MOLECULAR STUDIES OF FMRP FUNCTION IN YEAST

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The long-term goal of this project is to elucidate the natural function of FMRP and related gene products, with emphasis on exploring the proposed roles of these proteins as factors contributing to the regulation of gene expression of eukaryotic cells. In particular, we plan to study both expression and function of Scp160p, a candidate homologue of FMRP, in the genetically and biochemically amenable yeast Saccharomyces cerevisiae. In addition, we will use yeast as a model system in which to express and study wild-type and mutant forms of vertebrate FMRP. Current data derived both in vitro and in vivo from studies of mammalian cells indicate that both FMRP and the FMRP-related FXR proteins are ribonucleoproteins that associate with ribosomes and ostensibly mediate the metabolism, transport, stability, and/or translation of specific messages in cells. What remains unknown, however, is the specific impact of FMRP function on these biological pathways, the mechanism by which this impact occurs, and the identities and relationships of gene products subject to FMRP-mediated regulation. We propose to address these questions with regard to both yeast Scp160p and vertebrate FMRP by accomplishing the following specific objectives" (1) to characterize Scp160p subcellular localization in yeast both biochemically and microscopically and, coupled with deletion and mutagenesis protocols, to initiate structure/function studies of this protein; (2) to study the phenotypes of both loss and overexpression of Scp160p in yeast; 93) to identify biochemically both protein and RNA species with which Scp160p associated in vivo, as well as to identify genetically other loci in yeast whose products interact, either physically or functionally, with Scp160p, and (4) to express vertebrate FMRP proteins in yeast, to study both the localization and macromolecular interactions of these proteins with endogenous yeast proteins and/or FNAs, and to probe the capacity of these vertebrate FMRP sequences to complement deletion of the corresponding yeast endogenous sequences. Combined, these studies should provide information that is meaningful not only in terms of the basic science of expression and function of FMRP-like sequences in yeast, but also because insights gained from this work should provide a useful framework for interpreting the potentially much more complex data derived from studies of FMRP function in mammalian cells and tissues.