Over the quarter century of this grant funding, we have been developing tools to investigate the fate of single mRNA molecules in living cells. Most recently we have been able to image the real time translation of proteins in living cells, and the degradation of mRNAs at a precise moment during the cell cycle. We now would like to integrate all these processes into one coherent scheme, by imaging a single mRNA molecule from birth to death. This provides technical challenges, due to the photobleaching of fluorescent proteins and the difficulty of tracking mRNAs in a crowded cytoplasm. We propose in this application to address these obstacles by developing new approaches to reduce photobleaching in order to track mRNAs for longer times, equivalent to their lifetimes. We will reversibly photoactivate the mRNAs as they are transcribed in the nucleus, and then follow the few mRNAs as they export into the cytoplasm, translate and degrade. We will likewise simultaneously follow proteins bound to the mRNA in the nucleus that may regulate these processes. This will allow us to follow a single mRNA and proteins bound to it throughout its entire life cycle.
Major questions in cell biology can only be answered with the development of new technology. We have developed methods to image single molecules of messenger RNA and proteins in living yeast cells in order to understand how information is regulated in the cell. We have found that these molecules regulate cell growth by making proteins at exactly the right time and place and then disappear, promoting the precise timing needed for cell division. The goal of this proposal is to develop methods to visualize the life cycle of a single mRNA molecule in real time in living cells.
