REGULATION OF EXOSOME-MEDIATED MAMMALIAN mRNA TURNOVER

描述（由申请人提供）：mRNA周转的控制在决定细胞生长、分化和发育过程中的基因表达水平方面起着重要作用。异常的mRNA周转在包括癌症和免疫疾病在内的疾病的发展中起着关键作用，强调了严格控制mRNA周转的重要性。负责快速mRNA衰变的主要元件是富含AU的元件（ARE）。虽然它们在15年前就被发现了，但战神决定mRNA快速衰减的机制仍然不清楚。哺乳动物中mRNA衰变的机制步骤和负责的酶机制尚未完全阐明。我们纯化并表征了人外泌体，一种核糖核酸酶复合物，并证明了它是快速降解含ARE的RNA底物所必需的。这项工作的广泛的长期目标是确定外泌体介导的mRNA衰变的机制，重点是可能被操纵以靶向特定疾病相关转录物进行降解的方面。 为了实现我们的长期目标，了解ARE结合蛋白（ARE-BPs）在调节外泌体介导的mRNA降解（具体目标1）中的作用至关重要。该目的涉及KSRP结构域的功能表征，KSRP是一种与外泌体共纯化的新型ARE-BP，其与外泌体和ARE相互作用。在这个目标中，我们还将测试一种靶向特定mRNA降解的策略，这将提供以后可能用作治疗策略的信息。理解外泌体辅因子的作用对于阐明外泌体在mRNA周转中的功能机制也是至关重要的，并且可能最终提供改变外泌体活性的靶点（具体目标2）。这一目标涉及在体外和体内mRNA衰变的外泌体辅因子的鉴定和功能表征。 最后，还需要证明mRNA衰变的机制步骤以及外泌体亚基丢失对人类细胞系中有或没有战神的不稳定mRNA衰变的影响（特异性aim 1）。 拟议的研究将有助于阐明哺乳动物mRNA降解的机制，并为治疗干预提供靶点。

DESCRIPTION (provided by applicant): Control of mRNA turnover plays an important role in determining levels of gene expression during cell growth, differentiation, and development. Aberrant mRNA turnover plays a critical role in the development of diseases including cancer and immune disorders, emphasizing the importance of stringent control of mRNA turnover. A major element responsible for rapid mRNA decay is the AU-rich element (ARE). Although they were found more than 15 years ago, the mechanism by which AREs dictate rapid mRNA decay is still unclear. The mechanistic steps of mRNA decay and the responsible enzymatic machinery in mammals are not completely elucidated. We purified and characterized the human exosome, a complex of ribonucleases, and demonstrated that it is required for rapid degradation of ARE-containing RNA substrates. The broad long-term goal of this work is to determine the mechanism of exosome-mediated mRNA decay with an emphasis on aspects that might be manipulated to target specific disease-related transcripts for degradation. To accomplish our long range goals, it is critical to understand the role of ARE-binding proteins (ARE-BPs) in the regulation of exosome-mediated mRNA degradation (specific aim 1). This aim involves the functional characterization of domains of KSRP, a novel ARE-BP that co-purifies with the exosome, that interact with the exosome and the ARE. In this aim, we will also test a strategy to target a specific mRNA for degradation which will provide information that might be later used as a therapeutic strategy. Understanding the role of exosome cofactors is also critical for elucidating the mechanism of exosome function in mRNA turnover and may ultimately provide a target for altering exosome activity (specific aim 2). This aim involves the identification and functional characterization of exosome cofactors in mRNA decay in vitro and in vivo. Finally, it is also necessary to demonstrate the mechanistic steps of mRNA decay and the effect of loss of exosome subunits on decay of unstable mRNAs with or without AREs in human cell lines (specific aim1). The proposed research should help elucidate the mechanisms by which mammalian mRNAs are degraded and provide targets for therapeutic intervention.