Monitoring mechanisms in mammalian ribosome biogenesis

描述（由申请人提供）：核糖体是进行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期间。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，核糖体的生物合成产生一定比例的缺陷产物和动力学捕获的中间体。细胞如何区分正确构建和错误构建的核糖体？这项研究的主要目的是通过阐明哺乳动物细胞中核糖体合成质量控制的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）不同。这些差异之一是哺乳动物中核糖体形成的缺陷诱导p53介导的核仁应激反应，这在机制上尚未完全理解。因为前核糖体的框架，像核糖体本身一样，是由RNA组成的，所以核糖核酸酶在分解有缺陷的核糖体前体中起着关键作用。在这里，我们希望建立途径，通过该途径，外切核糖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.确定哺乳动物外泌体在降解错误组装的前60 S亚基中的作用。我们将确定外泌体是否在错误组装的前60 S亚基的初级监视中起作用或作为清道夫，以及如何通过候选衔接子调节这些活动。2.确定前60 S亚基的结构特征，控制它是否会被处理或降解。我们的模型是前核糖体的某些成分作为守门人，控制核酸酶进入前rRNA。这将通过分析核酸外切酶Xrn 2和前60 S亚基中5.8S RNA-核糖体蛋白复合物之间的相互作用来进行测试。3.确定前rRNA衰变产物是否在核糖体组装因子突变和阻断核糖体成熟的抗癌药物诱导的核仁应激反应中发挥作用，这两种突变均显著增加核酸酶对前rRNA的分解。总之，这些研究将测试的假设，前rRNA的哺乳动物核糖核酸外切酶的监视服务的双重功能，使核糖体的准确合成在正常情况下，并启动压力信号时，系统变得过载。

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.