Monitoring mechanisms in mammalian ribosome biogenesis

描述(申请人提供)：核糖体是完成整个细胞蛋白质组合成的生物纳米机器。细胞需要大量的核糖体来制造蛋白质，特别是在活跃的生长和增殖时期。真核生物中的每个核糖体都是通过一个复杂的组装途径制造的，需要200多个辅助蛋白因子。像任何其他复杂的组装过程一样，核糖体的生物合成会产生一定比例的缺陷产物和动力学捕获的中间产物。细胞如何区分构建正确的核糖体和不正确的核糖体？这项拟议研究的主要目的是通过阐明哺乳动物细胞核糖体合成质量控制的潜在机制来回答这个问题。我们在研究中使用小鼠细胞，因为哺乳动物的监视机制在许多方面与其他模式生物(如酵母)的监视机制不同。其中一个不同之处在于，哺乳动物核糖体形成的缺陷导致了P53介导的核仁应激反应，但从机制上讲，这一点尚未完全理解。由于前核糖体的框架，就像核糖体本身一样，是由RNA组成的，核糖核酸酶在分解有缺陷的核糖体前体方面发挥着关键作用。在这里，我们希望建立一个途径，通过这个途径，外切核酸酶开始消除有缺陷的前核糖体的过程。我们的项目有三个具体目标。1.确定哺乳动物外切体在错误组装的Pre-60S亚基降解中的作用。我们将确定外体是在错误组装的60S前亚基的初级监控中发挥作用，还是作为清道夫发挥作用，以及这些活动可能如何通过候选适配子来调节。2.确定控制其是否被加工或降解的60S前亚基的结构特征。我们的模型是，前核糖体的某些组件充当把关人，控制核酸酶对前rRNA的访问。这将通过解剖60S前亚基中的外切酶Xrn2和5.8S RNA-核糖体蛋白复合体之间的相互作用来检验。3.确定核糖体组装因子突变和阻断核糖体成熟的抗癌药物是否在核仁应激反应中发挥作用，这两种药物都显著增加了核糖核酸酶对前rRNA的破坏。总之，这些研究将检验这一假设，即哺乳动物外切核糖核酸酶对前rRNA的监控具有双重功能，即在正常情况下使核糖体能够准确合成，并在系统超载时启动应激信号。

DESCRIPTION (provided by applicant): Ribosomes are biological nanomachines that carry out synthesis of the entire cellular proteome. Cells require a large number of ribosomes to make proteins, especially during periods of active growth and proliferation. Each ribosome in eukaryotes is manufactured through an elaborate assembly pathway that requires more than 200 accessory protein factors. Like any other complex assembly process, biosynthesis of ribosomes generates a certain fraction of defective products and kinetically trapped intermediates. How do cells distinguish between ribosomes that are built correctly and those that are not? The main objective of the proposed research is to answer this question by elucidating the mechanisms underlying quality control of ribosome synthesis in mammalian cells. We use mouse cells in our studies because surveillance mechanisms in mammals differ in many aspects from those in other model organisms such as yeast. One of such differences is that defects in ribosome formation in mammals induce a p53-mediated nucleolar stress response, which is mechanistically not completely understood. Because the framework of preribosomes, like the ribosome itself, is made of RNA, ribonucleases play a key role in dismantling defective ribosome precursors. Here, we wish to establish the pathway through which exoribonucleases start the process of elimination of the defective preribosomes. Our project has three specific aims. 1. Determine the role of the mammalian exosome in the degradation of misassembled pre-60S subunits. We will determine whether the exosome functions in primary surveillance of misassembled pre-60S subunits or acts as a scavenger and how these activities may be regulated through candidate adaptors. 2. Identify structural features of the pre-60S subunit that control whether it will be processed or degraded. Our model is that certain components of preribosomes act as gatekeepers that control nuclease access to pre-rRNA. This will be tested by dissecting the interactions between the exonuclease Xrn2 and the 5.8S RNA-ribosomal protein complex in pre-60S subunits. 3. Determine if pre-rRNA decay products play a role in the nucleolar stress response induced by mutations in ribosome assembly factors and by anticancer drugs that block ribosome maturation, both of which significantly increase pre-rRNA breakdown by nucleases. Together, these studies will test the hypothesis that pre-rRNA surveillance by mammalian exoribonucleases serves the dual function of enabling accurate synthesis of ribosomes under normal circumstances, and initiating stress signaling when the system becomes overloaded.