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

RNA聚合酶III(RNAPIII)转录短的、高度结构的非编码RNA，涉及不同的细胞 过程，包括翻译、转录调控和剪接。RNAPIII的生物医学相关性 人类的活性是广泛的：tRNA、5S rRNA、vault RNA、7SL RNA和与NF90相关的小RNA (SNAR)已被证明在肿瘤、神经退行性脑组织、病毒- 感染的B细胞、寄生虫感染的巨噬细胞和HER2阳性的乳腺癌细胞。而这些 其他例子突出了在与健康相关的不同背景下的异常活动，越来越多的证据 提示RNAPIII转录的多个基因亚类在心脏环境中发挥重要作用 发育和疾病：tRNA和其他小RNA水平在心脏早期阶段发生变化 分化和耗尽对低氧的反应，7SK RNA的丢失足以诱导心肌肥厚， RMRP RNA在心肌肥厚的小鼠模型和缺血性心力衰竭患者中升高，以及 RNAPIII转录的Y RNA为氧化应激心肌细胞提供心脏保护。尽管如此 发现，目前对动态非编码RNA水平在这些基因中的作用知之甚少 或者RNA水平的差异是否由RNAPIII转录驱动，部分原因是几个独特的 与短的、高度结构化和重复的RNA的测序和比对有关的挑战。这个 拟议的研究试图通过开发新的高通量基因组方法来解决这些不足 在干细胞到心肌细胞的背景下分析RNAPIII转录和应用这些策略 差异化。此外，功能实验将测试动态RNAPIII模式在这些 背景，并将开发一种遗传-背景校正方法，以使未来能够对 RNAPIII在不同细胞环境中的转录，解释了拷贝数变异的差异 (CNV)在不同基因组来源的样本中。拟议的研究是我进步的下一个合乎逻辑的步骤 成为一名独立资助的调查员，拥有一项积极而成功的研究计划， RNAPIII转录在不同细胞环境中的作用和潜在的调控机制 人类疾病。该项目将在人类干细胞和心脏生物学、CNV检测方面提供关键培训 方法论，以及计算生物学和统计学，这些都是建立我的研究计划所必需的关键技能 并完成我作为独立组长的长期目标。总而言之，这项研究的结果有望 建立科学界非常感兴趣的改进的基因组工具，并产生重要的 洞察RNAPIII转录基因在细胞分化中的作用和调控。

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.