Characterization of the Role of Processing Bodies in Mammalian Mrna Decay.

mRNA stability pathways represent an important step in gene expression as they prevent over-expression of unwanted proteins that can disrupt cellular homeostasis. Recently it was discovered that some unstable mRNAs co-localize with mRNA decay factors in discrete foci called processing bodies (PBs) indicating that PBs may be sites of mRNA decay and/or translational silencing. However, little was known about what destines an mRNA to become sequestered in a PB, what delivers an mRNA to a PB, and why mRNAs localize there in the first place. To answer these important questions I developed in situ hybridization techniques to follow the localization of an unstable AU-rich element (ARE) containing mRNA reporter under various cellular conditions. I found that ARE-containing mRNAs form messenger ribonucleoproteins (mRNPs) that can assemble into PBs and that this assembly is mediated by the ARE-binding proteins TTP/BRF-1/2. Importantly, I discovered that ARE-mRNAs assemble decay mRNPs more efficiently when the mRNA is translationally silent due to the introduction of a 5' UTR hairpin. In addition, I found that ARE-mRNAs accumulate strongly in decay mRNPs (PBs) when mRNA decay enzymes are limiting due to ARE-binding protein over-expression or mRNA decay factor depletion. Based on these findings, I postulated that I could create relatively homogenous PBs by trapping the cells endogenous mRNAs in polysomes while turning on expression of an exogenous translationally silent ARE-containing mRNA reporter. Using this method, I demonstrated that PBs can be nucleated by an ARE-mRNA reporter under conditions of limiting mRNA decay. These findings are important to the field of mRNA stability because they demonstrate that translation and decay mRNP formation are mutually exclusive processes that are in constant competition in the cell. Moreover, my results suggest that the PB might not be important for mRNA decay as was once thought. In contrast, PBs might have an important function as a buffering system that sequesters excess mRNA decay substrates in a translationally silent location to prevent unwanted translation until mRNA decay enzymes become available. I have used my knowledge of PB assembly as a tool to understand how mRNAs with premature termination codons, which are rapidly degraded by the nonsense-mediated mRNA decay pathway (NMD), are assembled into decay mRNPs. The NMD pathway is elicited when the RNA helicase Upf1 recognizes a prematurely terminating ribosome and targets the mRNA to a translationally silent mRNP that degrades the mRNA. However, it was previously unknown which other NMD factors are required for the formation of a decay mRNP, or what the function of Upf1's helicase activity is in this process. Here I have determined which NMD factors are critical for NMD decay mRNP formation. I have also found that Upf1 utilizes ATPase activity to remodel NMD decay mRNPs prior to exonucleolytic decay of the mRNA. Surprisingly, I have found that endonucleolytic cleavage of NMD substrates can efficiently occur in the absence of Upf1-mediated mRNP remodeling. These findings suggest a revised model for the NMD pathway in mammals in which Upf1 recruits NMD factors to form a silent NMD decay mRNP that promotes endonucleolytic cleavage of at least a subset of NMD substrates. Next, Upf1 uses ATP-dependent mRNP remodeling to make the mRNA easily accessible to decapping factors and exonucleases. I have also extended my studies to an mRNA decay pathway that degrades mRNAs with microRNA (miRNA) binding sites in their 3' UTRs. I show that mRNAs that are targeted for decay or translational silencing by the siRNA or miRNA pathways, respectively, in human...