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.
Project description:Chromatin insulators are functionally conserved DNA-protein complexes that are situated throughout the genome and organize independent transcriptional domains. Previous work implicated RNA as an important cofactor in chromatin insulator activity, although the mechanisms by which RNA affects insulator activity are not yet understood. Here we identify the exosome, the highly conserved major cellular 3’ to 5’ RNA degradation machinery, as a physical interactor of CP190-dependent chromatin insulator complexes in Drosophila. High resolution genome-wide profiling of exosome by ChIP-seq in two different embryonic cell lines reveals extensive and specific overlap with the CP190, BEAF-32, and CTCF insulator proteins. Colocalization occurs mainly at promoters but also well-characterized boundary elements, such as scs, scs’, Mcp, and Fab-8. Surprisingly, exosome associates primarily with promoters but not gene bodies, arguing against simple cotranscriptional recruitment to RNA substrates. We find that exosome is recruited to chromatin in a transcription dependent manner, preferentially to highly transcribed genes. Similar to insulator proteins, exosome is also significantly enriched at divergently transcribed promoters. Directed ChIP of exosome in cell lines depleted of insulator proteins shows that CTCF is specifically required for exosome association at Mcp and Fab-8 but not other sites, suggesting that alternate mechanisms must also contribute to exosome chromatin recruitment. Taken together, our results reveal a novel relationship between exosome and chromatin insulators throughout the genome. ChIP-seq of exosome components. RNA-seq after control and exosome subunit knockdown in Drosophila cell lines.
Project description:We have sequenced messenger RNA isolated from seedling tissue for 19 accessions of Arabidopsis thaliana (with biological replication). The 19 accessions for which RNA-Seq reads were collected have served as the founders for the MAGIC lines, a high-resolution recombinant inbred line mapping resource. RNA sequencing data was used to examine differential gene expression among the accessions.