Illuminating multiplexed RNA dynamics to interrogate splicing in health and disease

核糖核酸(RNA)在许多细胞过程中起着关键作用。一个典型的例子就是Alternative 剪接，其中大的百万吨剪接体复合体从前信使中移除内含子区域 RNA(前-mRNA)，并重新加入外显子，在细胞核中形成成熟的mRNA。剪接体由 蛋白质和非编码的RNA组分。它的组装包括复杂的成熟步骤，高度 调节以确保在健康细胞中在正确的时间产生正确的成熟的mRNA分子。 实现所有mRNA的正确剪接受到严格的监管，需要复杂的相互作用 剪接元件的细胞线索和时空动力学。基因突变或环境因素 应激源是可能影响剪接过程和结果的扰动。这些都与人类有关 疾病状态，如癌症和神经疾病。总而言之，复杂时空的核心作用 适当剪接的RNA动力学需要询问生活在亚细胞水平上的不同RNA 时间到了。参与剪接的RNA物种的复杂性需要强大和通用的标记策略来 同时可视化多个RNA分子，并与其他感兴趣的生物分子相关。一把钥匙 这项研究计划的目标是开发这样一个健壮的工具箱，用于使用 先进的荧光显微镜。荧光寿命成像显微镜(FLIM)作为一种 尤其是多功能方法，因为它与添加复杂的成像方式兼容。一个中环 提议的工作中将包括的特征是同时可视化多个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细胞模型系统中研究RNA。备择 剪接与一种被称为U-小体的胞浆RNA-蛋白质颗粒的形成有关。 剪接体RNA(称为U-SnRNAs)是U-小体的决定性成分，与几种蛋白质一起 与剪接反应有关的基因。在不同的细胞模型中观察到了U小体，指出 在基因调控中起着核心作用，但关于它们的确切组成和功能的细节仍然难以捉摸。这 研究计划将结合U-小体的有针对性的研究和新开发的多重RNA 描述U小体机制作用的荧光标记工具。U形体的组成和它们 将定义扰动时的亚细胞动力学，以描述潜在的细胞机制。一个可能的 将研究U形体动力学和选择性剪接调节之间的联系。作为一个长期目标， U-小体在剪接动力学和调控中的作用可能会在生物细胞系统和 微扰，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，表明来自这一研究计划的见解可能有助于 未来人类健康可能的治疗和疾病管理策略。

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.