AIM 2将检查UPF1的RNAi对PTC刺激的β-珠蛋白mRNA和NMD的降解，主导负蛋白的表达，检查了该过程与下游内含子剪接的关系，并检查了其与先锋循环转换的关系。 AIM 3将表征β-球蛋白mRNA，重点是最近确定的独特基因，该基因被认为可以编码这种酶。这将需要重组蛋白的表达，其生化特性的表征以及通过RNA干扰对PTC刺激的β-珠蛋白mRNA的降解降解来研究失活MPMR1的影响的实验。长期目标是通过更好地理解导致含PTC的mRNA降解的酶机制来确定PTC引起的β-甲性贫血和其他疾病的新治疗靶标。