The long term objective of this research project is to understand how eukaryotic messenger RNA molecules are moved from their sites of transcription within the nucleus to their sites of translation in the cytoplasm. Nucleocytoplasmic transport of mRNA is an essential function of the eukaryotic cell, and likely requires the structural and functional integrity of the nucleus, including the nucleoskeleton, nuclear envelope and nuclear pore complexes. An analysis of this mRNA export process in the yeast, Saccharomyces cerevisiae, has been underway in this laboratory for approximately three years. An assay that permits the detection of poly(A)+ RNA in yeast cells by in situ hybridization was developed and used to screen temperature sensitive yeast strains. Genes that encode proteins that may be important for mRNA export have been identified by using this RNA localization assay to isolate a new class of yeast mutants (RAT mutants for Ribonucleic Acid Trafficking). We lack detailed knowledge of many aspects of mRNA trafficking. Therefore, it is probable that among the genes identified will be some that encode critical components of nuclear structures and the nuclear pore. Identifying and understanding the functions of these proteins is essential for understanding mRNA trafficking and is the fundamental goal of this research. A combination of genetic, molecular biological, cytological and biochemical approaches is proposed to extend the initial studies on these genes, to investigate the roles in mRNA trafficking of the proteins they encode, and to identify other cellular components involved in mRNA trafficking.
Four specific aims are proposed: 1) The initial characterization of the rat mutants will be completed. This includes cloning, mapping, and sequence analysis (if the gene has not previously been sequenced) of each RAT gene. mRNA localization defects in mutant strains will be confirmed by cell fractionation. RNA and protein synthesis and RNA processing will be analyzed in mutant strains at permissive temperature and at various times after a shift to the restrictive temperature. The protein product of each RAT gene will be localized with antibodies. 2) A limited number of genes and gene products will be chosen for further study. For these genes, additional ts alleles will be isolated and examined and proteins which interact with their gene products will be identified through use of the two hybrid protein-protein interaction system of S. Fields or through the isolation of extragenic suppressors. 3) The sensitivity of the mRNA localization assay will be increased so that individual mRNA species can be localized. 4) The effect on nucleocytoplasmic mRNA export of mutation in a number of other yeast genes will be determined by performing the in situ poly(A)+ RNA localization assay on additional mutant yeast strains.
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