Revealing the dynamics of RNA metabolism with nucleotide recoding chemistry

通过核苷酸重新编码化学揭示 RNA 代谢的动态

• 批准号: 10200846
• 负责人: Matthew David Simon
• 金额: $ 36.09万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200846
• 关键词: Affect; Biochemical; Biological; Cells; Chemistry; Complement; Complex; Data; Development; Diagnosis; Dimensions; Disease; Disulfides; End Point Assay; Enzymes; Experimental Designs; Gene Expression; Genetic Transcription; Hydrogen Bonding; Individual; Kinetics; Knock-out; Knowledge; Label; Measurement; Measures; Messenger RNA; Metabolic; Methods; MicroRNAs; Mission; Monitor; Mutation; Nature; Nucleotides; Pathway interactions; Population; Population Dynamics; Protocols documentation; Public Health; RNA; RNA Degradation; RNA Polymerase II; RNA Processing; RNA Splicing; RNA Stability; RNA analysis; RNA chemical synthesis; RNA metabolism; Regulation; Reproducibility; Research; Site; Small RNA; System; TP53 gene; Techniques; Thiouridine; Transcript; Transcription Initiation; United States National Institutes of Health; Untranslated RNA; Viral; Work; analog; base; cell type; experimental study; flexibility; fly; human disease; improved; mRNA decapping; promoter; response; sequencing platform; transcriptome sequencing

PROJECT SUMMARY Cellular RNA levels in both healthy and diseased cells are highly dynamic, yet most analyses of RNA concentrations (RNA-sequencing, RNA-seq) capture only a static snapshot of cellular RNA. RNA levels are controlled at diverse steps in RNA metabolism including transcriptional initiation, transcriptional pausing, RNA processing, and RNA degradation. While RNA-seq provides a global method to identify RNAs that are up or down-regulated in response to biological perturbations, specialized experiments are required to determine which specific steps of RNA metabolism are affected. The specialized nature of these experiments, however, limits their wide-spread use. Thus, there is a pressing need to develop a flexible experimental platform that can be easily adapted to study the kinetics of a broad range of the regulated steps in RNA metabolism. The overall objective of this work is to add a temporal dimension to RNA- seq, transforming it from a static endpoint assay into a robust technique to measure the kinetics of RNA metabolism and reveal the diverse ways these kinetics are regulated and impacted by disease. This platform is based on metabolic labeling and improvements in nucleotide chemistry to study RNA dynamics at a range of timescales. The full potential of these approaches will be realized once they are integrated a robust and unified platform to examine RNA metabolism across a range of timescales (min to days), and transcript sizes (miRNA to long mRNA). Aim 1 is to determine optimal ways to globally distinguish changes in RNA synthesis from changes in RNA stability. The statistical power of these experiments will be systematically examined to define optimal experimental designs. Differences in RNA synthesis and degradation will be measured in cell lacking an enzyme that promotes mRNA decapping in comparison with wild type cells. The direct targets of this enzyme are expected to be post-transcriptionally stabilized but unchanged transcriptionally. Aim 2 will extend this system to measure the dynamics of short RNAs, including target directed miRNA degradation. Aim 3 includes the measurement the transient RNA expression and RNA processing, and the integration of these approaches to study the p53 tumor suppressor pathway. Successful completion of these aims will establish nucleotide recoding chemistry as a general platform to reveal RNA dynamics across distinct levels of gene expression.

项目总结 健康细胞和疾病细胞中的细胞RNA水平都是高度动态的，但大多数对 RNA浓度(RNA测序、RNA序列)仅捕获细胞的静态快照 核糖核酸。RNA水平在RNA新陈代谢的不同步骤中受到控制，包括转录 启动、转录暂停、RNA处理和RNA降解。而RNA-seq 提供一种全局方法来识别上调或下调的RNA 生物扰动，需要专门的实验来确定哪些具体步骤 核糖核酸新陈代谢受到影响。然而，这些实验的专业性限制了 它们的广泛使用。因此，迫切需要开发一种灵活的实验 可容易地改装成研究大范围调节步骤的动力学的平台 在RNA新陈代谢中。这项工作的总体目标是为RNA增加一个时间维度- SEQ，将其从静态终点分析转变为一种稳健的动力学测量技术 并揭示了这些动力学被调节和影响的不同方式 疾病。这个平台是基于代谢标记和核苷酸化学的改进 研究一系列时间尺度上的RNA动力学。这些方法的全部潜力将是 一旦它们集成在一起，就实现了一个强大而统一的平台来检查RNA新陈代谢 跨越一系列时间尺度(从分钟到天数)和转录本大小(从miRNA到长mRNA)。目标1 是确定在全球范围内区分RNA合成的变化和 RNA的稳定性。这些实验的统计能力将被系统地检验以 确定最优试验设计。在RNA合成和降解方面的差异将是 与野生型相比，在缺乏促进信使核糖核酸降解的酶的细胞中测量 键入单元格。这种酶的直接靶标有望在转录后稳定下来。 但转录上没有变化。目标2将扩展该系统以测量空头的动态 RNA，包括靶向的miRNA降解。目标3包括测量 瞬时RNA表达和RNA加工，以及这些方法的集成 研究P53抑癌基因通路。这些目标的成功实现将确立 核苷酸重新编码化学作为揭示不同类型的RNA动态的通用平台 基因表达水平。