PROJECT SUMMARY / ABSTRACT Illuminating multiplexed RNA dynamics to interrogate splicing in health and disease 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 in healthy cells. Splicing patterns are subject to intense regulation, requiring a sophisticated interplay of cellular cues and spatiotemporal dynamics of splicing components. Perturbations of the splicing process may be caused by genetic mutations or environmental stressors. These are linked to 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 for broad applicability. 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. Multiplexed tagging and tracking of RNAs with our Riboglow platform using FLIM requires a FLIM microscope. This application requests purchase of an upgrade of an existing confocal microscopy system with fluorescence lifetime imaging microscopy capabilities. More broadly, these fluorescence lifetime imaging capabilities will allow researchers across different fields to investigate biomolecules in a variety of relevant cell model systems. The requested FLIM technology will benefit colleagues in my home department and neighboring departments and I am excited to build this advanced fluorescence microscopy capacity.