ppGpp Is Present in and Functions to Regulate Sleep in Drosophila

Discovery of molecules in living systems and demonstration of their functional roles are pivotal in furthering our understanding of the molecular basis of biology. ppGpp (guanosine-5’-diphosphate, 3’-diphosphate) has been detected in prokaryotes for more than five decades. Here we report that a genetic screen followed by chemical analysis revealed the presence of ppGpp in Drosophila. It can be detected in germ-free Drosophila and its level is controlled by an enzyme encoded by the mesh1 gene in Drosophila. Loss of function mutations in mesh1, which encoded the ppGpp degrading enzyme led to longer sleep latency and less total sleep. These phenotypes could be rescued by wild type mesh1, but not by the enzymatically defective mutant Mesh1E66A, functionally implicating ppGpp. Ectopic expression of RelA, the E. coli synthetase for ppGpp, phenocopied mesh1 knockout mutants, whereas overexpression of mesh1 resulted in the opposite phenotypes, supporting that ppGpp is both necessary and sufficient in sleep regulation. mesh1 is expressed in a specific population of neurons, and a chemoconnectomic screen followed by genetic intersection experiments implicate the pars intercerebralis (PI) as the site of ppGpp function. Our results have thus revealed that ppGpp is present in animals after long lag since its discovery in bacteria. Furthermore, we have demonstrated that ppGpp in a specific subset of neurons plays a physiological role in regulating sleep. We speculate that ppGpp may play function in mammals.

degrading enzyme led to longer sleep latency and less total sleep. These phenotypes  It was 52 years ago when guanosine-5'-diphosphate, 3'-diphosphate (guanosine 37 tetraphosphate, ppGpp) and guanosine-5'-triphosphate, 3'-diphosphate (guanosine 38 pentaphosphate, pppGpp) were implicated in gene regulation in Escherichia coli (E. S1D) and 16S rDNA PCR ( Figure S1E). ppGpp was detected in both fed and starved Mesh1 gene is mainly involved in regulating sleep latency. 128 We assessed awakening according to a procedure reported recently (Tabuchi et al., 129 2018). Awakening number at the beginning of nighttime sleep (dusk) ( Figure 3G) and To examine the expression pattern of the mesh1 gene, we crossed M1KOGal4 with 164 each of the following four UAS lines: UAS-mCD8-GFP for neuronal overviews (Lee 165 and Luo, 1999), UAS-redStinger for nuclei (Barolo, Castro and Posakony, 2004), 166 UAS-denmark for dendrites (Nikolai et al., 2010) and UAS-syt.eGFP for axon 167 9 terminals (Zhang, Rodesch and Broadie, 2002). 168 mesh1 was found in neurons in the brain and the ventral nerve cord ( Figure 5A 169 and 5E). In the brain, mesh1 expressing neurons were detected in the PI and the   ((Cashel and Gallant, 1969;Dalebroux and Swanson, 2012;Gourse et al., 180 2018;Hauryliuk et al., 2015;Magnusson, Farewell and Nyström, 2005;Potrykus et 181 al., 2008;Wang, Sanders and Grossman, 2007), it is possible that ppGpp functions in 182 many, even possibly all cells in animals. To test whether ppGpp functions in specific 183 cells, we increased and decreased ppGpp levels in different populations of cells, by 184 using ppGpp synthesizing and hydrolyzing enzymes.

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The E. coli RelA gene encodes a synthetase for ppGpp (Laffler and Gallant, 1974). 186 We confirmed that the RelA from E. coli used by us indeed increased ppGpp in vitro 187 ( Figure S1A). The biochemical activity of RelA protein is opposite to that of the 188 Mesh1 protein, the effect of RelA overexpression in flies should be similar to mesh1 189 10 knockout mutation. In flies with UAS-RelA driven either by tub-Gal4 for expression 190 in all cells (O'Donnell et al., 1994) or by elav-Gal4 for expression in neurons 191 (Robinow and White, 1991), sleep latency ( Figure 6A) and awakening number were 192 increased ( Figure 6B). By contrast, UAS-RelA driven by repo-Gal4 for expression in 193 glial cells (Halter et al., 1995) did not affect sleep ( Figure 6A and 6B). These results    (Cannel et al., 2016;Chen andDahanukar, 2018), Dilp2 (Crocker et al., 2010;224 Semaniuk et al., 2018;Yurgel et al., 2019) and SIFa (Martelli et al., 2017;Park et al., 225 2014), and one line for receptor R19G10 (an RYamide receptor) (Collins et al., 2011;226 Ohno et al., 2017). We crossed each of these PI drivers to a line of knockin flies with

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We investigated whether ppGpp played a role in starvation induced sleep loss (SISL).

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Similar to humans (MacFadyen, Oswald and Lewis, 1973), flies also show SISL 249 (Keene et al., 2010). Baseline sleep in fed flies was recorded before flies were starved 250 for 24 hours (Yurgel et al., 2019). Sleep during starvation was compared to that before 251 starvation ( Figure 8A).       292 We have shown that ppGpp is present in Drosophila and that it is hydrolyzed by         In bacteria, the best-known direct target of ppGpp is RNA polymerase (RNAP) (e.g.,

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Comparison was based on t-test with *** denoting p < 0.001.    and CCT-Gal4 library was a collection previously generated in our lab (Deng et al., . isoCS and w 1118 were wild-type and white-eye wild-type lines.