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RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions (CAGE)

ABSTRACT: Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs,, methods to massively map the genomic interacting sites of multiple transcripts have been developed. However they present still some limitations. Here, we introduce RNA and DNA interacting complexes ligated and sequenced (RADICL-seq), a novel technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation based methodology that reduces the bias for nascent transcription, increases genomic coverage and unique mapping rate efficiency compared to existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and emphasizes the role of transcription in the establishment of chromatin structure.

ORGANISM(S): Mus musculus

PROVIDER: GSE132191 | GEO | 2019/12/05

REPOSITORIES: GEO

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RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions (RADICL-seq)

Project description:Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs,, methods to massively map the genomic interacting sites of multiple transcripts have been developed. However they present still some limitations. Here, we introduce RNA and DNA interacting complexes ligated and sequenced (RADICL-seq), a novel technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation based methodology that reduces the bias for nascent transcription, increases genomic coverage and unique mapping rate efficiency compared to existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and emphasizes the role of transcription in the establishment of chromatin structure.

2019-12-05 | GSE132190 | GEO

Nascent transcript O-MAP reveals the molecular architecture a single-locus subnuclear compartment built by RBM20 and the TTN noncoding RNA

Project description:We here adapt Oligonucleotide-mediated proximity-interactome mapping (O-MAP), a novel RNA-targeted microenvironment-mapping method, to biochemically characterize the molecular neighborhoods near individual genomic loci. By targeting O-MAP to introns within the TTN pre-mRNA, we have systematically mapped the chromatin loci, RNAs, and proteins within a cardiomyocyte-specific "RNA factory" nucleated on these TTN-derived noncoding transcripts. This revealed an unanticipated compartmental architecture that organizes trans-chromosomal domains into a hub of transcriptionally silenced chromatin, and which recruits dozens of unique RNA-binding and chromatin-scaffolding factors, including QKI and SAFB, along with their target transcripts. Loss of the cardiac-specific splicing factor RBM20, a master regulator of TTN splicing, remodels nearly every facet of factor architecture, suggesting new mechanisms in RBM20-driven dilated cardiomyopathy. This establishes O-MAP as a pioneering method for probing single-locus, microcompartment-level interactions that are opaque to conventional tools, and suggests new mechanisms by which coding genes can "moonlight" as nuclear-architectural noncoding RNAs.

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RADIP technology comprehensively identifies H3K27me3-associated RNA–chromatin interactions

Project description:Many RNAs associate with chromatin, either directly or indirectly. To investigate the function of these RNAs, many technologies for mapping regions where RNAs interact across the genome have been developed. Adding information on the proteins involved in these RNA–chromatin interactions is critical for further analysis. Here, we developed RADIP (RNA and DNA interacting complexes ligated and sequenced (RADICL-seq) with immunoprecipitation), a novel technology that combines RADICL-seq technology with chromatin immunoprecipitation to characterize RNA–chromatin interactions mediated by individual proteins. We applied an anti-H3K27me3 antibody to the RADIP technology and generated libraries from mouse embryonic stem cells. We identified various RNAs, including protein-coding RNAs and non-coding RNAs, that were associated with chromatin via H3K27me3 mediation and that may facilitate the spread of Polycomb repressive complexes over broad regions of the mammalian genome.

2024-11-20 | GSE260447 | GEO

Oligonucleotide-mediated proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ.

Project description:Within all cells, RNA molecules form complex networks of molecular interactions that are central to their function, but discovering these interactions remains challenging. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a straightforward method for elucidating the biomolecules near an RNA of interest, within its native cellular context. O-MAP uses programmable DNA probes to deliver proximity-biotinylating enzymes to a target RNA, enabling molecules within that RNA's subcellular microcompartment to be enriched by streptavidin pulldown. O-MAP induces exceptionally precise in situ biotinylation, and unlike alternative methods it enables straightforward optimization of its RNA-targeting accuracy. Using the 47S pre-ribosomal RNA and long noncoding RNA Xist as models, we develop O-MAP workflows for unbiased discovery of RNA-proximal proteins, transcripts, and genomic loci. This revealed unexpected co-compartmentalization of Xist and other chromatin-regulatory RNAs, and enabled systematic discovery of nucleolar-chromatin interactions across multiple cell lines. O-MAP uses exclusively off-the-shelf parts requiring no genetic- or cell-line engineering and is easily portable across diverse specimen-types and target RNAs. We therefore anticipate its application to a broad array of RNA phenomena.

2024-02-27 | MSV000094186 | MassIVE

ChIP-Chip

Project description:The regulation of protein-coding and noncoding RNAs is linked to nuclear processes including chromatin modifications and gene silencing. However, the mechanisms that distinguish RNAs and mediate their functions are poorly understood. We describe a nuclear RNA processing network in fission yeast with a core module comprising the Mtr4-like protein, Mtl1, and the zinc finger protein Red1. The Mtl1-Red1 core promotes degradation of mRNAs and noncoding RNAs, and associates with different proteins to assemble heterochromatin via distinct mechanisms. Mtl1 also forms Red1-independent interactions with evolutionarily conserved proteins named Nrl1 and Ctr1, which associate with splicing factors. Whereas Nrl1 targets transcripts with cryptic introns to form heterochromatin at developmental genes and retrotransposons, Ctr1 is required to process intron-containing telomerase RNA. Together with our discovery of widespread cryptic introns, including in noncoding RNAs, these findings reveal unique cellular strategies for recognizing regulatory RNAs and coordinating their functions in response to developmental and environmental cues.

2013-11-21 | E-MTAB-1962 | biostudies-arrayexpress

University of Washington CSF biomarker study for Parkinson disease

Project description:<p>There is a clear need to develop biomarkers for Parkinson disease (PD) diagnosis and monitoring disease progression. In this study we evaluated cerebrospinal fluid (CSF) proteins, which are known to be critically involved in PD or identified in our preliminary profiling studies, aptamers, and RNAs as potential PD biomarkers. Access to subjects for this study was via the Pacific Northwest Udall Center (PANUC) and the Alzheimer's Disease Research Center (ADRC) at the University of Washington and Oregon Health and Sciences University (OHSU). Using CSF samples from 30 well-characterized patients with PD and 30 age-, sex-matched healthy controls, we prepared RNA seq libraries and performed deep sequencing of all RNA species, including small and long RNA, mRNAs, noncoding RNAs and differentially spliced transcripts. We then tried several methods for RNAseq data analysis to optimize our analysis pipeline. We identified a total of 3381 transcripts corresponding to 182 long intergenic RNAs (LincRNAs), 11 microRNAs (miRNAs), 2861 protein-coding transcripts, 200 pseudogenes and 127 antisense RNAs; some of them were differentially expressed between PD and control groups. Selected differentially expressed RNAs have been validated in the same set of CSF samples using real-time PCR (RT-PCR). Further validations in independent, larger cohorts of samples are still ongoing. Our results obtained so far suggested that CSF proteins and RNAs could be used as good indexes for PD diagnosis and disease severity/progression. This study is a part of the NIDDS-funded Parkinson's Disease Biomarkers Program (PDBP).</p>

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Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin

Project description:Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin

| PRJNA210415 | ENA

RNA-Chromatin Interactome Reveals ncRNA Functions for Transcription Regulation in 3D Genome Organization

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.

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Binding of the Zika virus genome to endogenous human transcripts

Project description:This study aims to study binding events between the Zika virus RNA genome and endogenous transcripts during infection of a human cell line. We develop a novel psolaren-based cross-linking technique to preserve interactions between mRNA and the Zika genome. Interacting RNA molecules are ligated together prior to reversal of the cross-links and selection for Zika-containing fragments. Reverse transcription and paired-end high-throughput sequencing then allow us to identify the interacting transcripts. We generated three batches of libraries, where all samples in each batch were generated from the same pool of RNA. Within each batch, we have the livefire sample, where the protocol was performed as described above; a reverse-control sample, where the protocol was performed by reversing the cross-links prior to ligation; and a no-cross-link control, where the protocol was performed without any cross-linking. The latter two samples represent negative controls where no genuine interactions should be observed.

2018-07-13 | E-MTAB-6427 | biostudies-arrayexpress

Determination of local chromatin interactions using a combined CRISPR and peroxidase APEX2 system

Project description:The architecture and function of chromatin are largely regulated by local interacting molecules, such as transcription factors and noncoding RNAs. However, our understanding of these regulatory molecules at a given locus is limited because of technical difficulties. Here, we describe the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and an engineered ascorbate peroxidase 2 (APEX2) system to investigate local chromatin interactions (CAPLOCUS). We showed that with specific small-guide RNA targets, CAPLOCUS could efficiently identify both repetitive genomic regions and single-copy genomic locus with high resolution. Genome-wide sequencing revealed known and potential long-range chromatin interactions for a specific single-copy locus. CAPLOCUS also identified telomere-associated RNA. CAPLOCUS, followed by mass spectrometry, identified both known and novel telomere-associated proteins in their native states. Thus, CAPLOCUS may be a useful approach for studying local interacting molecules at any given chromosomal location.

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