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.

项目概要/摘要核糖体生物发生 (RB) 的调节在维持细胞稳态和支持细胞生长的限速步骤是RNA对核糖体RNA基因的转录。聚合酶 I (Pol I) 的失调可导致癌症、心脏肥大等疾病。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用途。许多途径在 rDNA 的调节中发挥作用。参与这种调节的两个哺乳动物因子是聚合酶相关因子 53。 (PAF53) 和 PAF49 本研究的目的是确定 PAF49 和 53 在 rDNA 转录中的作用。并描述了正常和正常情况下直接抑制这一过程的下游生理效应。这将是一个重要的比较，因为当 rDNA 被抑制时，正常细胞就会停滞，而癌症则被抑制。细胞死亡。为了快速降解 PAF49/53，将开发一种利用 CRISPR/Cas 9 和生长素诱导降解子的新系统这将使我们能够在哺乳动物细胞中进行 PAF49/53 的体外生化和“遗传”研究。我们发表的数据表明，PAF53 是 rDNA 转录和细胞增殖所必需的。 PAF53 的三个结构域对于支持野生型细胞生长水平都是必需的，但不足以支持。实验室还在 PAF53 中定义了本研究尚未发现的第二个 DNA 结合域。扩展 PAF53 的结构域分析并进一步表征其 DNA 结合活性。意义重大，因为它将有助于进一步了解 Pol I 的 rDNA 转录过程和生理学抑制这一过程的后果，即核仁应激和细胞死亡，也将有助于这一发现。可用于有效癌症治疗的新药物靶点。为了完成拟议的研究，我将接触新技术，例如交联质量光谱分析、活细胞成像和染色质免疫沉淀以改善我的科学交流。技能，我将参加 OddPols、AACR 和其他国家和地方会议并在会上发言，以及参加我还将准备多篇关于科学写作的研讨会，此外，我还将拍摄多篇手稿。我还将参加研讨会和专业发展研讨会以获得教学经验的机会。帮助将我的科学视野扩展到我的领域之外。这些机会将使我能够建立人际网络并参与其中。总体而言，我将与其他科学家一起进行培训，以便为未来的合作项目建立联系。在此奖学金期间获得的奖学金将有助于我成为一名有竞争力的博士后候选人和成功的博士后候选人独立研究科学家。