The Architecture and Function of RNPs Required for Ribosome Biogenesis

描述(申请人提供)：核糖体是翻译的细胞动力源，由rRNA、催化部分和数十种核糖体蛋白组装而成。在真核细胞中，前rRNA经历了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。在酿酒酵母中，过去15年的研究进展表明，这些RNA加工事件是由大量的小核仁核糖核蛋白(SnoRNP)和多达500个反式作用蛋白质和RNA-蛋白质复合体介导的。虽然到目前为止，参与制造核糖体的大多数因素已经被确定，但关于它们是如何组装在一起形成前核糖体的功能单位，以及每个单位如何对基因表达中的特定事件做出贡献，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前rRNA加工、RNA折叠和核糖体组装步骤。这项应用的目的是确定核糖体生物发生中涉及的一些大分子组件的结构和组织，以获得迫切需要的机制见解。我们为每一项战略产生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(NO)RNPs、RNPs和相关蛋白质亚复合体的蛋白质组分是如何组装的，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，我们提出了以下具体目标：1.检验古生物盒C/D sRNP作为di-sRNP组装并发挥功能的假设；2.阐明真核生物SSU核糖体生物发生中涉及的小RNP和蛋白质复合体的结构和组织；3.检验前rRNA加工机制有序、循序渐进组装的假说，以阐明这些大分子在核糖体生物发生中的功能。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发生研究的实验策略，而且对这些无处不在的RNA-蛋白质复合体的结构和组装知之甚少。这一结果将具有重要意义，因为核糖体生物发生是基因表达的基本步骤，所有细胞，特别是生长细胞，都将大量新陈代谢投入其中。核糖体生物发生的失调与人类癌症有关，SSU过程组蛋白的突变与新生儿肝硬变、不孕症和神经纤维瘤病有关。 公共卫生相关性：这项建议旨在回答有关蛋白质和RNA-蛋白质复合体的功能和组织的重要问题，这些复合体与癌症和几种罕见的人类遗传病的发病机制有关。更彻底地了解这些过程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.