Expression data from Drosophila S2 cells depleted of exosome subunits
ABSTRACT: The exosome complex and its' subunits are vital for proccessing and or degrading many cellular RNAs. We used microarrays to detail the global changes in mRNA levels in response to exosome subunit depletion. Overall design: RNA was extracted from dsRNA treated S2 cells and hybridized using Affymetrix microarrays. We aimed to obtain a master set of mRNAs surveyed by exosme subunits in vivo.
Project description:The exosome complex and its' subunits are vital for proccessing and or degrading many cellular RNAs. We used microarrays to detail the global changes in mRNA levels in response to exosome subunit depletion. RNA was extracted from dsRNA treated S2 cells and hybridized using Affymetrix microarrays. We aimed to obtain a master set of mRNAs surveyed by exosme subunits in vivo.
Project description:Expression profiling following depletion of Mediator Cdk8 module subunits Cdk8, Cyclin C (CycC), Med12 and Med13 72 hours after dsRNA treatment of Drosophila melanogaster S2 cells. Results provide insight into the role of individual Cdk8 module subunits in regulation of transcription. Overall design: 22 samples. 2 Cdk8 dsRNA, 4 CycC dsRNA, 4 Med12 dsRNA, 4 Med13 dsRNA, 8 control samples including 4 Luciferase (Luc) dsRNA and 4 GFP dsRNA
Project description:Expression profiling following depletion of Mediator Cdk8 module subunits Cdk8, Cyclin C (CycC), Med12 and Med13 72 hours after dsRNA treatment of Drosophila melanogaster S2 cells. Results provide insight into the role of individual Cdk8 module subunits in regulation of transcription. 22 samples. 2 Cdk8 dsRNA, 4 CycC dsRNA, 4 Med12 dsRNA, 4 Med13 dsRNA, 8 control samples including 4 Luciferase (Luc) dsRNA and 4 GFP dsRNA
Project description:Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex. Identification of targets for individual exosome subunits in wild-type and mutant yeast cells.
Project description:The exosome complex plays a central role in RNA metabolism, and each of its core subunits is essential for viability in yeast However, comprehensive studies of exosome substrates and functional analyses of its subunits in multi.CELlular eukaryotes are lacking Here we show that, in sharp contrast to yeast and metazoan exosome complexes, individual subunits of the plant exosome core are functionally specialized Using whole-genome oligonucleotide tiling microarray analyses of csl4 null mutant plants and conditional genetic depletions of RRP4 and RRP41, we uncovered unexpected functional plasticity in the plant exosome core as well as generated a set of high-resolution genome-wide maps of Arabidopsis exosome targets These analyses provide evidence for widespread polyadenylation- and exosome-mediated RNA quality control in plants and reveal novel aspects of stable structural RNA metabolism Finally, numerous novel exosome substrates were discovered, including a select subset of mRNAs, miRNA processing intermediates, and hundreds of noncoding RNAs, the vast majority of which have not been previously described This large collection of RNAs belong to a Òdeeply hiddenÓ layer of the transcriptome that is tightly repressed and can only be visualized upon inhibition of exosome activity These first genome-wide maps of exosome substrates will aid in illuminating new fundamental components and regulatory mechanisms of eukaryotic transcriptomes Keywords: Strand-specific gene expression analysis using whole-genome oligonucleotide tiling microarray analyses of three exosome subunits, using csl4 null mutant plants and conditional genetic depletions of RRP4 and RRP41. Overall design: Two biological replicates were done for each sample on each chromosomal strand (Watson and Crick) which meant a total of four whole-genome oligonucleotide tiling microarrays were used per sample. We studied a total of 8 biological samples for this project Overall, a total of 32 whole-genome oligonucleotide tiling microarrays were used for this study
Project description:The nuclear RNA exosome is an essential multi-subunit complex that controls RNA homeostasis. Congenital mutations in exosome genes are associated with neurodegenerative diseases. Here, we show that transient depletion of nuclear RNA exosome subunits in epithelial cells inhibits influenza virus replication. Similarly, viral biogenesis was suppressed in cells derived from mice with conditional ablation of the RNA exosome subunit Exosc3. Furthermore, patient-derived cells with a congenital EXOSC3 mutation were less susceptible to influenza virus infection. Using proteomics and next generation sequencing during infection, we show that the viral polymerase complex (PA, PB2, PB1) co-opts the nuclear RNA exosome complex and cellular RNAs en route to 3’ end degradation. Mechanistically, the nuclear RNA exosome coordinates the initial steps of viral transcription with RNAPII at host promoters. Exosome deficiency uncouples chromatin targeting of the viral polymerase complex and the formation of cellular:viral RNA hybrids, which are essential RNA intermediates that license transcription of antisense genomic viral RNAs. Overall, we discovered a critical nexus between an essential component of the influenza virus (polymerase) and an essential component of the cell (exosome), alteration of which leads to breakage of host-pathogen symmetry and a lose-lose scenario (viral impairment and neurodegeneration). Overall design: (1) Examination of influenza RNA levels in murine B cells conditionally ablated for the RNA exosome subunit Exosc3 (2) Examination of both host:viral chimeric mRNAs and the cellular transcriptome in human A549 cells transiently depleted for RNA exosome subunits DIS3 or EXOSC10; along with siRNA control (3) Examination of both host:viral chimeric mRNAs and the cellular transcriptome in human A549 cells transiently transfected with a plasmid encoding human DIS3 or an empty vector (EV) control
Project description:The Microprocessor complex (DGCR8/Drosha) is required for microRNA (miRNA) biogenesis but also binds and regulates the stability of several types of cellular RNAs. Of particular interest, DGCR8 controls the stability of mature small nucleolar RNA (snoRNA) transcripts independently of Drosha, suggesting the existence of alternative DGCR8 complex/es with other nucleases to process a variety of cellular RNAs. Here, we found that DGCR8 co-purifies with subunits of the nuclear exosome, preferentially associating with its hRRP6-containing nucleolar form. Importantly, we demonstrate that DGCR8 is essential for the recruitment of the exosome to snoRNAs and to human telomerase RNA. In addition, we show that the DGCR8/exosome complex controls the stability of the human telomerase RNA component (hTR/TERC). Altogether, these data suggests that DGCR8 acts as a novel adaptor to recruit the exosome complex to structured RNAs and induce their degradation. [i] Examination of the RNA binding profile of hRRP6 (also known as EXOSC10) via iCLIP. [ii] HeLa cells were transiently depleted of hRRP6 or DGCR8 using siRNAs. For a control an non-targetting (siNon) siRNA was used. Three biological replicates of each samples were sent for RNA sequencing.
Project description:The RNA exosome complex functions in both the accurate processing and rapid degradation of many different classes of RNA. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but it was unclear how many substrates follow each pathway in vivo. To address this we used UV crosslinking in growing cells to identify transcriptome-wide interactions of RNAs with the major nuclear exosome-cofactor Mtr4 and with individual exosome subunits (Rrp6, Csl4, Rrp41 and Rrp44) along the threaded RNA path. We performed comparative analyses on exosome complexes lacking the exonucleolytic activity of Rrp44, either carrying a mutation in the Rrp44 S1 RNA-binding domain, predicted to disfavor direct access to the Rrp44 exonuclease active site, or with multiple mutations in Rrp41, reported to block RNA passage through the central channel. Our analysis identified targets using preferentially channel-threading pathway such as mRNAs, 5S rRNA or scR1. Our results suggest as well that aborted tRNAs transcripts, released during transcription, would be rapidly degraded using this route. Alternatively, pre-tRNAs appears to access Rrp44 directly. Both routes seems to be involved for degradation or maturation of RNAPI transcripts. The Rrp41 mutations were found to block substrate passage to Rrp44 only for cytoplasmic mRNAs, apparently confirming the prediction of widening of the lumen in the nuclear, Rrp6-associated complex. Overall design: 13 samples were analysed from strains carrying HTP tagged protein. Duplicate CRAC experiments were carried out for each protein