The role of dTopors in Drosophila male meiosis

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Project Summary/Abstract The Human tumor suppressor Topors (Topoisomerase I-interacting Arginine Serine rich protein) is a dual Ubiquitin/Sumo E3 ligase. In Drosophila the homologous protein, dTopors, plays an essential role in both nuclear structure and meiotic chromosome behavior in males. Mutations In dtopors that specifically alter its ubiquitin ligase activity result in spermatocyte nuclear blebbing and a high frequency of meiotic anaphase bridges and subsequent nondisjunction. This unusual phenotype is also observed in males bearing a meiotic-specific mutation in the nucleoporin gene rae1 (ribonucleic acid export 1), suggesting that the two genes act in a common pathway. A combined genetic, cytological and biochemical approach will be used to define the relationship between dTopors and Rae1. The double mutant phenotype will be examined, and genetic tests will be used to ask if rae1 alleles enhance a dtopors hypomorph. The localization of dTopors and Rae1 protein will be examined in the reciprocal mutant background, and the proteins will be tested for interaction using a yeast two-hybrid assay and co-immunoprecipitation. Partner E2 ubiquitin ligase(s) of dTopors will be identified using an RNAi knockout screen along with yeast two-hybrid interaction tests. An existing second site suppressor of dtopors effect on nuclear structure will be identified by conventional recombination mapping, complementation tests and DNA sequencing. Downstream targets of dTopors ubiquitination in meiotic cells will be determined by comparative LC MS/MS mass spectrometry on wildtype versus ubiquitin null mutant testis protein after immunoprecipitation with ubiquitin-tag-specific antibodies. Together these experiments will reveal the relationship between Rae 1 and dTopors, and determine the downstream targets of dTopors relevant to nuclear structure and chromosome segregation.