| We have characterized a mutation in Drosophila melanogaster that results in male and female sterility. In both males and females, germline stem cells (GSCs) are present, however germ cells of a later developmental state are not detected. Analysis of mutant ovaries show that GSCs undergo apoptosis once they begin to differentiate. We have identified the mutated gene and found that it is caused by a roo retrotransposon insertion within the ORF of a gene we have named emperor's thumb (et). et has six predicted transcripts that all share a conserved protease domain. Inferred from sequence similarity, et is predicted to encode a deubiquitinating enzyme (DUB) that cleaves ubiquitin-protein bonds. Ubiquitin chains target the protein to the 26S proteosome for degradation. Therefore, et may function as a deubiquitinase, removing ubiquitin from a protein whose function it is to inhibit apoptosis.; et loss-of-function causes lethality at the 1 st-2nd instar larval stage. We find that overexpression of one protein form of et in the eye results in the survival of interommatidial lattice cells that normally undergo apoptosis. Using the Flp/Frt system, we find that et loss-of-function in the eye causes cell death associated with a decrease in DIAP1 levels---suggesting that et is required for maintaining the normal concentrations of DIAP1 protein. Therefore, et's DUB activity plays some role in antagonizing apoptosis. However, we find that overexpression of an alternate et transcript in the eye at 25°C causes apoptosis and late pupal lethality. At 18°C and 22°C, flies have rough and severely mutated eyes, respectively. We also show that Dronc and diap1 genetically interact with et, where loss-of-function of Dronc or gain-of-function of diap1 rescue the lethality caused by et overexpression. Also, a 50% reduction in diap1 enhances the et eye overexpression phenotype at 18°C. It has been shown that DIAP1 degradation is required for its anti-apoptotic function. We propose that the overexpression of Et inhibits the processing of DIAP1, stabilizing it, preventing its degradation and thereby preventing the concurrent degradation of active Dronc and Drice, leading to apoptosis. Therefore, rather counterintuitive, DIAP1 instability is required for its anti-apoptotic function. |
