REGULATION OF EXOSOME-MEDIATED MAMMALIAN mRNA TURNOVER

外泌体介导的哺乳动物 mRNA 周转的调节

• 批准号: 7035802
• 负责人: CHING-YI CHEN
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7035802
• 关键词: HeLa cells; RNA binding protein; RNA interference; chemical stability; cofactor; enzyme mechanism; gene mutation; genetic regulation; messenger RNA; nucleic acid metabolism; protein structure function; transfection

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.

描述（由申请人提供）：mRNA周转的控制在确定细胞生长、分化和发育过程中基因表达水平方面发挥着重要作用。异常的 mRNA 更新在癌症和免疫性疾病等疾病的发展中起着至关重要的作用，这强调了严格控制 mRNA 更新的重要性。导致 mRNA 快速衰减的一个主要元素是富含 AU 的元素 (ARE)。尽管 ARE 已在 15 年前被发现，但 ARE 决定 mRNA 快速衰减的机制仍不清楚。哺乳动物中 mRNA 降解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类外泌体（一种核糖核酸酶复合物），并证明它是快速降解含有 ARE 的 RNA 底物所必需的。这项工作的广泛长期目标是确定外泌体介导的 mRNA 降解的机制，重点是可操纵以靶向特定疾病相关转录本进行降解的方面。 为了实现我们的长期目标，了解 ARE 结合蛋白 (ARE-BP) 在调节外泌体介导的 mRNA 降解中的作用至关重要（具体目标 1）。这一目标涉及 KSRP 结构域的功能表征，KSRP 是一种新型 ARE-BP，可与外泌体共同纯化，并与外泌体和 ARE 相互作用。为此，我们还将测试一种针对特定 mRNA 进行降解的策略，这将提供以后可能用作治疗策略的信息。了解外泌体辅助因子的作用对于阐明外泌体在 mRNA 周转中的功能机制也至关重要，并可能最终提供改变外泌体活性的目标（具体目标 2）。该目标涉及体外和体内 mRNA 降解中外泌体辅助因子的鉴定和功能表征。 最后，还有必要证明 mRNA 衰变的机制步骤以及外泌体亚基的丢失对人类细胞系中含有或不含有 ARE 的不稳定 mRNA 衰变的影响（具体目标 1）。 拟议的研究应有助于阐明哺乳动物 mRNA 降解的机制，并为治疗干预提供目标。