Control of Cytokine mRNA Stability by AUF1

描述(由申请人提供)： 这一建议针对的是一种重要但知之甚少的基因控制机制，即哺乳动物细胞中短暂的细胞因子和原癌基因mRNAs的靶向降解，这是由这些mRNAs的3‘非翻译区(3’UTR)中的富A+U元件(ARE)促进的。这一续订申请建立在我们在前一个项目期间的工作基础上，并试图表征一种名为AUF1的ARE结合蛋白的功能，它促进ARE-mRNAs的快速衰退。 目的1通过质谱学鉴定和鉴定新的AUF1相互作用蛋白，验证体内相互作用，并研究这些相互作用在ARE-mRNA衰变中的功能重要性。到目前为止，只有几个AUF1相互作用蛋白被鉴定出来。由于AUF1缺乏酶活性，因此AUF1通过与其他蛋白质的相互作用促进或调节细胞因子和原癌基因ARE-mRNAs的降解。 目的2分析AUF1在原代培养的小鼠脾和胸腺淋巴细胞中促进或调节内源性和报告基因Are-mRNAs快速转换的作用。我们最近建立了AUF1基因敲除小鼠，并研究了AUF1主要在脾和胸腺淋巴细胞中的表达，这使得研究AUF1在其真实表达的细胞类型和器官中的分子和生化功能成为可能。 目的3将描述AUF1在小鼠体内的功能。人们对AUF1在动物体内的作用知之甚少。通过比较野生型和AUF1基因敲除小鼠，研究将探讨AUF1在促进或调节胸腺和脾中淋巴细胞细胞因子mRNAs稳定性方面的作用。研究还将检查AUF1基因敲除小鼠在不同相关实验环境中的表型。

DESCRIPTION (provided by applicant): This proposal is directed to an important but very poorly understood mechanism of gene control, the targeted degradation of short-lived cytokine and proto-oncogene mRNAs in mammalian cells, which is promoted by an A+U-rich element (ARE) in the 3' untranslated region (3'UTR) of these mRNAs. This renewal application builds on our work during the previous project period and seeks to characterize the function of one ARE-binding protein known as AUF1, which promotes rapid decay of ARE-mRNAs. Aim 1 will identify and characterize new AUF1 interacting proteins by mass spectrometry, verify interactions in vivo, and investigate the functional importance of these interactions in ARE-mRNA decay. Only several of the AUFl-interacting proteins have been identified to date. Since AUF1 lacks enzymatic activity, it is through interactions with other proteins that AUF1 facilitates or regulates decay of cytokine and proto-oncogene ARE-mRNAs. Aim 2 will analyze the function of AUF1 in promoting or regulating the rapid turnover of endogenous and reporter ARE-mRNAs in isolated primary mouse spleen and thymus lymphocytic cells, where AUF1 is predominantly expressed. Our recent development of an AUF1 knockout mouse, and studies which localized AUF1 expression largely to splenic and thymic lymphocytes, makes it possible to investigate the molecular and biochemical functions of AUF1 in its authentic expressing cell type and organ. Aim 3 will characterize the function of AUF1 in the mouse. There is little if any understanding of the role of AUF1 in animals. By comparing wild type and AUF1 knockout mice, studies will investigate the function of AUF1 in promoting or regulating the stability of lymphocytic cytokine mRNAs in the thymus and spleen. Studies will also examine the phenotype of the AUF1 knockout mouse in different relevant experimental settings.