Regulation of AU-rich element-mediated mRNA decay

描述（申请人提供）：mRNA稳定性的调节在基因表达的控制中起着关键作用。mRNA转换失调与多种病理过程有关。了解mRNA衰变的调控有助于了解某些疾病的发病机制。在哺乳动物细胞中，一个重要的顺式作用元件是富au元件（ARE），它通过一个被称为ARE介导的mRNA衰变（AMD）的过程指导mRNA的衰变。在过去的七年里，我们研究了KSRP（一种促进衰变的are结合蛋白）在AMD中的功能。为了进一步了解KSRP活性在AMD中的调控作用，并确定其体内生理mRNA靶点，我们启动了一项纯化计划，分离与KSRP相关的蛋白，并产生了KSRP - null小鼠。这项续期拨款将利用一系列生化和分子方法来实现以下具体目标。具体目的1是表征DDX1的功能，DDX1是一种被发现与KSRP共纯化的RNA解旋酶，在调节AMD和KSRP的亚细胞定位中。本研究还将大规模纯化KSRP复合物，通过质谱法鉴定与KSRP共纯化的蛋白，并研究它们在AMD调控中的作用。具体目的2是研究Ksrp在I型干扰素（IFN-1和IFN-2）基因表达的转录后控制和Ksrp敲除小鼠的病毒感染中的作用。本研究将探讨Ksrp是否调控Ifna和Ifnb mrna的衰变，以及Ksrp缺乏对病毒感染的影响。特异性目的3是在LPS刺激的巨噬细胞中鉴定Ksrp靶mrna。我们将通过对mRNA衰变的全基因组分析来鉴定在lps刺激的Ksrp-/-巨噬细胞中稳定的mRNA，并通过核糖核蛋白免疫沉淀和微阵列分析来鉴定与Ksrp相关的mRNA。我们的长期目标是了解KSRP调节AMD的机制，确定其体内mRNA靶点，并表征与靶点失调相关的表型。Ksrp基因敲除小鼠的产生为研究Ksrp对某些细胞因子mRNA的转录后调控提供了有价值的工具，这将有助于我们理解由mRNA衰变缺陷引起的炎症和免疫疾病的发病机制。

DESCRIPTION (provided by applicant): Regulation of mRNA stability plays a critical role in the control of gene expression. Dysregulated mRNA turnover has been implicated in various pathological processes. Understanding the regulation of mRNA decay should provide insights into the pathogenesis of certain disease. An important cis-acting element responsible for rapid mRNA decay in mammalian cells is the AU-rich element (ARE), which directs mRNA decay through a process referred to as ARE-mediated mRNA decay (AMD). We have investigated the function of KSRP, a decay-promoting ARE-binding protein, in AMD for the past seven years. To further understand the regulation of KSRP activity in AMD and identify it's in vivo physiological mRNA targets, we have initiated a purification scheme to isolate proteins that associate with KSRP and have generated Ksrp- null mice. This grant renewal will utilize a series of biochemical and molecular approaches to achieve the following specific aims. Specific aim 1 is to characterize function of DDX1, an RNA helicase that was found to co-purify with KSRP, in regulation of AMD and the subcellular localization of KSRP. This aim will also purify KSRP complexes in a large scale, identify proteins that co-purify with KSRP by mass spectrometry, and investigate their roles in the regulation of AMD. Specific aim 2 is to investigate the roles of Ksrp in post- transcriptional control of type I interferon (IFN-1 and IFN-2) gene expression and in virus infection using Ksrp knockout mice. This aim will examine whether Ksrp regulates the decay of Ifna and Ifnb mRNAs and the effect of Ksrp deficiency on virus infection. Specific aim 3 is to identify Ksrp target mRNAs in LPS- stimulated macrophages. We will identify mRNAs that are stabilized in LPS-stimulated Ksrp-/- macrophages by genome-wide analysis of mRNA decay and those associated with Ksrp by ribonucleoprotein immunoprecipitation and microarray analysis. Our long-term goals are to understand the mechanism by which KSRP regulates AMD, to identify it's in vivo mRNA targets, and to characterize phenotypes associated with the dysregulation of the targets. The generation of Ksrp knockout mice should provide a valuable tool to study the post-transcriptional regulation of certain cytokine mRNAs by Ksrp, which should contribute to our understanding of the pathogenesis of inflammatory and immunological diseases resulting from a defect in mRNA decay. PUBLIC HEALTH RELEVANCE: Aberrant regulation of messenger RNA (mRNA) stability has been implicated in various pathological processes. Understanding the regulation of mRNA degradation would provide insights into the disease pathogenesis. Our research should allow for the development of alternative treatments for immune and inflammatory diseases and cancer.