RNA tools for probing spliceosome dynamics

项目概要/摘要 选择性剪接是在转录后水平上使遗传信息多样化的重要机制。 测序技术的进展揭示了可变剪接模式的变化是多种基因的关键特征。 包括胚胎发育、适应性免疫反应和癌症在内的生物学相关系统 进展最近的一项RNAseq研究表明，数千种转录本的选择性剪接模式 在李斯特菌感染的巨噬细胞中发生了改变。虽然相关的蛋白质和机制尚未建立， 其结果可能是限制细胞内复制的保护性细胞反应。这个项目的中心目标是 一个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， 在单细胞水平上的可变剪接机制。有趣的是，独立研究发现， 剪接体成分在细胞分裂过程中被暂时隔离在称为U体的细胞溶质RNA蛋白颗粒中。 李斯特菌感染，这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体的形成在空间和时间上都是高度异质性的， 必须在单个细胞的基础上进行评估。荧光显微镜提供了长期的可能性， 标记蛋白质和荧光标记病原体的可视化，但是可视化细胞RNA的强大工具 是有限的。为了实现非编码RNA的可视化，一种用于荧光标记活细胞中RNA的通用工具 （目标1）。然后，该工具将用于量化U体的时空动态， 同时监测李斯特菌复制（目的2）。剪接体成分的贡献将被解剖 通过监测RNA动力学和李斯特菌复制作为剪接体组件将被操纵 实验性的最后，时间分辨定量质谱法将用于鉴定蛋白质 调节可变剪接景观的重塑的候选物（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 在显微镜分析中的细菌复制。总之，这项研究将作为一个独特的模型系统， 使用荧光在生理相关模型系统中在单细胞水平上的可变剪接调节 显微镜

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.