RNA tools for probing spliceosome dynamics

用于探测剪接体动力学的 RNA 工具

基本信息

批准号：
10540416
负责人：
Esther Braselmann
金额：
$ 24.31万
依托单位：
GEORGETOWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-13 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10540416
关键词：
Affect Alternative Splicing Bacterial Infections Benchmarking Binding 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 Goals Homeostasis Infection Investigation Label Listeria Listeriosis Macrophage 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 candidate identification crosslink design fluorophore genetic information genetic regulatory protein insight knock-down mRNA Precursor novel overexpression pathogen posttranscriptional response small molecule spatiotemporal success tool transcriptome transcriptome sequencing tumor progression

项目摘要

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。