Mechanism and Significance of Nonsense Mediated RNA Decay Regulation in Erythropo

描述（由申请人提供）：正在分化的红细胞，特别是在地中海贫血中，暴露于严重的细胞应激，包括氧化和缺氧应激。活性氧和缺氧都会导致eIF2a的磷酸化，eIF2a是蛋白质合成起始的重要翻译因子，遗传学研究表明，这种磷酸化的调控在红细胞生成中起着重要作用。我们已经确定缺氧细胞中无义介导的RNA衰变的抑制依赖于eIF2a磷酸化。无义介导的RNA衰变（NMD）是一个多步骤的途径，负责30%的所有人类突变mrna的降解，以及高达10%的正常细胞mrna。但由于NMD尚未被认为是一种受调控的途径，其在正常或地中海贫血红细胞中的活性尚未被密切研究，其在红细胞生成中的意义尚未确定。我们假设NMD在地中海贫血红细胞分化过程中受到细胞缺氧和活性氧的抑制。这种由eIF2a磷酸化介导的抑制改变了基因表达，增强了应激反应，提高了这些细胞的存活率。我们建议1)确定eIF2a是否被人类地中海贫血红细胞中的活性氧磷酸化，以及这种磷酸化是否足以抑制NMD。简单地说，我们将从外周血中扩增和分化红细胞，并检查对照和地中海贫血志愿者的骨髓活检。这些样品将被评估eIF2a磷酸化状态、应激反应基因的诱导和活性氧的种类。然后，我们将使用各种压力和工程细胞系来确定eIF2a磷酸化是否足以抑制NMD。2)确定eIF2a磷酸化和NMD抑制在正常和地中海贫血红细胞中的生物学意义。简而言之，我们将确定eIF2a被活性氧或细胞缺氧磷酸化是否与分化红细胞的凋亡相关。使用表达阵列，我们将确定当NMD在红系细胞中被基因删除和/或当NMD在这些细胞中被缺氧抑制时稳定的mrna。NMD靶向选择性剪接的mRNA，通过使用表达阵列识别mRNA剪接变体，我们将确定这些变体是否在缺氧和NMD抑制的红细胞中富集。3)根据初步数据，eIF2a磷酸化将NMD靶向mrna隔离到细胞质应激颗粒中，这些mrna不能被降解，通过追求我们的工作模型，确定缺氧诱导无义介导的RNA衰变抑制的机制。使用共聚焦显微镜，我们将确定NMD重要酶的定位，以及在eIF2a磷酸化的应激细胞中NMD降解的mrna。公共卫生相关性：红细胞的正常生长和发育对维持健康至关重要。在地中海贫血等疾病中，红细胞发育受损可能导致严重贫血和对输血的依赖。我们已经确定了一种控制信使RNA稳定性的新机制，信使RNA是细胞中从DNA到蛋白质的遗传信息的载体。因为这种机制是由正常的，尤其是地中海贫血的红细胞中的细胞压力调节的，我们将确定红细胞的存活是否受到这种新形式的基因调节的影响，并确定受这种机制调节的基因。

DESCRIPTION (provided by applicant): The differentiating erythroid cell, particularly in thalassemia, is exposed to severe cellular stresses including oxidative and hypoxic stress. Both reactive oxygen species and hypoxia lead to the phosphorylation of eIF2a, a translation factor vital for the initiation of protein synthesis, and genetic studies have demonstrated that the regulation of this phosphorylation plays an important role in erythropoiesis. We have determined that the inhibition of nonsense mediated RNA decay in hypoxic cells is dependent on eIF2a phosphorylation. Nonsense mediated RNA decay (NMD) is a multi-step pathway responsible for the degradation of 30% of all human mutated mRNAs, as well as up to 10% of normal cellular mRNAs. But because NMD has not been thought of as a regulated pathway, its activity in normal or thalassemic erythroid cells has not been closely studied, and its significance in erythropoiesis has not been determined. We hypothesize that NMD is inhibited by cellular hypoxia and reactive oxygen species in differentiating thalassemic erythroid cells. This inhibition, mediated by eIF2a phosphorylation, alters gene expression, augments the stress response, and improves the survival of these cells. We propose to 1) Determine if eIF2a is phosphorylated by reactive oxygen species in human thalassemic erythroid cells, and whether this phosphorylation is sufficient for the inhibition of NMD. Briefly, we will amplify and differentiate erythroid cells from peripheral blood, and also examine bone marrow biopsies, from control and thalassemic volunteers. These samples will be assessed for eIF2a phosphorylation status, induction of stress responsive genes, and reactive oxygen species. We will then use a variety of stresses and engineered cell lines to determine if eIF2a phosphorylation is sufficient to inhibit NMD. 2) Determine the biological significance of eIF2a phosphorylation and NMD inhibition in normal and thalassemic erythroid cells. Briefly, we will determine if eIF2a phosphorylation by reactive oxygen species or cellular hypoxia correlates with apoptosis in differentiating erythroid cells. Using expression arrays we will determine the mRNAs that are stabilized when NMD is genetically deleted in erythroid cells and/or when NMD is inhibited by hypoxia in these cells. NMD targets alternatively spliced mRNAs, and by using expression arrays that identify mRNA splice variants we will determine if these variants are enriched in hypoxic and NMD repressed erythroid cells. 3) Determine the mechanism of hypoxia-induced inhibition of nonsense mediated RNA decay by pursuing our working model, based on preliminary data, that eIF2a phosphorylation sequesters NMD targeted mRNAs to cytoplasmic stress granules, where these mRNAs cannot be degraded. Using confocal microscopy we will determine the localization of enzymes important for NMD, as well as NMD degraded mRNAs in stressed cells where eIF2a is phosphorylated. PUBLIC HEALTH RELEVANCE: The normal growth and development of red blood cells is vital to sustain health. Impairment of red cell development, in diseases such as thalassemia, may lead to a severe anemia and a dependence on transfusions. We have identified a novel mechanism that controls the stability of messenger RNA, the carrier of genetic information from DNA to proteins in a cell. Because this mechanism is regulated by cellular stresses found in the normal, and particularly thalassemic, erythroid cells, we will determine if erythroid cell survival is affected by this novel form of gene regulation, and identify the genes that are regulated by this mechanism.