The Architecture and Function of RNPs Required for Ribosome Biogenesis

描述（由申请人提供）：核糖体是翻译的细胞动力室，由rRNA、催化部分和大量核糖体蛋白组装而成。在真核细胞中，前体rRNA经历广泛的化学修饰和几次切割事件，因为它与核糖体蛋白组装成核糖体的大小亚基。在酵母中，S.在酿酒酵母中，过去15年的进展揭示了这些RNA加工事件由许多小核仁核糖核蛋白（snoRNP）和多达500种反式作用蛋白和RNA-蛋白复合物介导。虽然到目前为止，参与制造核糖体的大多数因素都已被确定，但对它们如何组装在一起以制造前核糖体的功能单元以及这些单元中的每一个如何有助于基因表达中的特定事件知之甚少。我们的长期目标是了解真核细胞中核糖体生物合成所必需的前rRNA加工，RNA折叠和核糖体组装步骤。本申请的目的是确定核糖体生物发生中涉及的一些大分子组装体的结构和组织，以获得急需的机械见解。我们为这些策略中的每一个生成的强有力的初步数据表明成功的可能性很高。拟议研究的基本原理是了解s（无）RNP，RNP和相关蛋白质亚复合物的蛋白质组分如何组装，以及它们如何相互作用以介导核糖体生物发生中的步骤。对于这项研究，我们提出了以下具体目标：1。验证古细菌盒C/D sRNP组装并作为di-sRNP发挥功能的假设; 2.阐明真核生物SSU核糖体生物合成中小RNP和蛋白复合物的结构和组织; 3.为了验证前rRNA加工机制以有序、逐步的方式组装的假设，以阐明这些大分子在核糖体生物合成中的功能。拟议的工作是创新的，因为我们将使用尚未应用于核糖体生物合成研究的实验策略，并且因为对这些无处不在的RNA-蛋白质复合物的结构和组装知之甚少。这一结果将是重要的，因为核糖体生物合成是基因表达的一个基本步骤，所有细胞，特别是生长中的细胞，都投入了大量的新陈代谢。核糖体生物发生的失调与人类癌症有关，SSU加工体蛋白的突变与新生儿肝硬化、不孕症和神经纤维瘤病有关。 公共卫生相关性：该提案旨在回答有关蛋白质和RNA-蛋白质复合物的功能和组织的重要问题，这些蛋白质和RNA-蛋白质复合物与癌症和几种罕见的人类遗传疾病的发病机制有关。对这些过程的更透彻的理解将有助于我们设计更好的恶性肿瘤治疗方法。

DESCRIPTION (provided by applicant): Ribosomes, the cellular powerhouses of translation, are assembled from rRNA, the catalytic moiety, and scores of ribosomal proteins. In eukaryotic cells, the pre-rRNA undergoes extensive chemical modifications and several cleavage events as it is being assembled with ribosomal proteins into the small and large subunits of the ribosome. In the yeast, S. cerevisiae, progress over the last 15 years has revealed that these RNA processing events are mediated by numerous small nucleolar ribonucleoproteins (snoRNPs) and as many as 500 trans-acting protein and RNA-protein complexes. While by now most of the factors involved in making ribosomes have been identified, little is known about how they are assembled together to make the functional units of the pre-ribosome, and how each of these units contributes to specific events in gene expression. Our long-term goal is to understand the pre-rRNA processing, RNA folding and ribosome assembly steps essential to ribosome biogenesis in eukaryotic cells. The objective of this application is to determine the architecture and organization of some of the macromolecular assemblies involved in ribosome biogenesis in order to gain much needed mechanistic insights. The strong preliminary data that we have generated for each of these strategies indicates a high likelihood of success. The rationale for the proposed research is to attain an understanding of how the protein components of s(no)RNPs, RNPs and associated protein subcomplexes are assembled and how they interact with each other to mediate steps in ribosome biogenesis. For this study, we propose the following Specific Aims: 1. To test the hypothesis that the archaeal box C/D sRNP assembles and functions as a di-sRNP; 2. To elucidate the architecture and organization of small RNPs and protein complexes involved in eukaryotic SSU ribosome biogenesis; 3. To test the hypothesis that the pre-rRNA processing machinery is assembled in an ordered, stepwise manner to elucidate how these large macromolecules function in ribosome biogenesis. The proposed work is innovative because we will use experimental strategies that have not yet been applied to the study of ribosome biogenesis, and because little is known about the architecture and assembly of these ubiquitous RNA-protein complexes. The results will be significant because ribosome biogenesis is a fundamental step in gene expression to which all cells, particularly growing ones, devote a significant amount of their metabolism. Dysregulation of ribosome biogenesis is linked to cancer in humans, and mutations in SSU processome proteins have been linked to neonatal cirrhosis, infertility and neurofibromatosis. PUBLIC HEALTH RELEVANCE: This proposal is designed to answer important questions about the function and organization of protein and RNA-protein complexes that have been implicated in the pathogenesis of cancer and several rare human genetic diseases. A more thorough understanding of these processes will help us design better therapies for malignancies.