Ionizing Radiation Control of Inflammatory Cytokine mRNA Stability and Expression

描述(申请人提供)：基因表达可以通过运输、翻译效率和稳定性的改变在mRNA水平上进行调节。除转录速率外，信使核糖核酸的衰变速率还决定了其胞质内的丰度。因此，mRNA衰减率的调节是基因表达的一个重要控制点。众所周知，不同的mRNAs在真核细胞中表现出不同的半衰期，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的几个小时。电离辐射(IR)可在组织中诱导急性促炎细胞因子反应，尤其是巨噬细胞和其他免疫细胞，而病理炎症反应是放射治疗和意外照射中IR的主要复杂影响因素。尽管IR对于理解如何促进病理性炎症反应具有明显的重要性，但对其发生机制的研究却非常少。这项应用旨在了解IR促进促炎细胞因子过度表达的分子机制，重点在于其通过拮抗促衰变因子AUF1的活性来抑制炎性细胞因子mRNAs的正常快速降解的能力。目的1将使用永生化野生型和AUF1-/-巨噬细胞和质谱仪来鉴定和鉴定IR诱导的AUF1相互作用蛋白，这些蛋白可能通过调节AUF1活性参与介导IR对炎症反应的控制。为了获得与临床环境相关的信息，进一步的研究将检查多种较低剂量的IR对AUF1介导的炎症ARE-mRNAs和AUF1相互作用蛋白衰变的影响。然后，研究将确定其与AUF1结合的蛋白质在单次高剂量和多次分次低剂量IR治疗中丢失或获得。这些蛋白质与AUF1的体内相互作用将在生理相关条件下得到验证。目的2研究IR介导的AUF1结合蛋白相互作用在内源性促炎细胞因子ARE-mRNAs快速衰退中的作用。研究将使用永生化野生型和AUF1-/-巨噬细胞，以及RNA沉默和相互作用蛋白的基因过度表达，以充分表征它们在IR介导的炎症细胞因子mRNAs稳定和过度表达中的分子功能。目的3将鉴定主要内源性炎症细胞因子ARE-mRNAs，它们是通过控制AUF1活性而受IR调节的。研究将在未经处理和IR处理的永生化野生型和AUF1-/-巨噬细胞中确定AUF1活性的全局ARE-mRNA靶点，并确定其他ARE结合蛋白如TTP、KSRP和稳定蛋白Hur在IR介导的炎症细胞因子ARE-mRNAs稳定中的参与程度。目的4研究IR通过作用于AUF1抑制炎性ARE-mRNAs衰变的分子机制以及已确定的与AUF1相互作用的关键蛋白。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(一种参与短期mRNAs降解的核糖核酸酶样细胞器结构)来阻止AUF1的功能。公共卫生相关性：这项赠款申请的目的是了解炎症细胞因子通过其编码的mRNAs的快速降解来控制的机制，以及这是如何受到电离辐射的调节的。有几种蛋白质已被证明与这些mRNAs特异结合，并促进它们的降解。这项申请旨在确定其中一种名为AUF1的蛋白质控制关键炎症细胞因子对电离辐射的快速降解的机制。

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.