| Regulated mRNA turnover is a critical component of the gene expression program. Cells rapidly change their expression profile by modulating the transition between active translation and mRNA decay. In response to environmental changes, mRNAs exit the translational pool and enter processing bodies, which are sites of storage and decay in the cell. While shifting of populations of mRNAs has been well documented in response to stress, it is not clear how this transition occurs mechanistically or if populations of mRNAs are specifically targeted for decay.;To uncover specific regulatory patterns at the level of turnover, I developed a high throughput non-invasive labeling system to measure mRNA turnover directly in Saccharomyces cerevisiae. Metabolic labeling of RNA with 4-thiouracil specifically biotinylates and purifies a population of mRNAs in vivo. This labeling system coupled with RNA sequencing technology provides a quantitative, global assay to monitor changes of mRNA turnover. Using this assay in S. cerevisiae will provide an easily manipulatable system to comprehensively and quantitatively measure mRNA turnover changes throughout a variety of conditions. This global profiling of mRNA turnover will ultimately reveal how this cellular process is regulated in vivo.;There are several known proteins that regulate the entry of mRNAs into the decay pathway. A critical regulator of this transition is the protein Dhh1, a member of the DEAD box family of RNA-dependent ATPases. Dhh1 has well documented roles in both translational repression and as an enhancer of decapping, a key step in the decay pathway. Here, I present preliminary results characterizing the regulation of Dhh1 itself. My results reveal that Dhh1 expression is regulated at both the mRNA and protein level. In addition, active ATP hydrolysis is required for proper localization and function of Dhh1 in vivo. This complex regulation of Dhh1 may provide an additional mechanism to control mRNA turnover in the cell. |
