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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

基本信息

批准号：
10627902
负责人：
Matthew David Simon
金额：
$ 36.08万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10627902
关键词：
Affect Biochemical Biological Cells Chemistry Code Complement Complex Data Democracy 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 cell type experimental study flexibility fly human disease improved mRNA decapping posttranscriptional promoter response sequencing platform transcriptome sequencing

项目摘要

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.

项目摘要健康细胞和患病细胞中的细胞RNA水平是高度动态的，但大多数对RNA水平的分析都是动态的。 RNA浓度（RNA测序，RNA-seq）仅捕获细胞的静态快照，核糖核酸RNA水平在RNA代谢的不同步骤中受到控制，包括转录起始、转录暂停、RNA加工和RNA降解。虽然RNA-seq 提供了一种全面的方法，以确定RNA的上调或下调，响应生物扰动，需要专门的实验来确定哪些具体步骤 RNA代谢受到影响。然而，这些实验的专业性质限制了其广泛使用。因此，迫切需要开发一种灵活的实验一个平台，可以很容易地适应研究的动力学范围广泛的管制步骤在RNA代谢中。这项工作的总体目标是为RNA增加一个时间维度， seq，将其从静态终点测定转化为测量动力学的稳健技术并揭示了这些动力学受到调节和影响的不同方式，疾病该平台基于代谢标记和核苷酸化学的改进来研究不同时间范围内的RNA动力学。这些方法的全部潜力将是一旦它们被整合，就可以实现一个强大而统一的平台来检查RNA代谢在一系列时间尺度（分钟到天）和转录物大小（miRNA到长mRNA）上。要求1 是确定最佳的方法，以全面区分RNA合成的变化， RNA稳定性这些实验的统计功效将被系统地检验，定义最优实验设计。RNA合成和降解的差异与野生型相比，在缺乏促进mRNA脱帽的酶的细胞中测量类型细胞。这种酶的直接靶点预期是转录后稳定的但在转录上没有变化。Aim 2将扩展该系统以测量短 RNA，包括靶向miRNA降解。目标3包括测量瞬时RNA表达和RNA加工，以及这些方法的整合，研究p53肿瘤抑制通路。这些目标的成功实现将建立核苷酸编码化学作为一个通用平台，以揭示不同的RNA动力学基因表达水平。