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B-CePs as cross-linking probes for the investigation of RNA higher-order structure.

ABSTRACT: Elucidating the structure of RNA and RNA ensembles is essential to understand biological functions. In this work, we explored the previously uncharted reactivity of bis-chloropiperidines (B-CePs) towards RNA. We characterized at the molecular level the different adducts induced by the fast reacting compound B-CeP 1 with RNA. Following an approach based on solution thermal melting coupled with ESI mass spectrometry (STHEM-ESI), we proved the ability of B-CePs to induce inter-molecular cross-links between guanines in double stranded RNA. These results open the possibility of using B-CePs as structural probes for investigating higher-order structures, such as the kissing loop complex established by the dimerization initiation site (DIS) of the HIV-1 genome. We confirmed the potential of B-CePs to reveal the identity of RNA structures involved in long-range interactions, expecting to benefit the characterization of samples that are not readily amenable to traditional high-resolution techniques, and thus promoting the elucidation of pertinent RNA systems associated with old and new diseases.

SUBMITTER: Sosic A

PROVIDER: S-EPMC8266612 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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

Structure-specific ribonucleases for MS-based elucidation of higher-order RNA structure.

Project description:Supported by high-throughput sequencing technologies, structure-specific nucleases are experiencing a renaissance as biochemical probes for genome-wide mapping of nucleic acid structure. This report explores the benefits and pitfalls of the application of Mung bean (Mb) and V1 nuclease, which attack specifically single- and double-stranded regions of nucleic acids, as possible structural probes to be employed in combination with MS detection. Both enzymes were found capable of operating in ammonium-based solutions that are preferred for high-resolution analysis by direct infusion electrospray ionization (ESI). Sequence analysis by tandem mass spectrometry (MS/MS) was performed to confirm mapping assignments and to resolve possible ambiguities arising from the concomitant formation of isobaric products with identical base composition and different sequences. The observed products grouped together into ladder-type series that facilitated their assignment to unique regions of the substrate, but revealed also a certain level of uncertainty in identifying the boundaries between paired and unpaired regions. Various experimental factors that are known to stabilize nucleic acid structure, such as higher ionic strength, presence of Mg(II), etc., increased the accuracy of cleavage information, but did not completely eliminate deviations from expected results. These observations suggest extreme caution in interpreting the results afforded by these types of reagents. Regardless of the analytical platform of choice, the results highlighted the need to repeat probing experiments under the most diverse possible conditions to recognize potential artifacts and to increase the level of confidence in the observed structural information.

| S-EPMC6911265 | biostudies-literature

Wapl controls higher-order chromatin structure in interphase chromosomes

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

2013-08-09 | E-GEOD-41603 | biostudies-arrayexpress

RNA duplex map in living cells reveals higher order transcriptome structure

2016-04-04 | GSE74353 | 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 long noncoding RNA (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. VIDEO ABSTRACT.

| S-EPMC5029792 | biostudies-literature

Wapl controls higher-order chromatin structure in interphase chromosomes

Project description:This SuperSeries is composed of the SubSeries listed below.

2013-08-09 | GSE41603 | GEO

Protein unties the pseudoknot: S1-mediated unfolding of RNA higher order structure.

Project description:Ribosomal protein S1 plays important roles in the translation initiation step of many Escherichia coli mRNAs, particularly those with weak Shine-Dalgarno sequences or structured 5' UTRs, in addition to a variety of cellular processes beyond the ribosome. In all cases, the RNA-binding activity of S1 is a central feature of its function. While sequence determinants of S1 affinity and many elements of the interactions of S1 with simple secondary structures are known, mechanistic details of the protein's interactions with RNAs of more complex secondary and tertiary structure are less understood. Here, we investigate the interaction of S1 with the well-characterized H-type pseudoknot of a class-I translational preQ1 riboswitch as a highly structured RNA model whose conformation and structural dynamics can be tuned by the addition of ligands of varying binding affinity, particularly preQ1, guanine, and 2,6-diaminopurine. Combining biochemical and single molecule fluorescence approaches, we show that S1 preferentially interacts with the less folded form of the pseudoknot and promotes a dynamic, partially unfolded conformation. The ability of S1 to unfold the RNA is inversely correlated with the structural stability of the pseudoknot. These mechanistic insights delineate the scope and limitations of S1-chaperoned unfolding of structured RNAs.

| S-EPMC7038950 | biostudies-literature

Wapl controls higher-order chromatin structure in interphase chromosomes (gene expression)

Project description:Mammalian genomes contain several billion base pairs of DNA which are packaged in chromatin fibers. At selected gene loci, cohesin complexes have been proposed to arrange chromatin fibers into higher-order structures, but it is poorly understood how cohesin performs this task, how important this function is for determining the structure of chromosomes, and how this process is regulated to allow changes in gene expression. Here we show that the cohesin release factor Wapl controls chromatin structure and gene regulation at numerous loci throughout the mouse genome. Conditional deletion of the Wapl gene leads to stable accumulation of cohesin on chromatin, chromatin compaction, altered gene expression, cell cycle delay, chromosome segregation defects and embryonic lethality. In Wapl deficient chromosomes, cohesin accumulates in an axial domain, similar to how condensins form a M-bM-^@M-^\scaffoldM-bM-^@M-^] in mitotic chromosomes. We propose that Wapl controls chromatin structure and gene regulation by determining the residence time with which cohesin binds to DNA. 4 biological replicates for each genotype (Wapl +/F; Wapl -/F) treated with/without 4-OHT =16 samples

2013-08-09 | E-GEOD-41460 | biostudies-arrayexpress

Wapl controls higher-order chromatin structure in interphase chromosomes (ChIP-Seq)

Project description:Mammalian genomes contain several billion base pairs of DNA which are packaged in chromatin fibers. At selected gene loci, cohesin complexes have been proposed to arrange chromatin fibers into higher-order structures, but it is poorly understood how cohesin performs this task, how important this function is for determining the structure of chromosomes, and how this process is regulated to allow changes in gene expression. Here we show that the cohesin release factor Wapl controls chromatin structure and gene regulation at numerous loci throughout the mouse genome. Conditional deletion of the Wapl gene leads to stable accumulation of cohesin on chromatin, chromatin compaction, altered gene expression, cell cycle delay, chromosome segregation defects and embryonic lethality. In Wapl deficient chromosomes, cohesin accumulates in an axial domain, similar to how condensins form a “scaffold” in mitotic chromosomes. We propose that Wapl controls chromatin structure and gene regulation by determining the residence time with which cohesin binds to DNA. ChIP-Seq using two different antibodies (CTCF, Smc3); one (CTCF) and two (Smc3) replicates; two different genotypes (Wapl +/delta, Wapl -/delta). The control sample is a single-replicate INPUT for each genotype.

2013-08-09 | E-GEOD-41602 | biostudies-arrayexpress

Application of 2D-NMR with room temperature NMR probes for the assessment of the higher order structure of filgrastim.

Project description:The higher order structure (HOS) of biotherapeutics is a critical quality attribute that can be evaluated by nuclear magnetic resonance (NMR) spectroscopy at atomic resolution. NMR spectral mapping of HOS can be used to establish HOS consistency of a biologic across manufacturing changes or to compare a biosimilar to an innovator reference product. A previous inter-laboratory study performed using filgrastim drug products demonstrated that two-dimensional (2D)-NMR 1HN-15NH heteronuclear correlation spectroscopy is a highly robust and precise method for mapping the HOS of biologic drugs at natural abundance using high sensitivity NMR 'cold probes.' Here, the applicability of the 2D-NMR method to fingerprint the HOS of filgrastim products is demonstrated using lower sensitivity, room temperature NMR probes. Combined chemical shift deviation and principal component analysis are used to illustrate the performance and inter-laboratory precision of the 2D-NMR method when implemented on room temperature probes.

| S-EPMC6057790 | biostudies-literature

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