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RNA duplex map in living cells reveals higher order transcriptome structure

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Zhipeng Lu

PROVIDER: E-GEOD-74353 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

2016-04-04 | GSE74353 | GEO

Optimized photochemistry and enzymology enable efficient analysis of RNA structures and interactions in cells

Project description:RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Direct determination of RNA structures and interactions in living cells remain a major challenge in biology. Crosslinking and proximity-ligation-based approaches, such as PARIS (Psoralen Analysis of RNA Interactions and Structures), offer many advantages, such as the ability to directly capture complex RNA duplexes in cells with single molecule accuracy, near base pair resolution in high throughput and without size limit. Based on a systematic optimization of the photochemistry and enzymology, here we present PARIS2, a re-invented method for capturing RNA duplexes with >3000-fold improved efficiency. PARIS2 captures ribosome small subunit binding sites on mRNAs, thus reporting translation process. PARIS2 also captures the snRNP binding sites on various types of RNA targets. We applied PARIS2 to enterovirus D-68, a re-emerging viral pathogen associated with severe neurological symptoms and discovered dynamic/alternative conformations in the 5’UTR. The new photochemical and enzymological improvements and the PARIS2 method should be widely applicable to basic RNA research and RNA-related diseases.

2021-03-04 | GSE149493 | GEO

PARIS analysis of mouse Xist RNA structure

Project description:PARIS analysis of mouse Xist RNA structure

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Determination of RNA Secondary Structure By RNA-Sequencing of HEK293T Cells

Project description:RNA molecule fold into three dimensional structures in order to fill important roles in the cell such as translation and post-transcriptional regulation. These three dimensional structures, in turn, depend on the pattern of hydrogen bonds between ribonucleotides. We employ a technique based on high throughput RNA Sequencing (RNA-Seq) to uncover this patter on a transcriptome wide scale. We take advantage of two ribonucleases that have structure specific activity. RNAse S1 specifically digests single stranded RNA while RNAse V1 is specific to double stranded RNA. We aliquot each of our sample into to reactions. One is thoroughly treated with RNAse S1, digesting away single stranded RNA and leaving behind dsRNA. The other is treated with RNAse V1, which has the inverse effect. The surviving dsRNA and ssRNA molecules are then subject to high throughput sequencing. The resulting reads are used to generate a structure score, revelaing which regions of each transcript are involved in base pairing and which are unpaired.

2016-05-17 | E-GEOD-72681 | biostudies-arrayexpress

High-resolution chromosomal maps of Xist RNA reveal a two-step spreading mechanism during X-inactivation

Project description:The Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation (XCI), the process by which mammals compensate for unequal numbers of sex chromosomes. During XCI, Xist coats the future inactive X (Xi) and recruits Polycomb Repressive Complex 2 (PRC2) to the X-inactivation center (Xic). Currently unclear is how Xist spreads silencing on a 150 Mb scale. Here we generate high-resolution maps of Xist binding across a developmental time course using CHART-seq. In female cells undergoing XCI de novo, Xist follows a two-step mechanism in which it initially targets gene-rich islands before spreading to intervening gene-poor domains. Xist is depleted from genes that escape XCI but frequently concentrates near escapee boundaries. Xist binding was linearly proportional to PRC2 density and H3 lysine 27 trimethylation (H3K27me3), suggesting co-migration of Xist and PRC2. Interestingly, when the Xi is acutely stripped of Xist in post-XCI cells, Xist recovers quickly within both gene-rich and -poor domains on a time scale of hours instead of days, suggesting a previously primed Xi chromatin state. We conclude that Xist spreading takes on distinct stage-specific forms: During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist spreads rapidly to both gene-rich and -poor regions. Capture hybridization analysis of RNA targets (CHART) and input samples of (differentiating) mouse embryonic stem (ES) cells and immortalized mouse embryonic fibroblasts (MEF) using paired-end 75 nt reads on Illumina HiSeq2500, with 2 replicates per sample (# of samples). RNA-seq of the same cell lines with 50 nt reads on Illumina HiSeq2000, with 2 replicates per sample (2 samples, 4 datasets total). CHIP-seq: Data from GSE36905 was aligned and processed as CHART-seq samples. Resulting coverage tracks (EZH2/K27me3) are linked directly to GSE48649 (bedGraphs linked below).

2013-11-04 | E-GEOD-48649 | biostudies-arrayexpress

Probing Xist RNA structure in cells using Targeted Structure-Seq

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

m6A RNA methylation promotes XIST-mediated transcriptional repression

Project description:XIST is a long non-coding RNA (lncRNA) that mediates transcriptional silencing of X chromosome genes. Here we show that XIST is highly methylated with at least 78 N6-methyladenosine (m6A) residues, a reversible base modification whose function in lncRNAs is unknown. We show that m6A formation in XIST, as well as cellular mRNAs, is mediated by RBM15 and its paralog RBM15B, which bind the m6A-methylation complex and recruit it to specific sites in RNA. This results in methylation of adenosines in adjacent m6A consensus motifs. Furthermore, knockdown of RBM15 and RBM15B, or knockdown of the m6A methyltransferase METTL3 impairs XIST-mediated gene silencing. A systematic comparison of m6A-binding proteins shows that YTHDC1 preferentially recognizes m6A in XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m6A. These data reveal a pathway of m6A formation and recognition required for XIST-mediated transcriptional repression. Three to four biological HEK293T replicates were used to perform iCLIP of endogenous YTH proteins, RBM15, and RBM15B. Crosslinking induced truncations were identified using CIMS-CITS pipeline.

2016-09-07 | E-GEOD-78030 | biostudies-arrayexpress

Chemical modifications mark alternatively spliced and actively degrading messenger RNAs in Arabidopsis and humans

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2015-10-31 | E-GEOD-66224 | biostudies-arrayexpress

m6A RNA methylation promotes XIST-mediated transcriptional repression

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