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) 在许多细胞过程中发挥着关键作用。一个经典的例子就是替代 剪接，其中大型兆道尔顿剪接体复合物从前信使中去除内含子区域 RNA（前mRNA）并重新连接外显子，在细胞核中形成成熟的mRNA。剪接体由 蛋白质和非编码RNA成分。它的组装包括复杂的成熟步骤，这些步骤非常复杂 进行调节以确保健康细胞在正确的时间产生正确的成熟 mRNA 分子。 实现所有 mRNA 的正确剪接都受到严格的调控，需要复杂的相互作用 剪接成分的细胞线索和时空动态。基因突变或环境因素 压力源是可能影响剪接过程和结果的扰动。这些都与人类息息相关 癌症和神经系统疾病等疾病状态。总之，复杂时空的核心作用 正确剪接的 RNA 动力学要求需要在亚细胞水平上实时探究不同的 RNA 时间。参与剪接的 RNA 种类非常复杂，需要稳健且通用的标记策略来 同时可视化多个 RNA 分子并相对于其他感兴趣的生物分子。一把钥匙 该研究项目的目标是开发一个强大的工具箱，用于多重 RNA 可视化，使用 先进的荧光显微镜。荧光寿命成像显微镜（FLIM）作为一种 特别通用的方法，因为它与添加复杂的成像模式兼容。一个中央 拟议工作中将包含的功能是同时可视化多个 RNA 的能力， 包括在剪接中发挥作用的小非编码 RNA。更广泛地说，这些 RNA 成像工具将允许 不同领域的研究人员在各种相关细胞模型系统中研究 RNA。选择 剪接与一种胞质 RNA 蛋白颗粒（称为 U 体）的形成有关。 剪接体 RNA（称为 U snRNA）是 U 体以及多种蛋白质的定义成分 与剪接反应有关的。在不同的细胞模型中观察到 U 体，表明 在基因调控中发挥着核心作用，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 U 体和新开发的多重 RNA 的针对性研究 荧光标记工具描述 U 体的机制作用。 U-体成分及其 将定义扰动时的亚细胞动力学来描述潜在的细胞机制。一个可能的 将研究 U 体动力学和选择性剪接调控之间的联系。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扩展到生物细胞系统和 扰动，揭示了以前未知的新的基因调控层。 U型车身部件已 与几种人类疾病状态有关，表明该研究项目的见解可能有助于揭示 未来人类健康可能的治疗和疾病管理策略。