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(6) or visualize it in vivo(1). In addition, a lack of experimental techniques has limited our ability to understand the regulatory mechanisms of gene expression in Eukaryotic cells. This research will use state of the art fluorescence microscopy (See Figure 1) to determine the complete kinetic profile of RNA processing events and correlate them with splicing factor dynamics. To overcome previous temporal limitations, we will combine 3D orbital tracking(7, 8) microscopy and a newly developed fluorescent labeling strategy that allows simultaneous visualization of introns, exons and splicing factors inside of living cells. This will allow us to follow the fate of individual Eukaryotic pre-mRNA molecules as they undergo transcription, splicing, and decay, in real time and enable complete kinetic characterization of the synthesis and processing of individual RNA molecules as well as single molecule temporal correlation of splicing factor binding to RNA or neighboring regions of DNA. In previous work, due to the rapid photobleaching of cells, 10-20 measurements were averaged together to determine transcriptional and splicing kinetics(1). With 3D orbital tracking, the information garnered in three previous experiments on two separate microscopes will be available in a single cell measurement at a 100 times higher sampling rate. This higher sampling rate will also allow measurement of splicing factor binding allowing us to determine the molecular mechanism of a splicing factor mutation found to occur in multiple human cancers(3-5, 9). The project will utilize novel time resolved microscopy techniques based on fluorescence correlation spectroscopy cutting across traditional boundaries of computational physics, optics and cell biology. It will implement recent advances in microscopy in new ways to visualize fundamental spatiotemporal processes involved in epigenetic regulation at the single molecule level in living cells. This research will directly benefit patients suffering from AML, hairy cell leukemia's by showing the molecular mechanism leading to disease. It will also open new research avenues into the molecular basis of neurological and muscle diseases with origins in alternative splicing misregulation such as Parkinson's disease, Autism, ALS and Cardomyopathies(10)
Fluorescence imaging of transcription in living cells allows us to visualize the spatiotemporal dynamics of gene regulation in the nucleus. The goal of this research is to develop and implement tools to monitor all relevant length and time scales required for researchers to characterize complex and transient biological processes related to human health and disease.
| Panchapakesan, Shanker Shyam S; Ferguson, Matthew L; Hayden, Eric J et al. (2017) Ribonucleoprotein purification and characterization using RNA Mango. RNA 23:1592-1599 |
