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RNA Polymerase I Associated Factors: Novel Targets in Cancer Therapy

RNA 聚合酶 I 相关因素：癌症治疗的新靶点

基本信息

批准号：
10327609
负责人：
Rachel McNamar
金额：
$ 3.43万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10327609
关键词：
American Association of Cancer Research Auxins Binding Proteins Biochemical Biochemical Genetics Biogenesis Biological Biological Assay Biological Models C-terminal CRISPR/Cas technology Cell Cycle Arrest Cell Death Cell Proliferation Cell division Cell physiology Cells Chemicals Chemotherapy-Oncologic Procedure Communication Complex DNA DNA Binding DNA Binding Domain DNA Polymerase III DNA-Directed RNA Polymerase Data Development Dimerization Disease Educational process of instructing Educational workshop Essential Genes Exposure to Fellowship Future Genes Genetic Transcription Genetic study Heart Hypertrophy Heterodimerization Homeostasis In Vitro Investigation Knowledge Laboratories Malignant Neoplasms Mammalian Cell Manuscripts Maps Mass Spectrum Analysis Modality Morphology Normal Cell Nucleolar Proteins Pathology Pathway interactions Pharmaceutical Preparations Physiological Play Polymerase Process Protein Biosynthesis Publications Publishing RNA Polymerase I Regulation Research Ribosomal DNA Ribosomal RNA Ribosomes Role Scientist Stress Structure Supporting Cell System Tail Techniques Testing Training Transcription Initiation Work Writing Yeasts base cancer cell cancer therapy cell growth chromatin immunoprecipitation crosslink experience experimental study improved knock-down live cell imaging melting new therapeutic target novel protein protein interaction rRNA Genes rRNA Precursor recruit skills symposium therapeutic target transcription factor

项目摘要

PROJECT SUMMARY/ABSTRACT The regulation of ribosome biogenesis (RB) plays a central role in maintaining cellular homeostasis and supporting cell growth. The rate-limiting step in this process is transcription of the ribosomal RNA genes by RNA polymerase I (Pol I). Dysregulation of RB can contribute to pathologies such as cancer, cardiac hypertrophy, and ribosomopathies. Further, many chemotherapeutic drugs inhibit either rDNA transcription or rRNA processing, but have many off-target effects that limit their usefulness. Many pathways play a role in the regulation of rDNA transcription. Two mammalian factors that are involved in this regulation are Polymerase Associated Factor 53 (PAF53) and PAF49. The purpose of this study is to determine the role(s) of PAF49 and 53 in rDNA transcription and characterize the downstream physiological effects of directly inhibiting this process in both normal and cancer cells. This will be an important comparison since normal cells arrest when rDNA is inhibited while cancer cells die. To rapidly degrade PAF49/53, a novel system that utilizes CRISPR/Cas 9 and an auxin inducible degron will be used. This will allow us to carry out in vitro biochemical and “genetic” studies of PAF49/53 in mammalian cells. Our published data demonstrates that PAF53 is required for rDNA transcription and cell proliferation. In addition, the three domains of PAF53 are each necessary but not sufficient to support wild-type levels of cell growth. Our lab has also defined a second DNA-binding domain in PAF53 that had not been discovered. This study will expand upon the domain analysis of PAF53 and further characterize its DNA-binding activity. The work proposed is significant as it will aid in further understanding the process of rDNA transcription by Pol I and the physiological consequences of inhibiting this process, i.e. nucleolar stress and cell death. It will also contribute to the discovery of novel drug targets that could be utilized in effective cancer treatments. To complete the proposed research, I will be exposed to new techniques such as crosslinking mass spectrometry, live-cell imaging, and chromatin immunoprecipitation. To improve my scientific communication skills, I will attend and present at the OddPols, AACR and other national and local conferences, as well as attend workshops on scientific writing. I will also prepare multiple manuscripts for publication. Further, I will take multiple opportunities to gain teaching experience. I also will attend seminars and professional development workshops to help extend my scientific purview beyond my field. These opportunities will allow me to network and engage with fellow scientists in order to build connections for future collaborative projects. Overall, the training I will receive during this fellowship will help prepare me to be a competitive postdoctoral candidate and a successful independent research scientist.

项目总结/摘要核糖体生物合成（RB）的调节在维持细胞内稳态和维持细胞增殖中起着重要作用。支持细胞生长。在这个过程中的限速步骤是核糖体RNA基因的转录，聚合酶I（Pol I）。RB的调节异常可导致病理学，如癌症、心脏肥大和心肌梗死。核糖体病此外，许多化疗药物抑制rDNA转录或rRNA加工，但有许多脱靶效应限制了它们的有用性。许多途径在rDNA的调控中起作用转录。两种哺乳动物因子参与了这种调节，它们是聚合酶相关因子53 （PAF 53）和PAF 49。本研究的目的是确定PAF 49和53在rDNA转录中的作用并表征了在正常和非正常细胞中直接抑制该过程的下游生理效应，癌细胞这将是一个重要的比较，因为当rDNA被抑制时，正常细胞会停止，而癌症细胞会停止。细胞死亡。为了快速降解PAF 49/53，将开发一种利用CRISPR/Cas 9和生长素诱导的降解决定子的新系统。采用这将使我们能够在哺乳动物细胞中进行PAF 49/53的体外生物化学和“遗传”研究。我们发表的数据表明，PAF 53是rDNA转录和细胞增殖所必需的。此外，本发明还提供了一种方法， PAF 53的三个结构域都是必需的，但不足以支持野生型水平的细胞生长。我们实验室还确定了PAF 53中尚未发现的第二个DNA结合结构域。本研究将扩展了PAF 53的结构域分析，并进一步表征其DNA结合活性。工作提出的这将有助于进一步理解Pol I的rDNA转录过程和生理学过程，抑制这一过程的后果，即核仁应激和细胞死亡。这也将有助于发现新的药物靶点，可以用于有效的癌症治疗。为了完成拟议的研究，我将接触到新的技术，如交联质量光谱法、活细胞成像和染色质免疫沉淀。为了提高我的科学交流能力技能，我将出席并出席OddPos，AACR和其他国家和地方会议，以及出席科学写作研讨会。我还将准备多个手稿出版。此外，我将采取多个获得教学经验的机会。我还将参加研讨会和专业发展讲习班帮助我扩展我的科学视野这些机会将使我能够建立网络并参与其中与其他科学家合作，为未来的合作项目建立联系。总的来说，培训我会在这个奖学金期间收到将帮助我准备成为一个有竞争力的博士后候选人和一个成功的独立研究科学家。