Regulated processing, turnover, export, and surveillance of messenger RNA molecules are critical to maintain accurate gene expression in eukaryotes. An integral player in these distinct RNA metabolic pathways is the exosome, an essential protein complex comprising numerous 3' to 5' exoribonucleases. Yet, the exact mechanisms underlying how the exosome functions in these pathways are unclear. In this regard, our preliminary data show that Drosophila exosome subunits are differentially distributed in vivo, suggesting that exosome complexes have specialized functions corresponding to their subcellular compartmentalization. In particular, the dDisS subunit is predominantly nuclear and nucleoperipheral whereas the dCsl4 subunit in enriched in cytoplasmic foci. In this proposal, we explore the hypothesis that exosome substrate specificity is achieved by compartmentally and compositionally distinct exosome complexes through three specific aims: (1) define the interplay among dDis3, dCsl4, and the exosome complex in vivo, using high-resolution fixed and live cell fluorescence techniques; (2) demonstrate that proper dDisS and dCsl4 subcellular localization is critical for regulated mRNA decay in the nucleus and the cytoplasm, respectively, by examining endogenous and reporter mRNA stability; and (3) identify and characterize the compartmentalized dDis3- and dCsl4-precipitated exosome complexes and associated factors using chromatographic and mass spectrometric approaches. These studies are bound to yield important insight into how proper dDisS and dCsl4 subcellular distribution correlates with exosome complex structure and function. The long-term objective of the study is to identify specialized exosome subunit functions and interactions in the context of mRNA metabolic pathways. An in-depth understanding of how the exosome processes and degrades mRNAs is fundamentally important to human health, as aberrant gene expression is an etiological factor in numerous disease states. ? ? ?
