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 year, my laboratory has examined the role of the GATOR complex in the regulation of metabolism and oocyte development using the model organism Drosophila melanogaster. Our work has provided novel insights into the tissue specific regulation of TORC1 activity by the GATOR/TSC pathway. In single-cell eukaryotes, metabolic inputs instruct meiotic entry and early meiotic progression. However, how metabolic pathways influence meiotic progression in metazoans remains poorly understood. Over the last year, we have shown that the TORC1 regulators GATOR1 and GATOR2 mediate a critical response to meiotic DSBs during Drosophila oogenesis. We find that meiotic DSBs trigger the activation of a GATOR1 dependent pathway that downregulates TORC1 activity in the early meiotic cycle. Further studies established that low TORC1 activity, as established by the GATOR1 complex, is essential to the maintenance of oocyte genome stability. Specifically, GATOR1 mutant oocytes delay the repair of meiotic DSBs and hyperactivate p53, a transcription factor that is activated in response to genotoxic stress. Germline depletions of the TORC1 inhibitor TSC1 result in a similar array of ovarian defects. These data confirm that the GATOR1 oocyte defects are due to inappropriately high TORC1 activity during the early meiotic cycle. In contrast to the GATOR1 complex, the GATOR2 component mio ensures that the downregulation of TORC1 activity by GATOR1 does not become permanent, thus allowing for the continued growth of the oocyte in later stages of oogenesis. Unexpectedly, during the course of our studies, we found that in GATOR1 mutants, meiotic DSBs trigger the expression of retrotransposons. Epistasis analysis indicates that GATOR1 acts in a pathway that is independent of p53 to inhibit retrotransposons expression during the early meiotic cycle. Thus, our data indicate that the GATOR1 complex regulates two pathways that impact germline genome stability, the repair of meiotic DBSs and the inhibition of retrotransposon expression. 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. We completed a high throughput RNAi based screen for genes that are upstream activators or downstream effectors of the GATOR1 complex. The screen was based on the epigenetic relationship between GATOR1 and GATOR2. In Drosophila, mutations in the GATOR2 components seh1, cause the constitutive activation of the GATOR1 complex in the female germline, resulting in the permanent inhibition of TORC1 activity and a block to oocyte growth and development. In order to identify genes that, when co-depleted with seh1, rescue the seh1RNAi ovarian phenotypes we screened approximately 3000 RNAi lines from the Transgeneic RNAi Project (TRiP) that were optimized for germline expression. From this screen, we identified approximately 90 genes that when depleted, allow for the growth of seh1 depleted ovaries. We predict that many of these genes will be upstream activators or downstream effectors of the GATOR1 complex. Importantly, as anticipated, the screen identified multiple known regulators of TORC1 including Tsc1, Tsc2 and PTEN. Notably, we identified a new gene, the putative RNA binding protein DDX42, as an important effector of a stress response in the female germline. Additionally, we determined that the Ragulator, as well as RagA and RagC, recruit the TORC1 inhibitor TSC to lysosomes to affect the downregulation of TORC1 activity upon GATOR1 activation. To better understand the role of the GATOR complex, as well as the identified GATOR interacting proteins, we have used CRISPR/CAS9 methodologies to generate reagents that allow for the examination of the intracellular localization of TORC1, GATOR1, GATOR2 and TSC components in vivo.
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