Studying RNA-DNA interactome by Red-C identifies noncoding RNAs associated with various chromatin types and reveals transcription dynamics
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ABSTRACT: Noncoding RNAs (ncRNAs) participate in various biological processes, including regulation of transcription and sustaining genome 3D organization. Here, we present a method called Red-C, which exploits proximity ligation to identify contacts with the genome for all RNA molecules present in the nucleus. Using Red-C, we uncover RNA-DNA interactome of human K562 cells and identify hundreds of ncRNAs enriched in active or repressed chromatin. We found two miRNAs, MIR3648 and MIR3867 transcribed from rRNA locus, that are associated with inactive chromatin genome-wide. These miRNAs favor bulk heterochromatin over Polycomb repressed one and interact preferentially with late-replicating genomic regions. Analysis of RNA-DNA interactome also allowed us to trace the kinetics of mRNA production. Our data support the model of co-transcriptional intron splicing, however, surprisingly, contradict the hypothesis of circularization of actively transcribed genes.
Project description:Non-coding RNAs (ncRNAs) are transcribed throughout the genome and provide regulatory inputs to gene expression through their interaction with chromatin. Yet, the genomic targets and functions of most ncRNAs are unknown. Here we use chromatin-associated RNA sequencing (ChAR-seq) to map the global network of ncRNA interactions with chromatin in human embryonic stem cells, and the dynamic changes in interactions during differentiation into definitive endoderm. We uncover general principles governing the organization of the RNA-chromatin interactome, demonstrating that nearly all ncRNAs exclusively interact with genes in close three-dimensional proximity to their locus, and provide a model predicting the interactome. We uncover RNAs that interact with many loci across the genome, and unveil thousands of unannotated RNAs that dynamically interact with chromatin. By relating the dynamics of the interactome to changes in gene expression, we demonstrate that activation or repression of individual genes is unlikely to be controlled by a single ncRNA.
Project description:RNA is an essential component of the chromatin, influencing genome organization and expression. To investigate the role of lncRNAs in epigenetic activation, we profiled the RNA interactome of SWI/SNF complexes in proliferating and senescent conditions, when cells undergo widespread chromatin rearrangements. The isolation of transcripts associated to SMARCB1 core subunit, together with its chromatin binding profiling, showed prevalent association to active regions where it differentially binds ncRNAs locally transcribed.
Project description:Noncoding RNAs (ncRNAs) are an emerging class of regulatory molecules with a broad range of regulatory functions believed to be mediated by ncRNA-chromatin interactions. Genome-wide understanding of ncRNA functions requires precise mapping of all ncRNAs and their target loci. Current methods for studying chromatin-associated ncRNA lack specificity or are limited to singly assessing RNAs. We devised an unbiased strategy to identify all RNA Interactions with Chromatin by Paired-End-Taging (RICh-PET) and applied this approach to characterize the Drosophila RNA-chromatin interactome. We discovered that ncRNAs primarily target promoters and enhancers in open chromatin regions in colocalization with RNAPII and other TFs, suggesting combinatorial regulatory instructions for each locus. Enzymatic nuclear digestion followed by examination of specific chromatin loci indicated that ncRNAs collectively promote chromatin accessability, RNAPII-mediated interactions and overall 3D-genome organization. Our study demonstrates that RICh-PET and related methods represent a powerful suite of tools to interrogate the genome biology of ncRNAs.
Project description:RNA is an essential component of the chromatin, influencing genome organization and expression. To investigate the role of lncRNAs in epigenetic activation, we profiled the RNA interactome of SWI/SNF complexes in proliferating and senescent conditions, when cells undergo widespread chromatin rearrangements. The isolation of transcripts associated to SMARCB1 core subunit, together with its chromatin binding profiling, showed prevalent association to active regions where it differentially binds ncRNAs locally transcribed. Among them, LINC00565 interacts with SMARCB1 exclusively in proliferating conditions, exerting a pro-oncogenic role in tumors with intact SWI/SNF complexes. LINC00565 is transcribed from an enhancer region and through binding to SMARCB1, controls the activation of GAS6 oncogene as part of a topologically organized region, as well the activation of a larger set of genes by favoring SMARCB1 binding. Our results indicate that LINC00565 influences the ability of the SWI/SNF complexes to drive epigenetic activation of specific promoters, suggesting a SWI/SNF-RNA cooperation to achieve transcriptional activation.
Project description:RNA is an essential component of the chromatin, influencing genome organization and expression. To investigate the role of lncRNAs in epigenetic activation, we profiled the RNA interactome of SWI/SNF complexes in proliferating and senescent conditions, when cells undergo widespread chromatin rearrangements. The isolation of transcripts associated to SMARCB1 core subunit, together with its chromatin binding profiling, showed prevalent association to active regions where it differentially binds ncRNAs locally transcribed. Among them, LINC00565 interacts with SMARCB1 exclusively in proliferating conditions, exerting a pro-oncogenic role in tumors with intact SWI/SNF complexes. LINC00565 is transcribed from an enhancer region and through binding to SMARCB1, controls the activation of GAS6 oncogene as part of a topologically organized region, as well the activation of a larger set of genes by favoring SMARCB1 binding. Our results indicate that LINC00565 influences the ability of the SWI/SNF complexes to drive epigenetic activation of specific promoters, suggesting a SWI/SNF-RNA cooperation to achieve transcriptional activation.
Project description:Non-poly(A) RNA molecules including noncoding RNAs (ncRNAs) comprise the major portion of the total transcribed molecules in the cell. In addition to the mRNAs the ncRNAs also function as ribonucleoprotein particles (RNPs) and carry out biological functions including synthesis of new proteins, RNA processing, genome remodelling and regulation of transcription. We therefore envisaged a comprehensive transcriptome-wide identification of coding and non-coding RNA-binding proteins (RBPs) in the Leishmania spp. Towards this we applied the recently reported orthogonal organic phase separation (OOPS) method in combination with tandem mass tag (TMT) labelling-based quantitative proteomic mass spectrometry (MS) and report herein the most comprehensive identification of RBPs in Leishmania mexicana (L. mexicana) parasites. This study identified novel RNA binding property of thousands of L. mexicana proteins, significantly expanding the RBP landscape of the parasite. Furthermore, we showed that the classical Hsp90 inhibitor tanespimycin differentially regulates the RNA-binding property of hundreds of L. mexicana RBPs, shedding light into hitherto unknown large-scale downstream molecular effects of the small molecule inhibitor in the parasite.
Project description:The majority of the eukaryotic genome is transcribed into non-coding RNAs (ncRNAs), which are important regulators of different biological processes in the cell nucleus as part of the machinery controlling chromatin structure. However, the full extent of ncRNAs function has remained elusive. Here we deciphered the function of the microRNA Mirlet7d as a key regulator of the expression of bi-directionally transcribed genes. We found that nuclear Mirlet7d binds ncRNAs expressed from these genes. The Mirlet7d-ncRNA duplexes are further bound by C1D, which in turn targets the RNA exosome complex and EZH2 to the bi-directionally active loci. The exosome degrades the ncRNAs, whereas EZH2 induces heterochromatin and transcriptional silencing. Moreover, this multicomponent RNA-protein complex, which we called MiCEE, tethers the regulated genes to the perinucleolar region, thereby being required for proper nucleolus organization. Our study demonstrates that the MiCEE complex mediates epigenetic silencing of bi-directionally expressed genes and global genome organization.
Project description:We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics of newly synthesized proteins (pulsed stable isotope labeling with amino acids in cell culture, pSILAC) in combination with mRNA and non-coding RNA expression analyses by next generation sequencing (RNA-, miR-Seq) in the colorectal cancer (CRC) cell line SW480. Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down), lncRNAs (270 up, 123 down) and proteins (542 up, 569 down). Changes in mRNA and protein expression levels showed a positive correlation (r = 0.50, p < 0.0001). More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the downregulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3â??-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed upregulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibited proliferation in SW480 cells. Furthermore, HMGB1, KLF12 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of HMGB1, KLF12 and CIT was detected in advanced stages of cancer. This study provides new insights and a comprehensive catalogue of p53-mediated regulations and p53 DNA binding in CRC cells.