REGULATION OF EXOSOME-MEDIATED MAMMALIAN mRNA TURNOVER

描述(由申请人提供)：在细胞生长、分化和发育过程中，信使核糖核酸周转率的控制在决定基因表达水平方面起着重要作用。异常的信使核糖核酸转换在包括癌症和免疫紊乱在内的疾病的发展中起着关键作用，强调了严格控制信使核糖核酸转换的重要性。导致信使核糖核酸快速衰退的一个主要因素是富AU元素(ARE)。尽管它们是在15年前被发现的，但Ares决定mRNA快速衰退的机制仍然不清楚。在哺乳动物中，信使核糖核酸降解的机制步骤和负责的酶机制还没有完全阐明。我们纯化并鉴定了人类外切体，这是一种核糖核酸酶的复合体，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显子介导的mRNA衰退的机制，重点放在可能被操纵以针对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-BP)在调节外切体介导的mRNA降解(特定目标1)中的作用是至关重要的。这一目标涉及KSRP结构域的功能表征，KSRP是一种新的ARE-BP，与外体共同纯化，与外体和ARE相互作用。在这个目标中，我们还将测试一种针对特定mRNA进行降解的策略，这将提供稍后可能用作治疗策略的信息。了解外切体辅助因子的作用对于阐明外切体功能在mRNA周转中的机制也是至关重要的，并最终可能为改变外切体活性提供一个靶点(特定目标2)。这一目标涉及到体外和体内mRNA衰退中外切体辅助因子的鉴定和功能鉴定。最后，还有必要证明在人类细胞系中，有或没有ARES的情况下，mRNA衰退的机制步骤和外体亚单位的丢失对不稳定的mRNAs衰退的影响(特异性AIM1)。这项拟议的研究应该有助于阐明哺乳动物mRNAs被降解的机制，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.