RNA tools for probing spliceosome dynamics

项目摘要/摘要 选择性剪接是在转录后水平上使遗传信息多样化的一种核心机制。 测序技术的进步揭示了选择性剪接模式的变化是各种 生物相关系统，包括胚胎发育、适应性免疫反应和癌症 进步。最近的一项RNAseq研究表明，数千个转录本的选择性剪接模式 在感染李斯特菌的巨噬细胞中发生改变。虽然涉及的蛋白质和机制还没有建立起来， 结果可能是限制细胞内复制的保护性细胞反应。这样做的中心目标是 建议使用这种感染模型系统来深入了解非编码RNA和 单细胞水平上的选择性剪接机制。耐人寻味的是，独立发现 剪接体成分在细胞质RNA-蛋白质颗粒中暂时隔离，称为U-小体。 李斯特菌感染，提示时空隔离可能有助于选择性剪接调控。 李斯特菌的感染和U形体的形成在空间和时间上都是高度异质性的，理想的情况是 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 标记的蛋白质和荧光标记的病原体的可视化，但用于可视化细胞RNA的强大工具 都是有限的。为了实现非编码RNA的可视化，这是一种对活细胞中的RNA进行荧光标记的通用工具 将被开发(目标1)。然后这个工具将被用来量化U形体的时空动力学和 同时监测李斯特菌复制(目标2)。剪接体组件的贡献将被剖析 通过监测RNA动态和李斯特菌复制作为剪接体组件将被操纵 试验性的。最后，将使用时间分辨的定量质谱学方法来鉴定蛋白质。 规范替代拼接格局重塑的候选方案(目标3)。这些候选因素将 通过击倒和评估对U形体动力学和细胞内的影响来进一步研究 显微镜检测中的细菌复制。总而言之，这项研究将作为一个独特的模型系统来解开 在生理相关模型系统中使用荧光在单个细胞水平上的选择性剪接调节 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.