Molecular mechanism of RNA polymerase I transcription and dysregulation in cancer

描述（由申请人提供）：真核RNA聚合酶I （Pol I）转录核糖体RNA，核糖体的关键成分。Pol I转录占细胞总RNA的大部分，其在人类细胞中的上调是癌症的一个标志，使其成为一个有吸引力的癌症治疗靶点。目前我们对Pol I转录复合物的结构组织和结构，Pol I转录起始机制和Pol I起始因子的分子功能，以及肿瘤抑制因子和癌基因对Pol I活性的调控等方面的认识还存在较大的空白。我的主要职业目标是成为美国顶尖学术机构的独立研究员，建立一个成功且资金充足的研究项目，并成为Pol I和转录领域的领导者。为了实现这些目标，我将遵循职业发展计划，通过参与拨款写作、领导能力和实验室管理研讨会，以及参加有关转录、核糖体生物发生、蛋白质组学和癌症等主题的科学会议，扩大我的科学和领导技能。本研究计划的长期目标是了解Pol I转录的机制及其在癌症中的失调。其基本原理是，在最基本和最基本的水平上理解Pol I转录机制将有助于更好地理解Pol I与癌症之间的联系，从而导致新的癌症治疗策略。我提议的研究将使用一种概念上和技术上创新的跨有机体和跨学科的方法，结合生物信息学、计算、分子、生化、遗传、蛋白质组学和酵母和人类系统中的结构方法。我最近的突破和范式转换的发现与tfiib相关的Pol I启动因子代表了Pol I领域现状的重大和实质性的偏离。在这些初步研究的指导下，我将测试三个特定目标：(1)确定真核RNA聚合酶I预起始复合物的分子结构，(2)确定Pol I特异性起始因子的分子功能，(3)确定肿瘤抑制因子p53下调Pol I转录的机制。为了实现这些目标，我将使用三种完善的互补方法来识别和绘制正常起始前复杂环境下的新型Pol I相互作用。这些方法包括化学交联和质谱联用、位点特异性紫外线光交联和位点特异性羟基自由基蛋白酶足迹，这些都是Pol I领域的新方法。为了补充这些研究并测试所观察到的相互作用的功能相关性，我将使用结构建模和分子，遗传和生化分析的组合来确定从酵母到人类保守的Pol I因子功能。这项研究具有重要意义，因为它将导致对Pol I转录机制的详细描述，并为理解Pol I与癌症之间的联系提供一个概念框架。最终，这项工作将阐明可用于靶向治疗的Pol I转录的关键步骤。

DESCRIPTION (provided by applicant): Eukaryotic RNA polymerase I (Pol I) transcribes ribosomal RNA, key components of ribosomes. Pol I transcription accounts for the majority of the total RNA in cells, and its upregulation in human cells is a hallmark of cancer, making it an attractive therapeutic target for cancer. There are major gaps in our understanding of (1) the structural organization and architecture of Pol I transcription complexes, (2) the mechanism of Pol I transcription initiation and the molecular function of Pol I initiation factors, and (3) the regulation of Pol I activity by tumor suppressors and oncogenes. My primary career goals are to become an independent investigator at a top academic institution in the US, to establish a successful and well-funded research program, and to become a leader in the Pol I and transcription fields. To accomplish these goals, I will follow a career development plan that will expand my scientific and leadership skills through participation in grant writing, leadership, and laboratory management workshops, and by attending scientific meetings with topics on transcription, ribosome biogenesis, proteomics, and cancer. The long-term objective of this research proposal is to understand the mechanism of Pol I transcription and how it is dysregulated in cancer. The rationale is that understanding the Pol I transcription mechanism at the most basic and fundamental levels will translate to a better understanding of the connection between Pol I and cancer, leading to new cancer therapeutic strategies. My proposed research will use a conceptually and technically innovative cross-organismal and interdisciplinary approach that employs a combination of bioinformatics, computational, molecular, biochemical, genetic, proteomic, and structural methods in the yeast and human systems. My recent breakthrough and paradigm shifting discovery of a TFIIB-related Pol I initiation factor represents a significant and substantive departure from the status quo in the Pol I field. Guided by these preliminary studies, I will test three specific aims: (1) Determine the molecular architecture of te eukaryotic RNA polymerase I preinitiation complex, (2) Determine the molecular functions of the Pol I-specific initiation factors, and (3) Determine the mechanism by which the tumor suppressor p53 downregulates Pol I transcription. To accomplish these aims, I will use three well-established and complementary approaches to identify and map novel Pol I interactions in the context of a normal preinitiation complex environment. These methods include combined chemical crosslinking and mass spectrometry, site-specific UV-photocrosslinking, and site-specific hydroxyl radical protease footprinting that are new to the Pol I field. To complement these studies and to test the functional relevance of the observed interactions, I will use structural modeling and a combination of molecular, genetic, and biochemical assays to identify Pol I factor functions conserved from yeast to human. The proposed research is significant because it will lead to a detailed description of the Pol I transcription mechanism and provides a conceptual framework for understanding the link between Pol I and cancer. Ultimately, this work will illuminate key steps in Pol I transcription that can be used in targeted therapies.