Abstract
microRNAs play an important role in the regulation of sleep. Previous studies have identified bantam as a sleep-promoting microRNA in Drosophila. We sought to characterize the role of bantam in sleep regulation by identifying which neurons require bantam for normal sleep maintenance and determining the cellular and molecular mechanisms through which bantam regulates sleep. Using split-GAL4 lines to express a bantam inhibitor (sponge) in a cell-specific manner, we found that bantam regulates sleep through multiple mushroom body output neurons (MBONs), a class of neurons previously implicated in sleep regulation. To determine whether bantam regulates the morphology of the MBONs, we imaged brains of flies co-expressing a fluorescent marker and the bantam sponge in MBONs of interest, and found that bantam does not regulate the gross morphology or cell number of the neurons tested in either the adult or developing fly. We employed two strategies to identify the mRNA targets of bantam: a bioinformatic pipeline to predict conserved targets of bantam, and RNA sequencing of γ2α’1 MBONs expressing the bantam sponge. Three potential sleep-relevant targets of bantam were identified: GAD1, Ptp69D, and fog. RNA sequencing identified 73 predicted targets of bantam that were upregulated in γ2α’1 MBONs expressing the bantam sponge, and gene ontology analysis predicted vesicle and active zone maintenance as potential biological pathways regulated by bantam in these MBONs. Our results identify a role for bantam in the adult regulation of sleep in specific subtypes of MBONs and identify GAD1, Ptp69D, and fog as potential mRNA targets of bantam.