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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.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE74353 | GEO | 2016/04/04

SECONDARY ACCESSION(S): PRJNA299809

REPOSITORIES: GEO

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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. 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

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

Nucleosome sliding by the Chd1 chromatin remodeler relies on the integrity of the DNA duplex

Project description:The experiments are done with three libraries by introducing base-pair perturbations on Widom 601: (1) poly(dA:dT) tract library (2) mismatch library (3) insertion library. And additional two more libraries based on native yeast genomic sequences: (4) Park97 mismatch library (5) Park97 insertion library. And we measured the nucleosome positioining in the base-pair resolution for before and after sliding by Chd1 chromatin remodeler.

2022-05-12 | GSE198440 | GEO

PARIS analysis of mouse Xist RNA structure

Project description:PARIS analysis of mouse Xist RNA structure

| PRJNA523035 | ENA

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

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation [irCLIP-seq]

Project description:We show that Spen, an Xist binding repressor protein essential for XCI, binds to ancient retroviral RNA transcribed genome-wide, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen inactivation leads to de-repression of endogenous retroviral (ERV) elements in embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that are highly similar to the A-repeat of Xist, a region critical for Xist-mediated gene silencing. ERV RNA and Xist A-repeat bind the RRM3 domain of Spen in a competitive manner and insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen.

2020-05-16 | GSE131376 | GEO

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation [ATAC-Seq]

2020-05-16 | GSE131374 | GEO

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation [RNA-Seq]

2020-05-16 | GSE131373 | GEO

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation [ChIP-Seq]

2020-05-16 | GSE131412 | GEO

The minor groove-binding agent ELB-21 forms multiple interstrand and intrastrand covalent cross-links with duplex DNA and displays potent bactericidal activity against methicillin-resistant Staphylococcus aureus.

Project description:Objectives The antistaphylococcal pyrrolobenzodiazepine dimer ELB-21 forms multiple adducts with duplex DNA through covalent interactions with appropriately spaced guanine residues; it is now known to form interstrand and intrastrand adducts with oligonucleotide sequences of variable length. We determined the DNA sequence preferences of ELB-21 in relation to its capacity to exert a bactericidal effect by damaging DNA. Methods Formation of adducts by ELB-21 and 12- to 14-mer DNA duplexes was investigated using ion-pair reversed phase liquid chromatography and mass spectrometry. Drug-induced changes in gene expression were measured in prophage-free Staphylococcus aureus RN4220 by microarray analysis. Results ELB-21 preferentially formed intrastrand adducts with guanines separated by three nucleotide base pairs. Interstrand and intrastrand adducts were formed with duplexes both longer and shorter than the preferred target sequences. ELB-21 elicited rapid bactericidal effects against prophage-carrying and prophage-free S. aureus strains; cell lysis occurred following activation and release of resident prophages. Killing appeared to be due to irreparable damage to bacterial DNA and susceptibility to ELB-21 was governed by the capacity of staphylococci to repair DNA lesions through induction of the SOS DNA damage response mediated by the RecA-LexA pathway. Conclusions The data support the contention that ELB-21 arrests DNA replication, eliciting formation of ssDNA-RecA filaments that inactivate LexA, the SOS repressor, and phage repressors such as Cl, resulting in activation of the DNA damage response and de-repression of resident prophages. Above the MIC threshold, DNA repair is ineffective. Data is also available from <ahref=http://bugs.sgul.ac.uk/E-BUGS-115 target=_blank>BuG@Sbase</a>

2011-04-15 | E-BUGS-115 | biostudies-arrayexpress

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