Identification and Characterization of Cytoplasmic Processing Bodies (P Bodies) in Saccharomyces Cerevisiae

by Sheth, Ujwal.

An important aspect of regulating gene expression is the interplay between mRNA translation and its degradation. In the work presented here, I, in some cases in collaboration with others, provide insights into how mRNA translation and decay are connected and how they interact with each other to regulate gene expression. This work can be summarized as follows: First, I identified cytoplasmic processing bodies (P bodies) in yeast, which are sites where mRNAs can be decapped and degraded in a 5' to 3' manner. We base our conclusion on three key observations. First, factors involved in the 5' to 3' decay pathway accumulate in P bodies. Second, P bodies change in size when the flux of mRNA decay pathways is perturbed. For example, they decrease in size when entry into decapping is inhibited, and increase in size when decapping is blocked. Third, mRNAs trapped in the process of decay accumulate in P bodies. Second, in a collaborative effort, I have further characterized P bodies. This work involved addressing the role of RNA in P body formation and the relationship of P bodies to translation. Our results suggest that P bodies are dynamic and their size is affected by a range of cellular perturbations. We also provide evidence that P bodies are sensitive to the translational status of the cell and represent sites where translationally repressed mRNAs accumulate, and where they can be subjected to 5' to 3' decay. Third, I have determined that Nonsense-mediated decay (NMD), a quality control mechanism that rapidly degrades aberrant mRNAs, involves targeting of aberrant mRNAs to P bodies. In addition, I have identified specific roles for Upf proteins in the 10 process of NMD: Upf1p is involved in targeting mRNAs to P bodies and Upf2p and Upf3p playing a role in degradation of the aberrant mRNAs within P bodies. The identification of P bodies has direct implications on regulation of mRNA decapping, of both normal and aberrant mRNAs. The similarities of P bodies with mRNA storage granules in other organisms imply that P bodies will play a major role in regulation of translationally repressed mRNAs. 11