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着关键作用。一个经典的例子是替代性 剪接，其中大的巨道尔顿剪接体复合物从前信使中移除内含子区域， RNA（前mRNA），并重新连接外显子，形成成熟的mRNA在细胞核中。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， 调节以确保在健康细胞中正确的时间产生正确的成熟mRNA分子。 实现所有mRNA的正确剪接受到强烈的调控，需要复杂的相互作用， 细胞线索和剪接组件的时空动态。基因突变或环境 应激源是可能影响剪接过程和结果的扰动。这些都与人类 像癌症和神经系统疾病。总之，复杂时空的核心作用 正确剪接的RNA动力学需要在亚细胞水平上询问不同的RNA， 时间参与剪接的RNA种类的复杂性需要稳健和通用的标记策略， 同时并相对于其他感兴趣的生物分子可视化多个RNA分子。一个关键 这项研究计划的目标是开发这样一个强大的工具箱，用于多重RNA可视化， 先进的荧光显微镜荧光寿命成像显微镜（FLIM）作为一种 特别是通用的方法，因为它与添加复杂的成像模态兼容。中央 将包括在拟议工作中的功能是同时可视化多个RNA的能力， 包括在剪接中起作用的小的非编码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的细胞模型系统中研究RNA。替代 剪接与一种称为U体的细胞溶质RNA蛋白颗粒的形成有关。 剪接体RNA（称为U snRNA）是U体的定义成分，沿着几种蛋白质 与剪接反应有关。在不同的细胞模型中观察到U体，指出 在基因调控中起着核心作用，但关于它们的精确组成和功能的细节仍然难以捉摸。这 研究计划将结合联合收割机有针对性的调查U体和新开发的多重RNA 荧光标记工具来描绘U体的机械作用。U型体成分及其 扰动时的亚细胞动力学将被定义为描绘潜在的细胞机制。一个可能 将研究U体动力学和可变剪接调节之间的联系。作为长期目标， U体在剪接动力学和调节中的作用可以在整个生物细胞系统中扩展， 扰动，揭示了一个以前未知的新的基因调控层。U型车身部件已被 与几种人类疾病状态有关，表明这项研究计划的见解可能有助于阐明 未来人类健康可能的治疗和疾病管理策略。