The circular RNA circ_ATP8B regulates ROS production and antiviral immunity [RNA-Seq]
Circular RNAs (circRNAs) are widely expressed in eukaryotes. However, only a subset has been functionally characterized. We have identified and validated a collection of circular RNAs in Drosophila melanogaster. We show that the circular RNA circ_ATP8B is induced by viral infection and that depletion of circ_ATP8B, but not its linear sibling, compromises viral infection both in cultured cells and in vivo. In addition, our analyses reveal that circ_ATP8B is enriched in the fly gut and that gut-specific depletion of circ_ATP8B attenuates viral replication in an oral infection model. Furthermore, we find circ_ATP8B-depletion resulted in increased levels of reactive oxygen species (ROS) and enhanced expression of Duox (Dual oxidase), which produces ROS. Genetic and pharmacological manipulation of circ_ATP8B-depleted flies that reduce ROS levels rescue the viral replication defects elicited by circ_ATP8B depletion. Notably, circ_ATP8B and Duox associate with each other, and that expression of various versions of circ_ATP8B that are competent in binding Duox, but not a mutant circ_ATP8B that is incapable of binding Duox, restores physiological levels of ROS in circ_ATP8B-depleted cells. Lastly, our data show that Gaq, a subunit of G protein required for optimal Duox activity, acts downstream of circ_ATP8B to regulate Duox activity. We conclude that circ_ATP8B regulates anti-viral immunity by modulating Duox-dependent ROS production. Overall design: This study aims to identify RNAs that are differentially expressed upon depletion of the circular RNA circ_ATP8B in vivo. Flies carrying a shRNA transgene against circ_ATP8B or a control shRNA transgene against GFP were crossed to flies carrying the ubiquitously expressed Actin (Act)-Gal4 driver. Total RNAs were extracted from 4-7 day old male and female Act>shGFP and Act>sh-circ_ATP8B flies and subjected to RNA-seq to identify genes that are impacted by circ_ATP8B depletion.