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.

项目摘要/摘要 替代剪接是在转录后水平上多样化遗传信息的中心机制。 测序技术的进步揭示了替代剪接模式的转变，作为多样性的关键特征 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RNASEQ研究表明，数千个转录本的替代剪接模式 在感染李斯特菌的巨噬细胞中改变。虽然涉及的蛋白质和机制尚未确定，但 保护性细胞对限制细胞内复制的反应可能是结果。这个中心目标 建议是使用此感染模型系统来了解非编码RNA和 单个细胞水平上替代剪接的机制。有趣的是，它是独立发现的 剪接体成分在称为u-Bodies的胞质RNA-蛋白质颗粒中瞬时隔离 李斯特菌感染，表明时空隔离可能有助于替代剪接调节。 李斯特菌感染和U体的形成在空间和时间上都是高度异质的，理想情况下 必须以单细胞为基础进行评估。荧光显微镜为长期提供了可能性 可视化标记的蛋白质和荧光标记的病原体，但可视化细胞RNA的强大工具 有限。为了可视化非编码RNA，这是一种多功能工具，可在活细胞中标记RNA 将开发（目标1）。然后，该工具将用于量化U-Bodies的时空动力学和 同时监视李斯特菌复制（AIM 2）。将阐述剪接体组件的贡献 通过监视RNA动力学和李斯特菌复制作为剪接组成分 实验。最后，将使用时间分辨的定量质谱法来鉴定蛋白质 调节替代剪接景观的重塑的候选人（AIM 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进一步研究 显微镜测定中的细菌复制。这项研究将成为揭开独特的模型系统 使用荧光的生理相关模型系统中单细胞水平上的替代剪接调节 显微镜。