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，由rRNA、催化部分和许多核糖体蛋白组装而成。在真核细胞中，pre-rRNA在与核糖体蛋白组装成核糖体的大小亚基时，经历了广泛的化学修饰和多次裂解事件。在酵母中，过去15年的进展表明，这些RNA加工事件是由许多小核核核糖核蛋白（snoRNPs）和多达500种反式作用蛋白和RNA-蛋白复合物介导的。虽然到目前为止，大多数参与制造核糖体的因素已经被确定，但对于它们如何组装在一起以制造前核糖体的功能单元，以及这些单元如何促进基因表达中的特定事件，人们知之甚少。我们的长期目标是了解真核细胞中核糖体生物发生所必需的pre-rRNA加工，RNA折叠和核糖体组装步骤。本应用程序的目的是确定核糖体生物发生中涉及的一些大分子组装的结构和组织，以便获得急需的机制见解。我们为每种策略生成的强有力的初步数据表明，成功的可能性很高。本研究的基本原理是了解s(no)RNPs、RNPs和相关蛋白质亚复合物的蛋白质成分是如何组装的，以及它们如何相互作用以介导核糖体生物发生的步骤。对于本研究，我们提出以下具体目标：1。验证古细菌盒C/D sRNP作为双sRNP组装和功能的假设；2. 阐明参与真核SSU核糖体生物发生的小RNPs和蛋白质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发生中的作用。这项工作是创新的，因为我们将使用尚未应用于核糖体生物发生研究的实验策略，因为我们对这些无处不在的rna -蛋白复合物的结构和组装知之甚少。这一结果意义重大，因为核糖体的生物发生是基因表达的一个基本步骤，所有细胞，尤其是生长中的细胞，都将大量的新陈代谢投入其中。核糖体生物发生的失调与人类癌症有关，SSU过程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。

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.