Ionizing Radiation Control of Inflammatory Cytokine mRNA Stability and Expression

描述（由申请人提供）：基因表达可通过转运、翻译效率和稳定性的改变在mRNA水平上进行调节。除了转录速率之外，mRNA衰减速率决定mRNA的细胞质丰度。因此，mRNA衰减速率的调节是基因表达的重要控制点。已经确定，不同的mRNA在真核细胞中显示不同的半衰期，范围从许多炎性细胞因子和生长因子mRNA的几分钟到大多数其他mRNA的几小时。电离辐射（IR）暴露在组织中诱导急性促炎细胞因子反应，特别是在巨噬细胞和其他免疫细胞中，并且病理性炎症反应是放射治疗和意外暴露期间IR的主要并发影响。尽管了解IR如何促进病理性炎症反应的明显重要性，但很少有研究来表征这种情况发生的机制。本申请旨在了解IR促进促炎细胞因子过表达的分子机制，重点在于其通过拮抗促衰变因子AUF 1的活性来抑制炎性细胞因子mRNA的正常快速降解的能力。目的1将使用永生化的野生型和AUF 1-/-巨噬细胞和质谱法来鉴定和表征IR诱导的AUF 1相互作用蛋白，其可能通过调节AUF 1活性参与介导IR控制炎症反应。为了获得与临床环境相关的信息，其他研究将检查多个较低剂量分数的IR对AUF 1介导的炎性ARE-mRNA和AUF 1相互作用蛋白的衰减的影响。然后，研究将鉴定在单次高剂量和多次分次较低剂量IR治疗下失去或获得与AUF 1结合的蛋白质。然后将在生理相关条件下验证这些蛋白质与AUF 1的体内相互作用。目的2将表征IR介导的AUF 1结合蛋白相互作用对内源性促炎细胞因子ARE-mRNA的快速衰减的功能。研究将使用永生化的野生型和AUF 1-/-巨噬细胞，以及相互作用蛋白的RNA沉默和cDNA过表达，以充分表征它们在IR介导的稳定和炎性细胞因子mRNA过表达中的分子功能。目的3将确定主要的内源性炎性细胞因子ARE-的mRNA，通过控制AUF 1活性的IR调节。研究将鉴定未处理和IR处理的永生化野生型和AUF 1-/-巨噬细胞中AUF 1活性的全局ARE-mRNA靶标，并确定其他ARE结合蛋白如TTP、KSRP和稳定蛋白HuR参与IR介导的炎性细胞因子ARE-mRNA稳定的程度。目的4将表征IR通过作用于AUF 1和鉴定的关键AUF 1相互作用蛋白来抑制炎性ARE-mRNA的衰变的分子机制。研究还将确定IR是否通过作用于P体形成和功能和/或外泌体（一种参与短寿命mRNA降解的核糖核酸酶细胞器样结构）来阻断AUF 1功能。公共卫生相关性：这项资助申请的目的是了解炎症细胞因子通过其编码mRNA的快速降解来控制的机制，以及如何通过电离辐射进行调节。几种蛋白质已被证明能特异性结合这些mRNA并促进其降解。本申请试图定义被称为AUF 1的那些蛋白质之一控制响应于电离辐射的关键炎性细胞因子的快速降解的机制。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The purpose of this grant application is to understand the mechanism by which inflammatory cytokines are controlled by the rapid degradation of their encoding mRNAs and how this is regulated by ionizing radiation. Several proteins have been shown to bind specifically to these mRNAs and to promote their degradation. This application seeks to define the mechanism by which one of those proteins known as AUF1, controls the rapid degradation of the key inflammatory cytokines in response to ionizing radiation.