RNA tools for probing spliceosome dynamics

用于探测剪接体动力学的 RNA 工具

• 批准号: 10222446
• 负责人: Esther Braselmann
• 金额: $ 2.01万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10222446
• 关键词: Affect; Alternative Splicing; Bacterial Infections; Benchmarking; Binding; Biological; Biological Assay; Biological Models; Cell Nucleus; Cells; Cellular Assay; Cobalamin; Code; Complex; Coupled; Cues; Cytoplasmic Granules; Data; Disease; Embryonic Development; Fluorescence; Fluorescence Microscopy; Gene Expression Regulation; Genes; Genetic Materials; Genetic Transcription; Goals; Homeostasis; Infection; Investigation; Label; Listeria; Listeriosis; Mammalian Cell; Mass Spectrum Analysis; Measures; Mentors; Microscopy; Monitor; Mutation; Outcome; Pathway interactions; Pattern; Phase; Phenotype; Physiological; Process; Property; Protein Isoforms; Proteins; RNA; RNA Splicing; Regulation; Reporting; Resolution; Shapes; Small RNA; Spliceosomes; System; Technology; Time; Transcript; Untranslated RNA; Visualization; adaptive immune response; aptamer; crosslink; fluorophore; genetic information; genetic regulatory protein; insight; knock-down; mRNA Precursor; macrophage; novel; overexpression; pathogen; response; small molecule; spatiotemporal; success; tool; transcriptome; transcriptome sequencing; tumor progression

Project Summary/Abstract Alternative splicing is a central mechanism to diversify genetic information on the post-transcriptional level. Advances in sequencing technologies revealed shifts in alternative splicing patterns as key features in a variety of biologically relevant systems including embryo development, the adaptive immune response and cancer progression. A recent RNAseq study demonstrated that alternative splicing patterns for thousands of transcripts are altered in macrophages infected with Listeria. While proteins and mechanisms involved are not established, a protective cellular response to limit intracellular replication may be a consequence. The central goal of this proposal is to use this infection model system to gain insights into dynamics of non-coding RNAs and mechanisms of alternative splicing on a single cell level. Intriguingly, it was independently discovered that spliceosome components are transiently sequestered in cytosolic RNA-protein granules called U-bodies during Listeria infection, suggesting that spatiotemporal sequestration may contribute to alternative splicing regulation. Infection with Listeria and formation of U-bodies are highly heterogeneous both in space and time and ideally must be assessed on a single-cell basis. Fluorescence microscopy offers the possibility for long-term visualization of tagged proteins and fluorescently labeled pathogens, but robust tools to visualize cellular RNAs are limiting. To enable visualization of non-coding RNAs, a versatile tool to fluorescently label RNA in live cells will be developed (Aim 1). This tool will then be utilized to quantify spatiotemporal dynamics of U-bodies and simultaneously monitor Listeria replication (Aim 2). Contributions of spliceosome components will be dissected by monitoring RNA dynamics and Listeria replication as spliceosome components will be manipulated experimentally. Lastly, a time resolved quantitative mass spectrometry approach will be used to identify protein candidates that regulate re-shaping of the alternative splicing landscape (Aim 3). These candidate factors will be further investigated by knockdown and assessing consequences for U-body dynamics and intracellular bacterial replication in the microscopy assay. Together, this study will serve as a unique model system to unravel alternative splicing regulation on a single cell level in a physiologically relevant model system using fluorescence microscopy.

项目概要/摘要 选择性剪接是转录后水平上遗传信息多样化的核心机制。 测序技术的进步揭示了选择性剪接模式的转变，这是多种疾病的关键特征 生物学相关系统，包括胚胎发育、适应性免疫反应和癌症 进展。最近的一项 RNAseq 研究表明，数千个转录本的选择性剪接模式 感染李斯特菌的巨噬细胞发生改变。虽然所涉及的蛋白质和机制尚未确定， 限制细胞内复制的保护性细胞反应可能是结果。此次活动的中心目标 提议是使用这个感染模型系统来深入了解非编码 RNA 的动态和 单细胞水平上的选择性剪接机制。有趣的是，独立发现 剪接体成分在称为 U 体的胞质 RNA 蛋白颗粒中短暂隔离 李斯特菌感染，表明时空隔离可能有助于选择性剪接调节。 李斯特菌感染和 U 体形成在空间和时间上都具有高度异质性，理想情况下 必须在单细胞的基础上进行评估。荧光显微镜提供了长期研究的可能性 标记蛋白质和荧光标记病原体的可视化，以及可视化细胞 RNA 的强大工具 是有限的。为了实现非编码 RNA 的可视化，这是一种荧光标记活细胞中 RNA 的多功能工具 将得到发展（目标 1）。然后，该工具将用于量化 U 体的时空动力学和 同时监测李斯特菌复制（目标 2）。剪接体成分的贡献将被剖析 通过监测 RNA 动力学和李斯特菌复制，因为剪接体成分将被操纵 实验性地。最后，时间分辨定量质谱方法将用于鉴定蛋白质 调节替代剪接景观重塑的候选者（目标 3）。这些候选因素将 通过敲低和评估 U 体动力学和细胞内的后果来进一步研究 显微镜检测中的细菌复制。总之，这项研究将作为一个独特的模型系统来揭示 使用荧光在生理相关模型系统中对单细胞水平进行选择性剪接调节 显微镜。