Nonsense Mediated RNA Decay Regulation in Erythropoiesis & Thalassemia

描述(由申请人提供)：分化中的红系细胞，尤其是地中海贫血患者，暴露于严重的细胞应激，包括氧化和低氧应激。活性氧和低氧都会导致eIF2a的磷酸化，eIF2a是启动蛋白质合成的关键翻译因子，遗传学研究表明这种磷酸化的调节在红细胞生成中起着重要作用。我们已经确定，抑制无意义介导的RNA在低氧细胞中的衰退依赖于eIF2a的磷酸化。无意义介导的RNA衰变(NMD)是一个多步骤的途径，负责降解30%的人类突变的mRNAs，以及高达10%的正常细胞mRNAs。但由于NMD尚未被认为是一种调节途径，其在正常或地中海贫血红系细胞中的活性尚未得到深入研究，其在红细胞生成中的意义也尚未确定。我们推测，在地中海贫血红系细胞分化过程中，NMD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，并提高了这些细胞的存活率。我们建议1)确定eIF2a是否被人地中海贫血红系细胞中的活性氧物种磷酸化，以及这种磷酸化是否足以抑制NMD。简而言之，我们将从外周血中扩增和分化红系细胞，并检查对照和地中海贫血志愿者的骨髓活检。这些样本将被评估eIF2a的磷酸化状态、应激反应基因的诱导和活性氧物种。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磷酸化和抑制NMD在正常和地中海贫血红系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的红系细胞的凋亡相关。利用表达阵列，我们将确定当红系细胞中的NMD基因缺失和/或当这些细胞中的NMD被缺氧抑制时，稳定的mRNAs。NMD靶向选择性剪接的mRNAs，通过使用识别mRNA剪接变体的表达阵列，我们将确定这些变体是否在缺氧和NMD抑制的红系细胞中富含。3)根据我们的工作模型，在初步数据的基础上，eIF2a磷酸化隔离NMD靶向于细胞质应激颗粒的mRNAs，这些mRNAs在那里不能被降解，以确定缺氧抑制无意义介导的RNA衰退的机制。使用共聚焦显微镜，我们将确定对NMD重要的酶的定位，以及在eIF2a被磷酸化的应激细胞中NMD降解的mRNAs。与公共健康相关：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，红细胞发育障碍可能会导致严重的贫血和对输血的依赖。我们已经确定了一种新的机制来控制信使RNA的稳定性，信使RNA是从DNA到细胞内蛋白质的遗传信息的载体。由于这一机制是由在正常，特别是地中海贫血的红系细胞中发现的细胞压力来调节的，所以我们将确定红系细胞的存活是否受到这种新形式的基因调控的影响，并确定受这种机制调控的基因。

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.