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. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM072820-03
Application #
7483607
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
2006-09-01
Project End
2011-08-31
Budget Start
2008-09-01
Budget End
2009-08-31
Support Year
3
Fiscal Year
2008
Total Cost
$285,037
Indirect Cost
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Kiss, Daniel L; Hou, Dezhi; Gross, Robert H et al. (2012) Dis3- and exosome subunit-responsive 3' mRNA instability elements. Biochem Biophys Res Commun 423:461-6
Hou, Dezhi; Ruiz, Miriam; Andrulis, Erik D (2012) The ribonuclease Dis3 is an essential regulator of the developmental transcriptome. BMC Genomics 13:359
Smith, Sarah B; Kiss, Daniel L; Turk, Edward et al. (2011) Pronounced and extensive microtubule defects in a Saccharomyces cerevisiae DIS3 mutant. Yeast 28:755-69
Kiss, Daniel L; Andrulis, Erik D (2011) The exozyme model: a continuum of functionally distinct complexes. RNA 17:1-13
Kiss, Daniel L; Andrulis, Erik D (2010) Genome-wide analysis reveals distinct substrate specificities of Rrp6, Dis3, and core exosome subunits. RNA 16:781-91
Mamolen, Megan; Smith, Alexandra; Andrulis, Erik D (2010) Drosophila melanogaster Dis3 N-terminal domains are required for ribonuclease activities, nuclear localization and exosome interactions. Nucleic Acids Res 38:5507-17
Graham, Amy C; Kiss, Daniel L; Andrulis, Erik D (2009) Core exosome-independent roles for Rrp6 in cell cycle progression. Mol Biol Cell 20:2242-53
Graham, Amy C; Davis, Stephanie M; Andrulis, Erik D (2009) Interdependent nucleocytoplasmic trafficking and interactions of Dis3 with Rrp6, the core exosome and importin-alpha3. Traffic 10:499-513
Mamolen, Megan; Andrulis, Erik D (2009) Characterization of the Drosophila melanogaster Dis3 ribonuclease. Biochem Biophys Res Commun 390:529-34