Nonsense Mediated RNA Decay Regulation in Erythropoiesis & Thalassemia

描述（由申请人提供）：分化中的红系细胞，特别是地中海贫血中的红系细胞，暴露于严重的细胞应激，包括氧化和缺氧应激。活性氧和缺氧都导致eIF 2a的磷酸化，eIF 2a是一种对蛋白质合成起始至关重要的翻译因子，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，缺氧细胞中无义介导的RNA衰变的抑制依赖于eIF 2a磷酸化。无义介导的RNA降解（NMD）是一种多步骤途径，负责降解30%的所有人类突变mRNA以及高达10%的正常细胞mRNA。但是，由于NMD尚未被认为是一个受调节的途径，其在正常或地中海贫血红系细胞中的活性尚未得到密切研究，其在红细胞生成中的意义尚未确定。我们推测，NMD是抑制细胞缺氧和活性氧在分化地中海贫血红系细胞。这种由eIF 2a磷酸化介导的抑制改变了基因表达，增强了应激反应，并改善了这些细胞的存活。我们建议1）确定eIF 2a是否被人地中海贫血红系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，我们将扩增和区分来自外周血的红系细胞，并检查来自对照组和地中海贫血志愿者的骨髓活检。将评估这些样本的eIF 2a磷酸化状态、应激反应基因的诱导和活性氧。然后，我们将使用各种应激和工程细胞系来确定eIF 2a磷酸化是否足以抑制NMD。2)确定正常和地中海贫血红系细胞中eIF 2a磷酸化和NMD抑制的生物学意义。简单地说，我们将确定是否eIF 2a磷酸化活性氧或细胞缺氧与细胞凋亡分化红系细胞。使用表达阵列，我们将确定当NMD在红系细胞中遗传缺失时和/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，并且通过使用鉴定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集。3)通过我们的工作模型确定缺氧诱导的无义介导的RNA衰变抑制的机制，基于初步数据，eIF 2a磷酸化将NMD靶向mRNA螯合到细胞质应激颗粒，在那里这些mRNA不能降解。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eIF 2a被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。由于这种机制是由正常的，特别是地中海贫血，红系细胞中发现的细胞应激调节，我们将确定红系细胞的生存是否受到这种新形式的基因调控，并确定由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.