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 link...