tRNAs are processed, modified, and actively transported to the cytoplasm. Although our understanding of these processing events has grown, the mechanism of tRNA nucleocytoplasmic transport remains largely unexplored. This proposal aims to develop novel in vivo assays using Green Fluorescent Protein (GFP) to determine the cellular factors affecting tRNA export in yeast. A tRNA chimera will be constructed in which the wild-type variable loop will be replaced by a binding element for either a GFP fusion protein or an oligonucleotide probe, allowing for direct, visual monitoring of tRNA nuclear export. Using this assay, genetic screens will be used to discover new factors regulating tRNA export. The yeast homolog of human exportin-t, Los 1p, may be a tRNA export factor, but it is nonessential, suggesting that other tRNA export factors must also exist. In vitro and in vivo structure-function studies of the interaction between the nuclear transport factor Los 1p and tRNA along with the characterization of any newly discovered protein-protein or RNA-protein complexes will be performed to complement and expand the initial in vivo studies. Because all the identified nuclear transport factors are conserved from yeast to mammalian cells, these studies will enhance our understanding of nuclear transport in all eukaryotes.
