Small nucleolar RNAs (snoRNAs) are prevailing components of the chromatin-associated transcriptome. Here we show that specific snoRNAs are required for the activation of immune response genes and for survival during viral infections in Drosophila melanogaster. We have studied snoRNA:U3:9B, a chromatin-associated snoRNA that binds to a large number of protein coding genes, including immune response genes. We have used CRISPR/Cas9 to delete snoRNA:U3:9B and study its function in vivo. SnoRNA:U3:9B-deficient larvae are viable but failed to develop into pupae when challenged by expression of a Sindbis virus replicon. SnoRNA:U3:9B is localized to immune genes in vivo and the chromatin decompaction and gene activation typically observed at immune genes following infection are abolished in snoRNA:U3:9B-deficient larvae, which suggests that this snoRNA acts locally to regulate chromatin accessibility. Mechanistically, snoRNA:U3:9B is required for the recruitment of the chromatin remodeler Brahma to a set of target immune genes. In summary, these results uncover an antiviral defense mechanism that relies on a snoRNA for the recruitment of a chromatin remodeling factor to immune genes to facilitate immune gene activation.

The epigenetic mechanisms regulating developmental gene expression are examples of a strategy to generate unique expression profiles with global regulators controlling several genes. In a simplified view, a common set of tools, that include DNA motif recognizing proteins (recruiters), binding/interacting surfaces (ARPs- actin related proteins), epigenetic writers (histone methyltransferases, acetylases), readers (chromatin remodeling proteins, PRC1 members) and erasers (demethylases, deacetylases) form complexes which not only regulate transcription, but also retain the transcriptional memory through mitosis. There are two arms of epigenetic regulation: covalent modification of DNA and the post-translational modification of histones. In this review, we discuss both of these aspects briefly to illustrate functional diversity. We discuss our efforts at utilization of the genome sequence data for de novo identification of new players and their functional validation in this remarkable process.

SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3′ end maturation by facilitating 3′ end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3′ end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.

Small nucleolar RNAs (snoRNAs) are prevailing components of the chromatin-associatedtranscriptome and many orphan snoRNAs are associated with protein coding genes in the genome ofDrosophila melanogaster. We have studied a specific chromatin-associated snoRNA, snoRNA:U3:9B,that binds to immune response genes. Using a Sindbis virus replicon model, we have shownthat snoRNA:U3:9B depletion in S2 cells leads to reduced immune response gene expression andreduced chromatin accessibility at target immune response genes. We have used CRISPR/Cas9 tocreate a snoRNA:U3:9B knock-out fly strain and revealed that snoRNA:U3:9B-deficient larvae areviable in control conditions, but fail to develop into pupae when challenged by expression of the Sindbisvirus replicon, which suggests that this snoRNA is essential for the activation of an effective antiviralresponse. In agreement with this proposal, the chromatin decompaction and gene activation normallyobserved at immune response gene loci in response to Sindbis replicon expression are abolished inthe snoRNA:U3:9B-deficient larvae, as shown by ATAC-qPCR and RT-qPCR analyses. Moreover,ChIRP-qPCR experiments have shown that snoRNA:U3:9B associates with the immune responsegenes in vivo, which suggests that the defects observed on chromatin compaction and gene expressionare due to direct regulatory events. In summary, our results reveal the existence of an epigeneticmechanism that requires snoRNA:U3:9B to modulate local chromatin accessibility and enable theinduction of immune response genes.

Chromatin-associated RNAs (caRNAs) modulate chromatin organization and function. The RNAexosome degrades different types of nuclear transcripts, but its role in chromatin has not beenaddressed. Here we have used Drosophila melanogaster S2 cells as a model system to identify therepertoire of caRNAs and establish the role of the exosome in their regulation. We have analyzed bothunique and repetitive sequences, and combining RNA-seq and ATAC-seq we show that thesimultaneous depletion of the exosome catalytic subunits RRP6 and DIS3 not only affects caRNAlevels but also changes the local chromatin accessibility at specific loci. We have identified a group ofexosome-sensitive genes that are involved in developmental regulation and are characterized by abalanced chromatin state in which Polycomb and Trithorax factors coexist. Our results reveal that RNAdegradation by the exosome is an important mechanism for the homeostasis of such balancedchromatin states. Given that eukaryotic genomes are repetitive to a large extent, we have also analyzed repetitive caRNAs (rep-caRNAs) and we show that the exosome is needed to control repcaRNAlevels and to maintain the degree of chromatin packaging in repetitive genomic regions. Thisrole is particularly relevant in the pericentromeric regions where the exosome is required to silenceLTR elements and maintain centromere organization.