Actin and nuclear myosin 1 (NM1) are regulators of transcription and chromatin organization. Using a genome-wide approach, we report here that beta-actin binds intergenic and genic regions across the mammalian genome, associated with both protein-coding and rRNA genes. Within the rDNA, the distribution of beta-actin correlated with NM1 and the other subunits of the B-WICH complex, WSTF and SNF2h. In beta-actin(-/-) mouse embryonic fibroblasts (MEFs), we found that rRNA synthesis levels decreased concomitantly with drops in RNA polymerase I (Pol I) and NM1 occupancies across the rRNA gene. Reintroduction of wild-type beta-actin, in contrast to mutated forms with polymerization defects, efficiently rescued rRNA synthesis underscoring the direct role for a polymerization-competent form of beta-actin in Pol I transcription. The rRNA synthesis defects in the beta-actin(-/-) MEFs are a consequence of epigenetic reprogramming with up-regulation of the repressive mark H3K4me1 (mono-methylation of lys4 on histone H3) and enhanced chromatin compaction at promoter-proximal enhancer (T0 sequence), which disturb binding of the transcription factor TTF1. We propose a novel genome-wide mechanism where the polymerase-associated beta-actin synergizes with NM1 to coordinate permissive chromatin with Pol I transcription, cell growth, and proliferation.

RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. Here we show that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. We show that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network revealed physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrated that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, our results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin.

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3 beta phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3 beta selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3 beta(-/-) mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3 beta directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3 beta-mediated phosphorylation of NM1 is required for pol I transcription activation.

We have raised antibodies against the profilin of Chironomus tentans to study the location of profilin relative to chromatin and to active genes in salivary gland polytene chromosomes. We show that a fraction of profilin is located in the nucleus, where profilin is highly concentrated in the nucleoplasm and at the nuclear periphery. Moreover, profilin is associated with multiple bands in the polytene chromosomes. By staining salivary glands with propidium iodide, we show that profilin does not co-localize with dense chromatin. profilin associates instead with protein-coding genes that are transcriptionally active, as revealed by co-localization with hnRNP and snRNP proteins. We have performed experiments of transcription inhibition with actinomycin D and we show that the association of profilin with the chromosomes requires ongoing transcription. however, the interaction of profilin with the gene loci does not depend on RNA. Our results are compatible with profilin regulating actin polymerization in the cell nucleus. however, the association of actin with the polytene chromosomes of C. tentans is sensitive to RNase, whereas the association of profilin is not, and we propose therefore that the chromosomal location of profilin is independent of actin.

Rrp6 is an exoribonuclease involved in the quality control of mRNA biogenesis. We have analyzed the association of Rrp6 with the Balbiani ring pre-mRNPs of Chironomus tentans to obtain insight into the role of Rrp6 in splicing surveillance. Rrp6 is recruited to transcribed genes and its distribution along the genes does not correlate with the positions of exons and introns. In the nucleoplasm, Rrp6 is bound to both unspliced and spliced transcripts. Rrp6 is released from the mRNPs in the vicinity of the nuclear pore before nucleo-cytoplasmic translocation. We show that Rrp6 is associated with newly synthesized transcripts during all the nuclear steps of gene expression and is associated with the transcripts independently of their splicing status. These observations suggest that the quality control of pre-mRNA splicing is not based on the selective recruitment of the exoribonuclease Rrp6 to unprocessed mRNAs.

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.

In the brain, sophisticated networks of RNA regulatory events tightly control gene expression in order to achieve proper brain function. We and others have previously shown that several miRNAs, encoded within the miR-379-410 cluster, are subjected to A-to-I RNA editing. In the present study we conclude these edited miRNAs to be transcribed as a single long consecutive transcript, however the maturation into functional forms of miRNAs is regulated individually. In seven of the miRNAs, subjected to editing, we analyze how editing relates to miRNA maturation. Of particular interest has been maturation of miR-381-3p and miR-376b-3p, both important for neuronal plasticity, dendrite outgrowth and neuronal homeostasis. Most of the edited miRNAs from the cluster, are highly edited in their unprocessed primary transcript, including miR-381-3p and miR-376b-3p. However, editing in miR-381-3p is almost entirely absent in the mature form, while editing is increased in the mature form of miR-376b-3p compared to the primary transcript. We propose that ADAR1 positively influences the maturation of pri-miR-381 in an editing independent manner. In pri-miR-376b we hypothesize that ADAR1 and ADAR2 competes for editing, and while ADAR2 inhibits miRNA maturation, ADAR1 editing is frequently present in the mature miR-376b-3p. We further show that miR-381-3p and miR-376b-3p regulate the dendritically expressed Pumilio 2 (Pum2) protein. By next generation RNA sequencing (NGS RNA-seq) on purified synaptoneurosomes, we show that miR-381-3p is highly expressed at the synapse, suggesting its functional role in locally regulating Pum2. Furthermore, we identify a set of highly expressed miRNAs at the synapse, which may act locally to target synaptic mRNAs.

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.