Novel Regulators of T Cell mRNA Decay

描述(由申请人提供)：本项目的长期目标是了解信使核糖核酸降解在调节T淋巴细胞基因表达中的作用。稳定状态的信使核糖核酸水平由转录和降解之间的平衡决定。这一建议是基于这样的假设，即特定的RNA调节序列和识别这些序列的特定反式作用蛋白在mRNA衰退水平上调节基因表达。我们最近利用微阵列技术测量了在静息或激活条件下在T细胞中表达的大约6000个转录本的mRNA衰减率，我们发现了数百个在mRNA衰变水平上受到调控的转录本。我们使用计算方法搜索在mRNA衰退水平上共同调节的转录本中发现的保守序列，发现大约100个在T细胞中表达的短寿命转录本在其3‘非翻译区含有保守的11聚体RNA序列UGUUGUUGUGU。我们将这个序列命名为富Gu元件(GRE)，并使用报告系统证明了这种RNA元件介导了HeLa细胞中的mRNA衰退。在初步实验中，我们还证明了CUG结合蛋白1特异性地与GRE结合，并介导GRE依赖的mRNA衰退。这项资助的目标是描述这些新的调控因子，并确定更多针对mRNA衰变的新调节因子。具体目的1是研究富GU元件(GRE)和CUG结合蛋白1(CUGBP1)在调节T细胞mRNA衰变中的作用。特异性目的2是研究新的保守的RNA序列元件在调节T细胞mRNA衰退中的作用，特异性目的3是表征新的RNA结合蛋白在调节T细胞mRNA衰退中的作用。我们将使用生化和分子生物学的方法来检测新的调节元件和RNA结合蛋白在调节T细胞mRNA衰退中的功能。这些实验将深入了解mRNA衰变调节重要生长调控基因表达的机制，包括细胞因子基因和原癌基因。了解mRNA衰变调节这些基因表达的机制将有助于深入了解人类的疾病状态，如自身免疫、免疫缺陷、癌症和传染病，并将导致新的抗癌和免疫调节药物的开发，这些药物通过靶向特定的mRNA衰变途径发挥作用。为了人类细胞的正常发育和功能，基因需要在精确的时间被打开，它们需要在精确的时间被关闭。细胞用来关闭基因的一个重要方法是消除信使RNA，信使RNA随后停止蛋白质的生产。信使RNA的消除是通过信使RNA衰变的过程在细胞中发生的。在这项拨款申请中提出的实验的目标是确定导致信使核糖核酸衰变的蛋白质，并了解信使核糖核酸衰变过程是如何调节的。许多人类疾病，如癌症或自身免疫性疾病的发生，是因为基因被打开，但随后又没有以正常方式关闭。了解基因是如何关闭的，将导致开发新的药物，用于关闭癌症或自身免疫等疾病患者的特定基因。

DESCRIPTION (provided by applicant): The broad long-term goal of this project is to understand the role of mRNA degradation in regulating T lymphocyte gene expression. Steady state mRNA levels are determined by the balance between transcription and degradation. This proposal is based on the hypothesis that specific RNA regulatory sequences and specific trans-acting proteins that recognize these sequences function to regulate gene expression at the level of mRNA decay. We recently used microarray technology to measure mRNA decay rates of approximately 6,000 transcripts expressed in T cells under conditions of rest or activation, and we identified hundreds of transcripts that were regulated at the level of mRNA decay. We used computational methods to search for conserved sequences found in transcripts that were co-regulated at the level of mRNA decay and found that approximately 100 short-lived transcripts expressed in T cells contained the conserved 11-mer RNA sequence UGUUUGUUUGU in their 3' untranslated region. We have termed this sequence the GU-rich element (GRE) and have shown using a reporter system that this RNA element mediates mRNA decay in HeLa cells. In preliminary experiments, we have also shown that the CUG-binding protein 1 binds specifically to the GRE and mediates GRE-dependent mRNA decay. The goals of this grant are to characterize these novel regulators and to identify additional novel regulators of mRNA decay. Specific aim 1 is to characterize the role of the GU-rich element (GRE) and the CUG-binding protein 1 (CUGBP1) in regulating T cell mRNA decay. Specific aim 2 is to characterize the role of novel conserved RNA sequence elements in regulating T cell mRNA decay, and specific aim 3 is to characterize the role of novel RNA-binding proteins in regulating T cell mRNA decay. We will use biochemical and molecular biological methods to test the function of novel regulatory elements and RNA-binding proteins in the regulation of T cell mRNA decay. These experiments will provide insight into the mechanisms by which mRNA decay regulates the expression of important growth regulatory genes, including cytokine genes and proto-oncogenes. Understanding the mechanisms by which mRNA decay regulates the expression of these genes will provide insight into human disease states such as autoimmunity, immunodeficiency, cancer, and infectious diseases and will lead to the development of new anti-cancer and immunomodulatory drugs that act by targeting specific mRNA decay pathways. In order for human cells to develop and function normally, genes need to be turned on at precise times and they need to be turned off at precise times. An important method that cells use to turn genes off is to eliminate messenger RNA, which then stops the production of proteins. The elimination of messenger RNA occurs in the cell through a process known as messenger RNA decay. The goal of the experiments proposed in this grant application is to define the proteins that are responsible for mRNA decay and to understand how the process of mRNA decay is regulated. Many human diseases such as cancer or autoimmune diseases occur because genes are turned on but then are not turned off in a normal manner. Understanding how genes are turned off will lead to the development of new drugs that will be used to turn off specific genes in patients with diseases such as cancer or autoimmunity.