PARIS analysis of mouse Xist RNA structure
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PROVIDER: PRJNA523035 | ENA |
REPOSITORIES: ENA
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Project description:Long noncoding RNAs (lncRNAs) are an important class of transcripts that regulate gene expression on many levels, yet their mechanisms of action remain poorly understood. Previous studies have indicated that these RNAs can serve as modular scaffolds to recruit a variety of protein complexes and coordinate their distinct functions. XIST, a founding member of the lncRNA family, controls the inactivation of an entire X chromosome in placental mammals. Here we develop and integrate several orthogonal structure-interaction analysis methods to demonstrate that XIST RNA-protein complex folds into a modular architecture that is conserved in evolution. The discrete XIST RNA domains interact with distinct sets of effector proteins to orchestrate the X chromosome inactivation (XCI). The modular architecture plays an essential role, in addition to the sequence motifs, in determining the specificity of RBP binding and m6A modification. Together, this work builds a comprehensive structure-function model for the XIST RNA-protein complex, and establishes a paradigm for mechanistic studies of lncRNA functions.
2020-04-23 | GSE126716 | GEO
Project description:The long non-coding RNA (lncRNA) Xist is a master regulator of X-chromosome inactivation in mammalian cells. Models for how Xist and other lncRNAs function depend on thermodynamically stable secondary and higher-order structures that RNAs can form in the context of a cell. Probing accessible RNA bases can provide data to build models of RNA conformation that provide insight into RNA function, molecular evolution, and modularity. To study the structure of Xist in cells, we built upon recent advances in RNA secondary structure mapping and modeling to develop Targeted Structure-Seq, which combines chemical probing of RNA structure in cells with target-specific massively parallel sequencing. By enriching for signals from the RNA of interest, Targeted Structure-Seq achieves high coverage of the target RNA with relatively few sequencing reads, thus providing a targeted and scalable approach to analyze RNA conformation in cells. We use this approach to probe the full-length Xist lncRNA to develop new models for functional elements within Xist, including the repeat A element in the 5'-end of Xist. This analysis also identified new structural elements in Xist that are evolutionarily conserved, including a new element proximal to the C repeats that is important for Xist function. Examination of dimethylsufate reactivity of Xist lncRNA and 18S rRNA in cells using targeted reverse transcription to determine reactivity, and comparisons with untreated control samples.
2016-02-09 | E-GEOD-67667 | biostudies-arrayexpress
Project description:The long non-coding RNA (lncRNA) Xist is a master regulator of X-chromosome inactivation in mammalian cells. Models for how Xist and other lncRNAs function depend on thermodynamically stable secondary and higher-order structures that RNAs can form in the context of a cell. Probing accessible RNA bases can provide data to build models of RNA conformation that provide insight into RNA function, molecular evolution, and modularity. To study the structure of Xist in cells, we built upon recent advances in RNA secondary structure mapping and modeling to develop Targeted Structure-Seq, which combines chemical probing of RNA structure in cells with target-specific massively parallel sequencing. By enriching for signals from the RNA of interest, Targeted Structure-Seq achieves high coverage of the target RNA with relatively few sequencing reads, thus providing a targeted and scalable approach to analyze RNA conformation in cells. We use this approach to probe the full-length Xist lncRNA to develop new models for functional elements within Xist, including the repeat A element in the 5'-end of Xist. This analysis also identified new structural elements in Xist that are evolutionarily conserved, including a new element proximal to the C repeats that is important for Xist function.
2016-02-09 | GSE67667 | GEO
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.
2016-04-04 | GSE74353 | GEO
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
Project description:Determine the tissue specific impact of Xist loss on dosage compensation in various somatic tissues of the mouse
2020-01-30 | E-MTAB-8161 | biostudies-arrayexpress
Project description:Purpose: Compare the transcriptome of homogeneous XIST+ and XIST- hES cell populations. Methods: We isolated homogeneous XIST+ and XIST- cell populations. The XIST+ cells correspond to cells with a XIST cloud and one ATRX pinpoint. The XIST- cells correspond to cells with no XIST cloud and one ATRX pinpoint. Results: We took advantage of the clonal pattern of X-chromosome inactivation in H9 cells and analyzed the data in an allelic manner. By comparing the RNA-Seq data with known H9 SNPs, we identified genomic positions which were relaxed from XCI in XIST- cells compared to XIST+ cells. Conclusions: Genic as well as unannotated transcripts are massively relaxed from XCI in H9 cells when XIST expression is lost, however, this reactivation is only partial and a large region around the centromere is protected from relaxation of silencing. Total RNA (rRNA depleted) profiles of XIST+ and XIST- human embryonic stem cells
2012-12-21 | E-GEOD-39757 | biostudies-arrayexpress
Project description:Long noncoding RNAs (lncRNAs) are an important class of transcripts that regulate gene expression on many levels, yet their mechanisms of action remain poorly understood. Previous studies have indicated that these RNAs can serve as modular scaffolds to recruit a variety of protein complexes and coordinate their distinct functions. XIST, a founding member of the lncRNA family, controls the inactivation of an entire X chromosome in placental mammals. Here we develop and integrate several orthogonal structure-interaction analysis methods to demonstrate that XIST RNA-protein complex folds into a modular architecture that is conserved in evolution. The discrete XIST RNA domains interact with distinct sets of effector proteins to orchestrate the X chromosome inactivation (XCI). The modular architecture plays an essential role, in addition to the sequence motifs, in determining the specificity of RBP binding and m6A modification. Together, this work builds a comprehensive structure-function model for the XIST RNA-protein complex, and establishes a paradigm for mechanistic studies of lncRNA functions.
2020-04-23 | GSE126715 | GEO
Project description:We reprot transcriptome states in mouse blastocyst cultured with various conditons as well as Xist deletion. By comparing the gene expression states of blastocyst grown in different culture conditions with Xist deletion, we identified the differentially expressed genes, which are dependent culture conditions and Xist RNA.
2019-11-06 | GSE133845 | GEO
Project description:Xist orchestrates X chromosome inactivation, a process that entails chromosome-wide silencing and remodeling of the 3-dimensional structure of the X chromosome. Yet, it remains unclear whether these changes in nuclear structure are mediated by Xist and whether they are required for silencing. Here we show that Xist directly interacts with the Lamin B Receptor (LBR), an integral component of the nuclear lamina, and that this interaction is required for Xist-mediated silencing. We show that this interaction recruits the inactive X to the nuclear lamina and by doing so enables Xist to spread to actively transcribed genes across the X. Our results demonstrate that lamina recruitment changes the accessibility of DNA thereby enabling Xist, and its silencing proteins, to spread across the X to silence transcription. We examined the genomic localization of the Xist lncRNA using RNA Antisense Purification (RAP) in male mouse ES cells where the endogenous Xist promoter is replaced by a tet-inducible one (pSM33) containing 1) wild-type Xist (WT), 2) A-repeat deletion Xist (dA), 3) LBR binding site deletion Xist (dLBS), 4) dLBS-Xist rescued with LMNB1 (LMNB1Res), 5) LBR CRISPRi knock down (LBRKD), or 6) SHARP CRISPRi knock down (SHARPKD).
2016-07-24 | E-GEOD-80510 | biostudies-arrayexpress