Ribosome Biogenesis in Diamond Blackfan Anemia

描述(申请人提供)：我们实验室长期以来一直对骨髓衰竭的分子发病机制感兴趣，这是一种以骨髓无法产生足够血细胞为特征的疾病。骨髓衰竭可能会影响所有三个主要的血细胞谱系(红细胞、白细胞和血小板)；在其他情况下，其中一个谱系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(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失、可变的先天性异常以及恶性倾向有关。DBA是一种遗传异质性疾病。在大约25%的DBA病例中发现了核糖体蛋白RPS19的突变。RPS19是一种普遍表达的蛋白质，从突变导致红细胞终末分化的特定缺陷的途径尚不清楚。在这个试验性和可行性项目中，我们将检验这一假设，即导致DBA患者临床表现的潜在机制是核糖体生物发生缺陷。我们提出：1)RPS19突变导致核糖体前RNA处理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活性的早期红系祖细胞是有害的；2)在患有DBA但没有RPS19突变的患者中，相同的途径受到影响。为了验证这一假设，我们将研究淋巴母细胞系中rRNA的加工和核糖体的生物发生，这些细胞系来自1)RPS19突变引起的DBA患者，2)正常人和3)没有RPS19突变的DBA患者。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，或者是否在患有DBA但没有RPS19突变的个体中发现类似的变化。我们将通过分析核糖体亚基、完整核糖体和多聚体的分布来研究核糖体生物发生的动力学。使用蛋白质组学的方法，我们将专门测试突变的RPS19蛋白是否参与核糖体的生物发生，以及是否是翻译活性多聚体的一部分。这些结果将显示DBA患者的细胞是否在rRNA处理和核糖体生物发生方面存在缺陷，这将使我们能够在未来的研究中重点关注这些途径。对正常和DBA造血细胞核糖体生物发生过程的了解可能会导致开发出令人兴奋的新工具，这些工具可用于a)更特异和敏感的诊断DBA，b)鉴定和表征无RPS19突变的DBA患者的分子缺陷(S)，以及c)确定新的靶点，开发可用于治疗DBA的特异性和新型试剂。

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.