SnoRNA:U3:9B is required for the activation of immune response genes in Drosophila melanogaster [RNA-Seq]

Small nucleolar RNAs (snoRNAs) are prevailing components of the chromatin-associated transcriptome. We have studied a specific chromatin-associated snoRNA, snoRNA:U3:9B, that binds to a large number of protein coding genes in the genome of Drosophila melanogaster, including immune response genes. We have used CRISPR/Cas9 to delete snoRNA:U3:9B. SnoRNA:U3:9B-deficient larvae were viable in control conditions but failed to develop into pupae when challenged by expression of a Sindbis virus replicon, which suggests that snoRNA:U3:9B is required for the activation of an effective antiviral response. Consistent with this hypothesis, the chromatin decompaction and gene activation typically observed at immune response genes in response to Sindbis replicon expression were abolished in snoRNA:U3:9B-deficient larvae. Moreover, snoRNA:U3:9B was required for the recruitment of the ATP-dependent helicase brm to a set of target immune genes. The association of snoRNA:U3:9B with immune response genes in vivo shown by ChIRP-qPCR suggests that the chromatin and gene expression defects observed in the snoRNA:U3:9B knock-out strains are due to direct regulatory events. In summary, our findings reveal an immune defence mechanism that relies on a snoRNA for the recruitment of chromatin remodelling factors to immune genes, thereby modulating local chromatin accessibility to facilitate immune gene activation. Total RNA was extracted using Trizol (Ambion). Total RNA samples consist in pooled biological triplicates. For chromatin-associated RNA extraction, S2 cells were crosslinked with formaldehyde for 10 min at RT and the cross-linking was stopped by addition of 1 M glycine for 10 additional min. RNAs from chromatin fraction were size fractionated into 0-500bp and > 500bp with AMPure beads (1.6X and 0.6X concentration respectively). 0-500bp were directly used for library preparation and > 500 bp RNAs were sonicated prior library preparation. Chromatin samples consist of 3 biological triplicates.