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RIA to verify MARIO in mapping RNA-RNA interactome and RNA structures in vivo

ABSTRACT: Many biological processes are regulated by RNA-RNA interactions 1, nonetheless it remains formidable to analyze the entire RNA interactome. We developed a method, MARIO (MApping Rna-rna Interactions in vivO), to map protein-assisted RNA-RNA interactions in vivo. By circumventing the selection for a specific RNA-binding protein 2-5, our approach vastly expands the identifiable portion of the RNA interactome. Using this technology, we mapped the RNA interactome in mouse embryonic stem cells, which was composed of 46,780 RNA-RNA interactions. The RNA interactome was a scale-free network, with several lincRNAs and mRNAs emerging as hubs. We validated an interaction between two hubs, Malat1 and Slc2a3 using single molecule RNA fluorescence in situ hybridization. Base pairing was observed at the interaction sites of long RNAs, and was particularly strong in transposonRNA-mRNA and lincRNA-mRNA interactions. This reveals a new type of regulatory sequences acting in trans. Consistent with their hypothesized roles, the RNA interaction sites were more evolutionarily conserved than other regions of the transcripts. MARIO also provided new information on RNA structures, by simultaneously revealing the footprint of single stranded regions and the spatially proximal sites of each RNA. The unbiased mapping of the protein-assisted RNA interactome with minimum perturbation of cell physiology will greatly expand our capacity to investigate RNA functions. Three (3) ESC samples were treated with one (1) type of antisense oligonucleotides as is described in Kretz M. et al. (Nature. 2013 Jan 10;493(7431):231-5) to show the RNA interaction among specific RNAs and verify the results from MARIO

ORGANISM(S): Mus musculus

SUBMITTER: Sheng Zhong

PROVIDER: E-GEOD-65849 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Mapping RNA-RNA interactome and RNA structures in vivo with MARIO

Project description:Many biological processes are regulated by RNA-RNA interactions 1, nonetheless it remains formidable to analyze the entire RNA interactome. We developed a method, MARIO (MApping Rna-rna Interactions in vivO), to map protein-assisted RNA-RNA interactions in vivo. By circumventing the selection for a specific RNA-binding protein 2-5, our approach vastly expands the identifiable portion of the RNA interactome. Using this technology, we mapped the RNA interactome in mouse embryonic stem cells, which was composed of 46,780 RNA-RNA interactions. The RNA interactome was a scale-free network, with several lincRNAs and mRNAs emerging as hubs. We validated an interaction between two hubs, Malat1 and Slc2a3 using single molecule RNA fluorescence in situ hybridization. Base pairing was observed at the interaction sites of long RNAs, and was particularly strong in transposonRNA-mRNA and lincRNA-mRNA interactions. This reveals a new type of regulatory sequences acting in trans. Consistent with their hypothesized roles, the RNA interaction sites were more evolutionarily conserved than other regions of the transcripts. MARIO also provided new information on RNA structures, by simultaneously revealing the footprint of single stranded regions and the spatially proximal sites of each RNA. The unbiased mapping of the protein-assisted RNA interactome with minimum perturbation of cell physiology will greatly expand our capacity to investigate RNA functions. Three (3) ESC samples with different treatment (different digestion size and/or crosslinking method) and one (1) MEF sample were included to test our new approach for RNA-interactome mapping and the different samples were analyzed to show RNA interactome differences between them.

2016-06-23 | E-GEOD-61489 | biostudies-arrayexpress

RIA to verify MARIO in mapping RNA-RNA interactome and RNA structures in vivo

2016-06-23 | GSE65849 | GEO

Mapping RNA-RNA interactome and RNA structures in vivo with MARIO

2016-06-23 | GSE61489 | GEO

RNA duplex map in living cells reveals higher order transcriptome structure

Project description:RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here we develop PARIS, a method based on reversible psoralen-crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher order architectures, and RNA:RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures, and discovered conserved long-range and alternative structures. XIST, a lncRNA essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. Cells are crosslinked with AMT and RNA is extracted from cells by RNase digestion. Crosslinked RNA fragments are selected from total RNA by 2D gel electrophoresis. Purified crosslinked RNA duplexes are proximity ligated and crosslinking is reversed. Then the chimeric RNA molecules are converted into cDNA libraries for high-throughput sequencing.

2016-04-04 | E-GEOD-74353 | biostudies-arrayexpress

Mapping RNA-RNA interactome and RNA structures in vivo

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2016-06-23 | E-GEOD-81388 | biostudies-arrayexpress

The pluripotent genome in three dimensions is shaped around pluripotency factors

Project description:It is becoming increasingly clear that the shape of the genome importantly influences transcription regulation. Pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) were recently shown to organize their chromosomes into topological domains that are largely invariant between cell types. Here, we applied 4C technology and combined ChIP-seq with Hi-C data to demonstrate that inactive chromatin is unusually disorganized in PSC nuclei. We show that gene promoters engage in contacts between topological domains in a largely tissue-independent manner while enhancers have a more tissue-restricted interaction profile. Most strikingly, genomic clusters of pluripotency factor binding sites find each other very efficiently, in a manner that is strictly PSC-specific, dependent on the presence of Oct4 and Nanog protein and inducible upon artificial recruitment of Nanog to a selected chromosomal site. We conclude that pluripotent stem cells have a unique higher-order genome structure shaped by pluripotency factors. We speculate that this interactome enhances the robustness of the pluripotent state. 4C-seq was performed on a range of viewpoints in pluripotent (2x ESC, iPSC) and differentiated (neural precursor cells and astrocytes) cell lines.

2013-07-24 | E-GEOD-37275 | biostudies-arrayexpress

RNA-seq to investigate the phase separation-dependent FUS RNA interactome

Project description:The protein Fused in Sarcoma undergoes liquid-liquid phase separation. To investigate whether this phase transition alters the RNA interactome we purified phase-separated FUS droplets and soluble FUS from HEK 293T cells transfected with GFP-tagged WT FUS or P525L FUS. Phase separated FUS was purified by fluorescence-activated particle sorting (droplets) and soluble FUS by co-Immunoprecipitation (IP) respectively followed by isolation of co-purified RNA. Here we show that phase separation affects the RNA interactome of FUS.

2021-06-23 | E-MTAB-8456 | biostudies-arrayexpress

X-chromosome dynamics revealed by the RNA interactome and chromosomal binding of CTCF

Project description:CTCF is a master regulator that plays a role in genome architecture and gene expression. A key aspect of CTCF’s mechanism involves bringing together distant genetic elements for intra- and inter-chromosomal interactions. Evidence from epigenetic processes, such as X-chromosome inactivation (XCI), suggests that CTCF may carry out its functions through interacting RNAs. Using genome-wide approaches to investigate the relationship between CTCF’s RNA interactome and its epigenomic landscape, here we report that CTCF interacts with thousands of transcripts in mouse embryonic stem cells (mESC), many in close proximity to CTCF’s genomic binding sites. Biochemical analysis demonstrates that CTCF is a high-affinity RNA binding protein that contacts RNA directly and specifically. In the XCI model, CTCF binds the active and inactive X-chromosomes allele-specifically. At the X-inactivation center, Tsix RNA binds CTCF and targets CTCF to a region associated with X-chromosome pairing. Our work implicates CTCF-RNA interactions in long-range chromosomal interactions in trans and adds a new layer of complexity to CTCF regulation. The genome-wide datasets reported here will provide a useful resource for further study of CTCF-mediated epigenomic regulation. CTCF RNA interactome was identified by UV-crosslinking and immunoprecipitation followed by high-throughput sequencing (CLIP-seq), and was compared to CTCF's epigenomic landscape as obtained by chromatin immunoprecipitation (ChIP-seq).

2015-01-09 | E-GEOD-58242 | biostudies-arrayexpress

TOP1M-NM-1 functions on nucleosome density

Project description:DNA topoisomerases release supercoils in DNA introduced during replication or transcription. How DNA topoisomerases impact transcription in the context of eukaryotic chromatin is poorly understood. In this study, using a floral stem cell model in Arabidopsis, we uncovered a role of TOP1M-NM-1 in Polycomb Group (PcG) protein-mediated histone lysine 27 trimethylation at, and transcriptional repression of, the stem cell maintenance gene WUSCHEL (WUS). The strong genetic interactions between a top1M-NM-1 mutant and mutations in PcG genes and the overwhelming enrichment of PcG targets among genes affected in expression in the top1a mutant revealed a role of TOP1M-NM-1 in PcG-mediated regulation. Intriguingly, not only the repression of some PcG target genes but also the expression of others requires TOP1M-NM-1. The mechanism that unifies the opposing effects of TOP1M-NM-1 on PcG target genes appears to lie in its role in decreasing nucleosome density. Genome-wide nucleosome mapping shows that TOP1M-NM-1 is required for the depletion of nucleosomes at regulatory regions of genes, which probably allows the binding of factors that either recruit PcG, as we show for AGAMOUS at the WUS locus, or counteract PcG-mediated regulation. This study uncovers a strong and previously unknown connection between TOP1M-NM-1 and PcG. 4 samples in total, two replicates from Ler and top1a-2 plants

2014-10-14 | E-GEOD-53950 | biostudies-arrayexpress

Highly connected enhancer communities control lineage-determining genes in human mesenchymal stem cells

Project description:Adipocyte differentiation is driven by waves of transcriptional regulators that reprogram the en-hancer landscape and change the wiring of the promoter interactome. Here, we establish enhancer-capture Hi-C to interrogate the extent and role of enhancer-to-enhancer interactions during adi-pocyte differentiation of human mesenchymal stem cells. We find that enhancers form an elaborate network that is highly dynamic during differentiation and tightly coupled to changes of enhancer activity. These interactions are important for enhancer function, since transcription factors at baited enhancers stabilize transcription factor binding at target enhancers, a phenomenon we term cross-interaction stabilization of transcription factors (CIST). Moreover, highly interconnected enhancers (HICE) act as integration hubs orchestrating differentiation by formation of insulated 3D enhancer communities, inside which, HICE, and other enhancers, converge on phenotypically important gene promoters. Collectively, these results indicate that dynamic enhancer interactions play a key role in regulation of enhancer function, and that HICE are important for both signal integration and compartmentalization of the genome.

2020-08-13 | GSE140782 | GEO

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