MECHANISM OF MACROMOLECULAR EXPORT FROM THE NUCLEUS

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DESCRIPTION (adapted from the applicant's abstract): Export of RNA and protein from the nucleus into the cytoplasm is a fundamental cellular process and a key step in the control of eukaryotic gene expression. The mechanisms governing these transport events are still poorly understood but kinetic competition studies in Xenopus oocytes have indicated that different RNA classes, including mRNA, snRNA, tRNA and rRNA use distinct export pathways. Since most, if not all RNAs, are associated with proteins in the nucleus it was suggested that RNA export events are mediated by proteins in the nucleus it was suggested that RNA export events are mediated by proteins containing appropriate nuclear export signals (NESs). This is supported by the identification of small transferable signals in proteins that cause their rapid and active export from the nucleus. The long-term goal of the proposed research is to characterize different RNA and protein export pathways in the yeast S. cerevisiae and to understand the nuclear export mechanisms at a molecular level. More specifically, three specific aims are proposed: (1) The P.I. will characterize the nuclear export factor export in 1 (Xpo1p/Crm1p) and its role in mRNA export. A variety of biochemical and genetic approaches will be used to identify factors which interact with Xpo1p. The role of the GTPase Ran in regulating the interaction of Xpo1p with other cellular factors will be examined and the adapter(s) that mediate(s) Xpo10,s role in mRNA export will be characterized. (2) The NES-mediated nuclear protein export machinery will be dissected. Genetic approaches will be explored to identify additional components of the NES-protein export machinery. This export pathway will be used as a model system to understand the molecular details of how macromolecules are transported through the nuclear pore complex into the cytoplasm. (3) Finally, the P.I. will determine whether distinct RNA export pathways exist in yeast. In situ labeling in fixed and live cells will be used to characterize the export of the different RNA classes in several mutant backgrounds. If distinct pathways are identified, factors that mediate and regulate these export events will be identified.