Ionizing radiation control of inflammatory cytokine mRNA stability and expression

摘要 基因的表达可以通过改变运输、翻译效率在mRNA水平上进行调节 和稳定性。除了转录的速度外，信使核糖核酸的衰变速度还决定细胞质 丰富的mRNAs。因此，mRNA衰减率的调节是基因中的一个重要控制点 表情。众所周知，不同的mRNA在真核细胞中表现出不同的半衰期，范围 从许多炎性细胞因子和生长因子mRNAs的几分钟到大多数其他mRNAs的数小时。 通过电离辐射(IR)暴露在组织中诱导急性促炎细胞因子反应， 尤其是在巨噬细胞和其他免疫细胞中，病理炎症反应是主要的 放射治疗和意外照射对IR的影响。尽管显然重要的是 关于IR如何促进病理性炎症反应的研究，目前还很少。 为描述这种现象发生的机制而进行的研究。此应用程序被定向到 了解IR促进促炎细胞因子过度表达的分子机制， 专注于通过拮抗其抑制炎性细胞因子mRNAs正常快速降解的能力 促衰变因子AUF1的活性。 目标1将使用永生化野生型和AUF1-/-巨噬细胞和质谱学来鉴定和 鉴定IR诱导的AUF1相互作用蛋白，这些蛋白可能参与介导IR调控 通过调节AUF1活性的炎症反应。为了获得与临床环境相关的信息， 其他研究将考察多种较低剂量的IR对AUF1介导的心肌细胞衰变的影响。 炎症性Are-mRNAs和AUF1-相互作用蛋白。然后，研究将确定与其结合的蛋白质 单次大剂量和多次分次小剂量IR治疗后，AUF1丢失或恢复。活体内 这些蛋白质与AUF1的相互作用将在生理相关条件下得到验证。 目的2将表征IR介导的AUF1结合蛋白相互作用在快速 内源性促炎细胞因子Are-mRNAs的衰变。研究将使用永生的野生动物和 AUF1-/-巨噬细胞，以及RNA沉默和相互作用蛋白的cDNA过度表达，以充分 表征它们在IR介导的炎症细胞因子稳定和过度表达中的分子功能 MRNAs。 目标3将确定受IR调节的主要内源性炎症细胞因子ARE-mRNAs 通过控制AUF1活性。研究将确定AUF1活性的全局ARE-mRNA靶点 和IR处理永生化野生型和AUF1-/-巨噬细胞，并确定其他 Are结合蛋白如TTP、KSRP和稳定蛋白Hur参与IR介导的稳定 炎性细胞因子是-mRNAs。 目的4将描述IR抑制炎症反应的分子机制。 通过作用于AUF1的mRNAs和与AUF1相互作用的关键蛋白确定。研究还将确定 IR是否通过作用于P小体的形成和功能，和/或作用于外体，从而阻断AUF1的功能 核糖核酸酶细胞器样结构，参与降解短寿命的mRNAs。

ABSTRACT Gene expression can be regulated at the mRNA level through alterations in transport, translational efficiency and stability. The rate of mRNA decay, in addition to the rate of transcription, determines cytoplasmic abundance of mRNAs. Regulation of mRNA decay rates is therefore an important control point in gene expression. It is well established that different mRNAs display diverse half-lives in eukaryotic cells, ranging from minutes for many inflammatory cytokine and growth factor mRNAs, to many hours for most other mRNAs. Acute pro-inflammatory cytokine responses are induced in tissues by ionizing radiation (IR) exposure, particularly in macrophages and other immune cells, and the pathological inflammatory response is a major complicating affect of IR during radiotherapy and accidental exposure. Despite the obvious importance for understanding how IR promotes a pathological inflammatory response, there has been remarkably little research conducted to characterize the mechanism by which this occurs. This application is directed to understanding the molecular mechanism by which IR promotes overexpression of pro-inflammatory cytokines, focusing on its ability to inhibit the normal rapid degradation of inflammatory cytokine mRNAs by antagonizing the activity of pro-decay factor, AUF1. Aim 1 will use immortalized wild type and AUF1-/- macrophages and mass spectrometry to identify and characterize IR-inducible AUF1 interacting proteins that are likely involved in mediating IR control of the inflammatory response through regulation of AUF1 activity. To obtain information relevant to the clinical setting, additional studies will examine the affect of multiple lower dose fractions of IR on AUF1-mediated decay of inflammatory ARE-mRNAs and AUF1-interacting proteins. Studies will then identify proteins whose binding to AUF1 is lost or gained with single high dose and multiple fractionated lower dose IR- treatment. The in vivo interaction of these proteins with AUF1 will then be verified under physiologically relevant conditions. Aim 2 will characterize the function of IR-mediated AUF1 binding protein interactions on the rapid decay of endogenous pro-inflammatory cytokine ARE-mRNAs. Studies will use immortalized wild type and AUF1-/- macrophages, as well RNA silencing and cDNA overexpression of interacting proteins, to fully characterize their molecular functions in IR-mediated stabilization and overexpression of inflammatory cytokine mRNAs. Aim 3 will identify the major endogenous inflammatory cytokine ARE-mRNAs that are regulated by IR through control of AUF1 activity. Studies will identify global ARE-mRNA targets of AUF1 activity in untreated and IR treated immortalized wild type and AUF1-/- macrophages, and determine the extent to which other ARE-binding proteins such as TTP, KSRP and stabilizing protein HuR are involved in IR-mediated stabilization of inflammatory cytokine ARE-mRNAs. Aim 4 will characterize the molecular mechanism by which IR inhibits the decay of inflammatory ARE- mRNAs by acting on AUF1 and the key AUF1 interacting proteins identified. Studies will also determine whether IR blocks AUF1 function by acting on P-body formation and function, and/or on the exosome, a ribonuclease organelle-like structure that is involved in the degradation of short-lived mRNAs.