Ribosome Biogenesis in Diamond Blackfan Anemia

钻石黑扇贫血症中的核糖体生物发生

• 批准号: 7129299
• 负责人: Monica Bessler
• 金额: $ 21.56万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-03 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7129299
• 关键词: biotechnology; cell differentiation; cell line; clinical research; congenital aplastic anemia; density gradient ultracentrifugation; erythrocytes; erythroid stem cell; family genetics; gene mutation; genetic disorder; genetic screening; genetic susceptibility; human genetic material tag; human subject; mass spectrometry; northern blottings; polysomes; proteomics; ribosomal RNA; ribosomal proteins; ribosomes; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): Our laboratory has a longstanding interest in the molecular pathogenesis of bone marrow failure, a condition characterized by the inability of the bone marrow to produce sufficient blood cells. Bone marrow failure may affect all three major blood cell lineages (erythrocytes, leukocytes and platelets); in other cases, one lineage is disproportionately affected. Here we propose to newly investigate the mechanisms that lead to Diamond Blackfan Anemia (DBA) a rare bone marrow failure syndrome presenting mainly in early infancy. DBA is associated with the decreased production or absence of erythroid precursors in the bone marrow, variable congenital anomalies, and a predisposition to malignancy. DBA is a genetically heterogeneous disease. Mutations in the ribosomal protein RPS19 have been identified in about 25% of cases with DBA. The pathway that leads from a mutation in RPS19, a ubiquitously expressed protein, to a specific defect in terminal differentiation of red cells is not understood. In this pilot and feasibility project we will test the hypothesis that the underlying mechanism responsible for the clinical manifestations in patients with DBA is a defect in ribosome biogenesis. We propose 1) that RPS19 mutations cause a defect in pre-ribosomal RNA processing and ribosome biogenesis that, although present in all growing cells from patients with DBA, is detrimental for the rapidly dividing and synthetically highly active early erythroid progenitor cell and 2) that the identical pathway is affected in patients who have DBA but no RPS19 mutation. To test this hypothesis we will investigate rRNA processing and ribosome biogenesis in lymphoblastoid cell lines established from 1) patients with DBA due to an RPS19 mutation, 2) normal individuals and 3) DBA patients who do not have an RPS19 mutation. By investigating rRNA precursors we will test whether rRNA processing proceeds in a normal fashion, whether observed abnormalities are specific for the RPS19 mutation, or whether similar alterations are found in individuals with DBA but no RPS19 mutation. We will investigate the dynamics of ribosome biogenesis by analysis of the profile of ribosomal subunits, intact ribosomes and polysomes. Using a proteomic approach we will specifically test whether the mutant RPS19 protein participates in the biogenesis of ribosomes and is part of translationally active polysomes. The results will show whether cells from patients with DBA have a defect in rRNA processing and ribosome biogenesis which will enable us to focus on these pathways in our future research. Knowledge of the course of ribosome biogenesis in normal and DBA hematopoietic cells might lead to the development of exciting new tools that may be used a) for more specific and sensitive diagnosis of DBA, b) for the identification and characterization of the molecular defect(s) in individuals with DBA but no RPS19 mutation and finally, c) for the identification of new targets for the development of specific and novel reagents that may be tested for the treatment of individuals with DBA.

描述（由申请人提供）：我们的实验室长期以来对骨髓衰竭的分子发病机制感兴趣，骨髓衰竭是一种以骨髓无法产生足够血细胞为特征的疾病。骨髓衰竭可能影响所有三种主要血细胞谱系（红细胞、白细胞和血小板）；在其他情况下，一种血统受到不成比例的影响。在这里，我们建议重新研究导致钻石黑扇贫血症（DBA）的机制，这是一种罕见的骨髓衰竭综合征，主要出现在婴儿早期。 DBA 与骨髓中红系前体细胞的产生减少或缺乏、可变的先天性异常以及恶性肿瘤的易感性有关。 DBA是一种遗传异质性疾病。约 25% 的 DBA 病例中已发现核糖体蛋白 RPS19 突变。 RPS19（一种普遍表达的蛋白质）的突变导致红细胞终末分化的特定缺陷的途径尚不清楚。在这个试点和可行性项目中，我们将测试以下假设：DBA 患者临床表现的根本机制是核糖体生物合成的缺陷。我们提出 1) RPS19 突变会导致核糖体前 RNA 加工和核糖体生物发生的缺陷，尽管存在于 DBA 患者的所有生长细胞中，但对快速分裂和合成高度活跃的早期红系祖细胞是有害的；2)在具有 DBA 但没有 RPS19 突变的患者中，相同的通路也会受到影响。为了检验这一假设，我们将研究淋巴母细胞系中的 rRNA 加工和核糖体生物发生，这些细胞系由 1) 由于 RPS19 突变而患有 DBA 的患者、2) 正常个体和 3) 没有 RPS19 突变的 DBA 患者建立。通过研究 rRNA 前体，我们将测试 rRNA 加工是否以正常方式进行，观察到的异常是否是 RPS19 突变所特有的，或者是否在具有 DBA 但没有 RPS19 突变的个体中发现类似的改变。我们将通过分析核糖体亚基、完整核糖体和多核糖体的概况来研究核糖体生物发生的动力学。我们将使用蛋白质组学方法专门测试突变型 RPS19 蛋白是否参与核糖体的生物发生以及是否是具有翻译活性的多核糖体的一部分。结果将表明 DBA 患者的细胞在 rRNA 加工和核糖体生物发生方面是否存在缺陷，这将使我们能够在未来的研究中重点关注这些途径。了解正常和 DBA 造血细胞中核糖体生物发生过程可能会导致令人兴奋的新工具的开发，这些工具可用于 a) 更特异和灵敏的 DBA 诊断，b) 用于分子缺陷的识别和表征）在具有 DBA 但没有 RPS19 突变的个体中，最后，c）用于识别新靶标，以开发可用于测试对 DBA 个体的治疗的特异性和新型试剂。