MECHANISM OF MEIOSIS-SPECIFIC RNA SPLICING IN YEAST

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The products of the MER1 and MER2 genes of S.cerevisiae are required for recombination and chromosome segregation during meiosis. The MER1 gene is transcribed only in meiotic cells. The MER2 gene is transcribed in both vegetative and meiotic cells but the transcript is spliced to yield a functional message only in meiosis. Splicing of the MER2 transcript requires the product of the MER1 gene. The long term goal of the proposed research is an understanding of the molecular mechanism of meiosis-specific, MER1-dependent RNA splicing. The MER2 gene will be mutagenized in vitro in order to identify cis- acting sequences that play a role in splicing regulation. In particular, the MER2 5' splice junction (GUUCGU) will be changed to the consensus 5' splice site sequence (GUAUGU). Splicing extracts prepared from wild-type vegetative cells (MER1-) and from cells engineered to express the MER1 gene during mitotic growth (MER1+) will be used to demonstrate MER1-dependent splicing of the MER2 transcript in vitro. Attempts will be made to determine whether the MER1 protein plays a direct role in splicing by (i) heat activation of extracts from a temperature-sensitive mer1 mutant, (ii) adding MER1 protein produced in bacteria to MER1- extracts and/or (iii) using anti- MER1 antibodies to immunodeplete MER1+ extracts. Spliceosomes will be isolated and examined for the presence of the MER1 protein. In addition, attempts will be made to demonstrate binding of the MER1 protein to the MER2 transcript by gel retardation assays; the binding site will be mapped by chemical modification/interference experiments. To identify trans-acting factors that play a role in MER2 splicing, second-site suppressors of mer1 mutants will be isolated. These experiments will employ a mer2::URA3 fusion gene whose expression depends on splicing; thus, mutants in which the MER2 transcript is spliced can be selected on medium lacking uracil. It is hoped that allele-specific suppressors can be isolated and that these will define genes whose products interact directly with the MER1 protein. mer1 strains carrying an intronless MER2 gene (cMER2) are defective in meiosis, suggesting that MER1 is required to splice the transcript of at least one other gene in addition to MER2. In order to identify MER1- dependent genes, a yeast genomic library will be screened for genes which, when overexpressed, suppress the meiotic defect of mer1 CMER2 strains. An alternative approach involves the prp26 mutant, which accumulates introns (released by splicing) as undegraded lariats. Lariats will be isolated from prp26 MER1 and prp26mer1 meiotic cells and used to screen a yeast genomic library. Clones that hybridize to introns from MER1, but not mer1, strains will be characterized further.