Translation is the process by which the genetic information in mRNAs is read and used by the ribosomes to generate proteins with specific cellular functions. A novel approach for monitoring translation of individual mRNAs has been developed for this project. Using this approach, a number of fundamental questions about translation will be addressed. For example, are single mRNAs translated constitutively or in a burst-like fashion and do mRNAs encoded by the same gene behave homogeneously or heterogeneously? This work has the potential to influence the current understanding of the central dogma of molecular biology. The research and educational activities associated with this project will provide students at various levels with training in cutting edge molecular techniques. This project also includes efforts to increase diversity in STEM and outreach to the local community.
The current proposal aims to address temporal dynamics and control mechanism of translation at the single molecule level in live cells. mRNA transcribed from the same gene may be bound by different protein factors, non-coding RNAs, or may adapt different conformations, which results in heterogeneity or noise in their behaviors. In agreement with this concept, it was previously observed that translation appears to be regulated at the level of single-mRNAs, as indicated by idiosyncratic "bursting" behavior. Ensemble biochemical approaches cannot be used because they average millions of cells and molecules. An in vivo, single-molecule technique is the only way to uncover the origins of this previously unexplored facet of gene expression regulation. Researchers have previously developed ways to visualize single mRNAs and their nascent peptides. However, further developments in experiment and theory are needed to study the temporal translation dynamics. First, single mRNAs and their translation status will be imaged for an extended period of time to capture the long-term translation dynamics. Second, the binding of trans-acting factors to mRNAs will be imaged. It is generally accepted that the fate of mRNA is determined by factors associated with it, such as RNA binding proteins and microRNAs. By correlating the trans-factor binding with translation activity, the mechanism of translation regulation could be uncovered. Third, a quantitative biophysical model of translation will be established to extract physiological parameters from the proposed single-molecule imaging experiments. After careful measurement of steady state, translation dynamics will be perturbed chemically or genetically to systematically vary cis- or trans- acting factors on mRNA, and to monitor the intramolecular mRNA conformation. A mechanistic model of translation will be built to elucidate the innate temporal heterogeneity.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
