The long-term objectives of this research program remain directed at defining the mechanisms by which mRNA molecules are exported from the nucleus and determining under what circumstances and by what mechanisms nucleocytoplasmic transport is regulated. Our understanding of nucleocytoplasmic transport of proteins and small RNAs (e.g. tRNAs) has grown substantially through the identification of transport signals and karyopherin receptors that recognize them. We know much less about mRNA export, which is considerably more complex, in part because it is tightly coupled to mRNA biogenesis and checkpoints exist that permit only completely- and accurately-processed mRNAs to be exported. mRNAs are exported as RNA/protein complexes (mRNPs). Many factors required uniquely for mRNA export have been identified but how they work together is not known. A major focus is to understand the role in mRNA export of the DEADbox protein (DBP) Dbp5/Rat8. DBFs play essential roles in virtually all aspects of RNA metabolism. Dbp5 shuttles and interacts directly with components of the nuclear pore complex (NPC). To address two centrals questions about RNA export, the following specific aims are proposed:
Aim 1) To test the hypothesis that Dbp5 acts when bound to the cvtoplasmic filaments of NPCs to couple ATP hydrolysis with mRNP translocation through the pore and removal of mRNP proteins. The interactions between Dbp5, proteins of the cytoplasmic filaments of the NPC, and accessory proteins will be mapped. Genetic and physical approaches will be used to determine whether Dbp5 must bind to NPC filaments to mediate mRNA export. The dynamics of interaction between Dbp5 and NPCs will be analyzed using fluorescence recovery after photobleaching (FRAP) and with mating assays. Whether Dbp5 can disassemble model RNA/protein complexes in vitro will be determined.
Aim 2) To test the hypothesis that they key to export lies in the composition and organization of mRNP complexes. The studies will compare export-competent mRNPs from wild-type cells and mRNPs that accumulate in export mutant strains. Other studies will determine how exported heat shock mRNPs differ from mRNPs that accumulate in nuclei following heat shock.
Aim 3) To determine whether Dbp5 functions not only at NPCs but also in the nucleus and what it does in the nucleus.
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