Illuminating multiplexed RNA dynamics to interrogate splicing in health and disease

阐明多重 RNA 动力学以探究健康和疾病中的剪接

• 批准号: 10713923
• 负责人: Esther Braselmann
• 金额: $ 28.62万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10713923
• 关键词: Affect; Alternative Splicing; Biological; Biological Models; Body Composition; Cell Nucleus; Cell model; Cell physiology; Cells; Complex; Cues; Cytoplasmic Granules; DNA Sequence Alteration; Disease; Disease Management; Fluorescence; Fluorescence Microscopy; Foundations; Future; Gene Expression; Gene Expression Regulation; Goals; Health; Human; Imaging Device; Introns; Investigation; Joining Exons; Label; Link; Malignant Neoplasms; Mammalian Cell; Messenger RNA; Outcome; Play; Process; Proteins; RNA; RNA Splicing; Reaction; Regulation; Research; Research Personnel; Role; Small Nuclear RNA; Spliceosomes; System; Time; Untranslated RNA; Visualization; Work; environmental stressor; fluorescence lifetime imaging; human disease; imaging modality; insight; interest; nervous system disorder; programs; spatiotemporal; tool

Ribonucleic acids (RNAs) play key roles in numerous cellular processes. A classic example is alternative splicing, where the large megadalton spliceosome complex removes intron regions from the pre-messenger RNA (pre-mRNA) and re-joins the exons to form the mature mRNA in the nucleus. The spliceosome consists of protein and non-coding RNA components. Its assembly includes intricate maturation steps that are highly regulated to ensure that the correct mature mRNA molecules are produced at the right time in healthy cells. Achieving correct splicing of all mRNAs is subject to intense regulation, requiring a sophisticated interplay of cellular cues and spatiotemporal dynamics of splicing components. Genetic mutations or environmental stressors are perturbations that may affect the splicing process and outcomes. These are linked to human disease states like cancer and neurological diseases. Together, the central role of complex spatiotemporal RNA dynamics for proper splicing calls for the need to interrogate diverse RNAs live on a subcellular level over time. The complexity of RNA species involved in splicing requires robust and versatile labeling strategies to visualize multiple RNA molecules simultaneously and relative to other biological molecules of interest. A key goal of this research program is to develop such a robust toolbox for multiplexed RNA visualization using advanced fluorescence microscopy. Fluorescence lifetime imaging microscopy (FLIM) emerges as a particularly versatile approach, as it is compatible with adding sophisticated imaging modalities. A central feature that will be included in the proposed work is the ability to visualize multiple RNAs simultaneously, including small non-coding RNAs with roles in splicing. More broadly, these RNA imaging tools will allow researchers across different fields to investigate RNAs in a variety of relevant cell model systems. Alternative splicing has been linked to formation of a type of cytosolic RNA-protein granules, called U-bodies. Spliceosome RNAs (called U snRNAs) are the defining components of U-bodies, along with several proteins that are implicated in the splicing reaction. U-bodies were observed across different cellular models, pointing to a central role in gene regulation, but details about their precise composition and function remain elusive. This research program will combine targeted investigation of U-bodies and the newly developed multiplexed RNA fluorescence tagging tools to delineate mechanistic roles of U-bodies. U-body compositions and their subcellular dynamics upon perturbation will be defined to delineate underlying cellular mechanisms. A possible link between U-body dynamics and alternative splicing regulation will be investigated. As a long-term goal, the role of U-bodies in splicing dynamics and regulation may be expanded upon across biological cell systems and perturbations, revealing a previously unknown new layer of gene regulation. U-body components have been linked with several human disease states, indicating that insights from this research program may shed light on possible treatment and disease management strategies of human health in the future.

核糖核酸（RNA）在许多细胞过程中起关键作用。一个经典的例子是替代 剪接，大型Megadalton剪接体复合物可以从预选前的内含子区域移开内含子区域 RNA（前MRNA）并重新加入外显子以形成成熟的mRNA。剪接体组成 蛋白质和非编码RNA成分。它的组装包括高度的复杂成熟步骤 调节以确保在健康细胞中正确的时间生成正确的成熟mRNA分子。 实现所有mRNA的正确剪接都需要严格的调节，需要复杂的相互作用 剪接组件的细胞提示和时空动力学。基因突变或环境 压力源是可能影响剪接过程和结果的扰动。这些与人有关 癌症和神经系统疾病等疾病状态。同时，复杂时空的核心作用 RNA动力学用于适当的剪接，要求需要审问各种RNA的亚细胞级别 时间。剪接涉及的RNA物种的复杂性需要鲁棒和多功能的标签策略才能 同时可视化多个RNA分子，并相对于其他感兴趣的生物分子。钥匙 该研究计划的目标是开发这样的强大工具箱，用于使用 晚期荧光显微镜。荧光寿命成像显微镜（FLIM）以A的形式出现 特别是多功能方法，因为它与添加复杂的成像方式兼容。中央 提议的工作中将包含的功能是能够同时可视化多个RNA的功能， 包括在剪接中具有角色的小型非编码RNA。更广泛地，这些RNA成像工具将允许 各个领域的研究人员研究了各种相关细胞模型系统中的RNA。选择 剪接与形成一种称为u-Bodies的胞质RNA-蛋白颗粒的形成。 剪接体RNA（称为u snRNA）是U-Bodies的定义成分，以及多种蛋白 与剪接反应有关。在不同的细胞模型中观察到U-体，指出 在基因调节中的核心作用，但有关其精确组成和功能的细节仍然难以捉摸。这 研究计划将结合对U体的有针对性研究和新开发的多重RNA 荧光标记工具来描述U-Bodies的机械作用。 U体型及其 扰动后的亚细胞动力学将定义为描述潜在的细胞机制。可能 将研究U体动力学与替代剪接调节之间的联系。作为一个长期目标， U体在剪接动力学和调节中的作用可能会在生物细胞系统之间扩展，并在 扰动，揭示了以前未知的基因调节层。 U体分已经是 与几种人类疾病状态有关，表明该研究计划的见解可能会揭示 未来可能的治疗和疾病管理策略。