The development and application of tools to characterize the level and function of RNA polymerase III transcription dynamics during cellular differentiation

表征细胞分化过程中 RNA 聚合酶 III 转录动力学水平和功能的工具的开发和应用

• 批准号: 10505936
• 负责人: Kevin Van Bortle
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-11-24 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10505936
• 关键词: 7SL RNA; Accounting; Address; Bioinformatics; Biological Assay; Biology; Biotin; Breast Cancer Cell; Cardiac; Cardiac Myocytes; Cardiac development; Cell physiology; Chemicals; Communities; Computational Biology; Copy Number Polymorphism; DNA; Data; Detection; Development; Disease; Down-Regulation; ERBB2 gene; Feasibility Studies; Funding; Future; Gene Dosage; Gene Expression Profile; Gene Structure; Genes; Genetic; Genetic Transcription; Genomics; Goals; Health; Heart Diseases; Heart Hypertrophy; Heart failure; Human; Hypoxia; In Vitro; Maps; Methodology; Methods; Myocardial Ischemia; Nerve Degeneration; Nuclear; Oxidative Stress; Parasites; Patients; Pattern; Phase; Pilot Projects; Play; RNA; RNA Polymerase III; RNA Splicing; RNA-Binding Proteins; Regulation; Research; Research Personnel; Ribosomal RNA; Role; Running; Sampling; Sequence Alignment; Small RNA; Structure; System; Testing; Tissue Sample; Tissues; Training; Transcriptional Regulation; Transfer RNA; Translations; Untranslated RNA; Ursidae Family; Variant; Viral; base; brain tissue; cardiogenesis; cardioprotection; cell type; experimental study; follow-up; gene correction; genome sequencing; genome-wide; genomic tools; human disease; human stem cells; improved; infected B cell; insight; interest; knock-down; macrophage; mouse model; novel; overexpression; programs; response; skills; statistics; stem cells; success; tool; transcriptome sequencing; tumor; whole genome

RNA polymerase III (RNAPIII) transcribes short, highly structured non-coding RNAs involved in diverse cellular processes, including translation, transcription regulation, and splicing. The biomedical relevance of RNAPIII activity in humans is wide-ranging: tRNA, 5S rRNA, vault RNA, 7SL RNA, and small NF90-associated RNA (snaR) have been shown to be either elevated or depleted in tumors, neurodegenerative brain tissues, viral- infected B cells, parasite-infected macrophages, and HER2-positive breast cancer cells, respectively. While these and other examples highlight aberrant activities in distinct health-related contexts, a growing body of evidence suggests that multiple RNAPIII-transcribed gene subclasses play important roles in the context of heart development and disease: tRNA and other small RNA levels are altered during early stages of cardiac differentiation and depleted in response to hypoxia, loss of 7SK RNA is sufficient to induce cardiac hypertrophy, RMRP RNA is elevated in mouse models of cardiac hypertrophy and in patients with ischemic heart-failure, and RNAPIII-transcribed Y RNA confers cardioprotection to oxidatively stressed cardiomyocytes. Despite these findings, little is currently known about the functional role of dynamic non-coding RNA levels within these contexts, or whether differences in RNA levels are driven by RNAPIII transcription due in part to several unique challenges related to the sequencing and alignment of short, highly structured and repetitive RNAs. The proposed study seeks to address these deficiencies by developing new high-throughput genomic methods for profiling RNAPIII transcription and applying these strategies in the context of stem cell-to-cardiomyocyte differentiation. In addition, functional experiments will test the role of dynamic RNAPIII patterns within these contexts, and a genetic-background correction method will be developed to enable future comparisons of RNAPIII transcription across diverse cellular contexts, accounting for differences in copy number variation (CNV) in samples of non-identical genomic origin. The proposed study is the next logical step in my progression to becoming an independently funded investigator with an active and successful research program identifying the role and underlying regulatory mechanisms of RNAPIII transcription within diverse cellular contexts and human disease. This project will provide critical training in human stem cell and cardiac biology, CNV detection methodology, and computational biology and statistics, critical skills necessary to establish my research program and accomplish my long-term goals as an independent group leader. Together, results of this study promise to establish improved genomic tools of significant interest to the scientific community, and to yield important insight on the role and regulation of RNAPIII-transcribed genes during cellular differentiation.

RNA 聚合酶 III (RNAPIII) 转录短的、高度结构化的非编码 RNA，参与多种细胞 过程，包括翻译、转录调控和剪接。 RNAPIII 的生物医学相关性 在人类中的活性范围广泛：tRNA、5S rRNA、vault RNA、7SL RNA 和小 NF90 相关 RNA (snaR) 已被证明在肿瘤、神经退行性脑组织、病毒- 分别是感染的 B 细胞、寄生虫感染的巨噬细胞和 HER2 阳性乳腺癌细胞。虽然这些 和其他例子强调了在不同的健康相关背景下的异常活动，越来越多的证据 表明多个 RNAPIII 转录基因亚类在心脏疾病中发挥重要作用 发育和疾病：tRNA 和其他小 RNA 水平在心脏疾病的早期阶段发生改变 分化并因缺氧而耗尽，7SK RNA 的损失足以诱导心脏肥大， RMRP RNA 在心脏肥大小鼠模型和缺血性心力衰竭患者中升高，并且 RNAPIII 转录的 Y RNA 为氧化应激的心肌细胞提供心脏保护作用。尽管有这些 研究结果表明，目前对于动态非编码 RNA 水平在这些基因中的功能作用知之甚少。 RNA 水平的差异是否是由 RNAPIII 转录驱动的，部分原因是几个独特的 与短、高度结构化和重复 RNA 的测序和比对相关的挑战。这 拟议的研究旨在通过开发新的高通量基因组方法来解决这些缺陷 分析 RNAPIII 转录并在干细胞到心肌细胞的背景下应用这些策略 差异化。此外，功能实验将测试动态 RNAPIII 模式在这些中的作用。 将开发一种遗传背景校正方法，以便将来能够进行比较 RNAPIII 跨不同细胞环境的转录，解释了拷贝数变异的差异 (CNV) 存在于不同基因组来源的样本中。拟议的研究是我进展中的下一个合乎逻辑的步骤 成为一名独立资助的研究人员，拥有积极且成功的研究计划，确定 RNAPIII 转录在不同细胞环境中的作用和潜在调控机制 人类疾病。该项目将提供人类干细胞和心脏生物学、CNV 检测方面的关键培训 方法论、计算生物学和统计学，以及建立我的研究计划所需的关键技能 并实现我作为独立小组领导者的长期目标。总之，这项研究的结果有望 建立科学界感兴趣的改进基因组工具，并产生重要的成果 深入了解 RNAPIII 转录基因在细胞分化过程中的作用和调节。