Spatiotemporal Dynamics of the Genome by 3D Orbital Tracking

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The expression of genetic information depends on the fate of RNA transcripts. In Eukaryotic cells, this fate is determined by the successful execution of several processes, including transcription (initiation, elongation, and release) splicing, nuclear export and degradation. These processes are not independent in space or time. The rate and completion of any one process may influence that of another, and kinetic studies conducted on isolated components can be misleading or incomplete. Unfortunately, the coordinated kinetics of RNA processing events remains poorly understood primarily due to a lack of experiments that can either reproduce it in vitro or visualize it in vivo. The main goal of our research is to understand the molecular mechanisms underlying the functioning of the living genome. Our main tool is live cell single molecule fluorescence microscopy. By fluorescently labeling protein and RNA molecules and DNA elements within living cells we can determine the spatiotemporal relationships between them that govern gene expression and RNA splicing at an active gene in a living cell. Our most recent work has measured the timing of gene activation by the transcription factor Glucocorticoid receptor after binding to dexamethasone and the temporal coordination of transcription[1,2] and splicing of intron 2 of a human beta globin reporter gene[3]. We do this using novel single molecule approaches and cutting edge live cell single molecule fluorescence microscopy. Our methods are based on fluorescence correlation spectroscopy which utilizes high temporal resolution to characterize changes in fluorescence intensity at a location in space and time and relates that to molecular concentrations, interactions and dwelltime using physical and computational models. Models are tested using Bayesian inference criterion. This research will directly benefit patients suffering from SARS, Breast cancer, AML and hairy cell leukemia's by showing the molecular mechanism leading to disease and clinical outcomes. It will also open new research avenues into the molecular basis of neurological and muscle diseases with origins in alternative splicing mis regulation such as Parkinson's disease, Autism, ALS and Cardiomyopathies.