Molecular mechanism of RNA polymerase I transcription and dysregulation in cancer

癌症中RNA聚合酶I转录和失调的分子机制

• 批准号: 8678333
• 负责人: Bruce Alan Knutson
• 金额: $ 21.15万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-17 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8678333
• 关键词: Accounting; Architecture; Biochemical; Biochemical Genetics; Biochemistry; Biogenesis; Bioinformatics; Biological Assay; Cancer Etiology; Cell Culture Techniques; Cell Nucleolus; Cell Proliferation; Cells; Characteristics; Chemicals; Complement; Complex; DNA Polymerase I; DNA Polymerase II; DNA Polymerase III; Defect; Development Plans; Disease; Educational workshop; Environment; Fibrinogen; Foundations; Funding; Future; Genetic; Genetic Transcription; Goals; Grant; Health; Hereditary Disease; Homologous Gene; Human; Hydroxyl Radical; In Vitro; Institution; Knowledge; Laboratories; Lead; Leadership; Link; Location; Malignant Neoplasms; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Methodology; Methods; Molecular; Molecular Genetics; Morphologic artifacts; Mutation; Normal Cell; Nuclear; Oncogenes; Peptide Hydrolases; Peptide Initiation Factors; Polymerase; Positioning Attribute; Production; Proliferating; Protein p53; Protein-Protein Interaction Map; Proteins; Proteomics; RNA; RNA Polymerase I; Regulation; Repression; Research; Research Personnel; Research Proposals; Ribosomal RNA; Ribosomes; Role; Sequence Analysis; Site; Structural Models; System; TATA-Box Binding Protein; Techniques; Technology; Testing; Therapeutic; Time; Training; Transcription Factor TFIIB; Transcription Initiation; Transcription Process; Translating; Tumor Suppressor Genes; Tumor Suppressor Proteins; Up-Regulation; Work; Writing; Yeast Model System; Yeasts; base; burden of illness; cancer cell; cancer proteomics; career; career development; cost; crosslink; human disease; in vivo; innovation; insight; interdisciplinary approach; loss of function mutation; meetings; novel; novel strategies; programs; promoter; protein crosslink; protein protein interaction; skills; therapeutic target; transcription factor

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.

描述（由申请方提供）：真核RNA聚合酶I（Pol I）转录核糖体RNA，核糖体的关键组分。Pol I转录占细胞中总RNA的大部分，并且其在人类细胞中的上调是癌症的标志，使其成为癌症的有吸引力的治疗靶点。在以下几个方面，我们的理解存在重大差距：（1）Pol I转录复合物的结构组织和架构，（2）Pol I转录起始的机制和Pol I起始因子的分子功能，以及（3）肿瘤抑制因子和癌基因对Pol I活性的调节。我的主要职业目标是成为美国顶级学术机构的独立调查员，建立一个成功的和资金充足的研究计划，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，通过参与资助写作，领导和实验室管理研讨会，以及参加转录，核糖体生物发生，蛋白质组学和癌症主题的科学会议，来扩展我的科学和领导技能。这项研究计划的长期目标是了解Pol I转录的机制以及它在癌症中的失调。其基本原理是，在最基本和基础水平上理解Pol I转录机制将转化为更好地理解Pol I与癌症之间的联系，从而产生新的癌症治疗策略。我提出的研究将使用概念和技术创新的跨生物体和跨学科的方法，采用生物信息学，计算，分子，生物化学，遗传，蛋白质组学和结构方法在酵母和人类系统的组合。我最近的突破和范式转变TFIIB相关的Pol I启动因子的发现代表了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，我将测试三个具体目标：（1）确定真核生物RNA聚合酶I前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，我将使用三个完善的和互补的方法来识别和映射新的Pol I在一个正常的preinitiation复杂的环境中的相互作用。这些方法包括组合化学交联和质谱法，位点特异性紫外光交联，和位点特异性羟基自由基蛋白酶足迹，是新的Pol I领域。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，遗传和生物化学测定的组合，以确定从酵母到人类保守的Pol I因子功能。这项研究意义重大，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。