TORC1 regulates metabolism and growth in response to a large array of upstream inputs. The GATOR complex, an upstream regulator of TORC1 activity, contains two sub-complexes, GATOR1 and GATOR2. The trimeric GATOR1 complex inhibits TORC1 activity in response to amino acid limitation. In humans, the GATOR1 complex has been implicated in a wide array of pathologies including cancer and hereditary forms of epilepsy. The GATOR2 complex inhibits the activity of GATOR1 to promote TORC1 activity. Relative to GATOR1, little is known about the regulation or mechanism of action of GATOR2. Over the last several years, my laboratory has explored the role of the GATOR complex in the regulation of metabolism and oocyte development using the model organism Drosophila melanogaster. The GATOR2 complex is comprised of five proteins, Mio, Seh1, Wdr24, Wdr59 and Sec13. In Drosophila, mutations in the GATOR2 components mio and seh1, cause the constitutive activation of the GATOR1 complex in the female germ line, resulting in the permanent inhibition of TORC1 activity and a block to oocyte growth and development. We have determined that germline RNAi depletions of any of the GATOR1 components in the mio and seh1 mutant backgrounds, relieve this permanent TORC1 inhibition and rescue the mio and seh1 ovarian phenotypes. Over the last year we have used this epistatic relationship to conduct a high throughput RNAi based screen to identify upstream regulators and downstream effectors of the GATOR1 complex during oogenesis. First, we determined that expressing a short hairpin RNA against the seh1 transcript, using the nanos-Gal4 germline specific driver, recapitulates the seh1 mutant ovarian phenotype. Moreover, co-depleting nprl2, nprl3, and iml1 dramatically rescues the seh1RNAi ovarian phenotype. In order to identify additional genes that, when co-depleted with seh1, rescue the seh1RNAi ovarian phenotypes we have used RNAi lines from the Transgeneic RNAi Project (TRiP) that have been optimized for germline expression. This co-depletion screen has identified an array of new GATOR interacting genes. Importantly, as anticipated, the screen identified multiple known regulators of TORC1 including Tsc1, Tsc2 and PTEN. Additionally, we identified many genes that function in translation, transcription and the DNA damage response that have not previously been implicated in metabolic regulation. These newly identified GATOR interacting genes will provide a framework for our future studies on the regulation and function of the GATOR complex during oogenesis. In single-cell eukaryotes, metabolic inputs instruct meiotic entry and early meiotic progression. However, the role of metabolism in the regulation of early meiotic events in metazoans remains poorly defined. Over the last year we demonstrated a requirement for the conserved GATOR1 in the regulation of meiotic double-stranded breaks in Drosophila and mice. In yeast, the down regulation of TORC1 activity by GATOR1 in response to amino acid starvation promotes meiotic entry and the repair of meiotic double-stranded breaks. In previous work we have found that GATOR1 and TORC1 activity regulate meiotic entry in metazoans. Over the last year we have gone on to demonstrate that in Drosophila, GATOR1 mutant oocytes accumulate an increased number of double-stranded breaks that persist beyond paychtene. This increase in the steady state number of double-stranded breaks is accompanied by a dramatic increase in p53 activity. Consistent with a conserved function for the GATOR1 complex during meiosis in mammals, depletions of the GATOR1 component DEPCD5/Iml1 result in an increased number of double-stranded breaks in mouse spermatocytes. Taken together, our data support the model that the TORC1 inhibitor GATOR1 plays a conserved role in the regulation of double-stranded breaks during meiotic recombination and identify a potential link between TORC1 activity and the maintenance of genome stability during gametogenesis in all eukaryotes.
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