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Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA [SHAPE-Map]

ABSTRACT: To refine the authentic CENP-C binding sites of lnc-CCTT and globally map lnc-CCTT secondary structure, we also performed SHAPE-MaP (selective 2’-hydroxyl acylation analyzes by primer extension and mutational profiling), which uses hydroxyl-selective electrophiles to modify the 2’-hydroxyl groups of unbound single-stranded nucleotides, in HeLa cells both ex vivo and in vivo. Lnc-CCTT secondary structure was modeled by combination SHAPE data from cell-free ex vivo with pairing probabilities. As expected, nucleotides 43-79 nt, a determinant for RNA-DNA triplex formation, exhibited a continuous single-strandedness, which may be prone to binding DNA. More importantly, only nucleotides 118-177 nt, which was folded into a stem-loop structure in the secondary structure, showed a significant reduced SHAPE reactivities in cell when comparing to cell-free state, suggesting this region could be attributed to interaction with protein components.

ORGANISM(S): Homo sapiens

PROVIDER: GSE149529 | GEO | 2022/04/20

REPOSITORIES: GEO

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Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA [irCLIP-Seq]

Project description:To fine-map the position of lnc-CCTT that directly interact with CENP-C, we performed irCLIP-seq (infrared crosslingking and immunoprecipitation followed by high throughput RNA sequencing), which utilize ultraviolet (UV) light to induce zero-length covalent bonds between RNA and the directly attached protein and an infrared-dye-conjugated and biotinylated ligation adaptor to isolate RNA fragments. irCLIP-seq identified a possible CENP-C binding region of lnc-CCTT ranging from nucleotides 127-177nt.

2022-04-20 | GSE149530 | GEO

Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA [RIP-Seq]

Project description:To identify potential lncRNAs that associate with CENP-C, RNA immunoprecipitation (RIP) using the CENP-C and control IgG antibody was performed in the whole-cell lysates of HeLa cells, followed by Illumina short-read sequencing (RIP-seq). We found three CENP-C-binding lncRNAs that meet the enrichment criteria (fold change > 2, p < 0.01), including AGAP2-AS1, GS1-124K5.4, and AC124789.1 (lnc-CCTT) . Consistent with the results of RIP-seq, RIP-qPCR assay in whole-cell lysates showed that all three lncRNAs could bind to CENP-C, while lnc-CCTT showing the most robust association of CENP-C. ). It is well-established that the localization of lncRNAs within the cell is the primary determinant of their molecular functions. RT-qPCR (quantitative PCR with reverse transcription) assay of subcellular fractionation and RNA fluorescence in situ hybridization (RNA-FISH) revealed that AGAP2-AS1 and GS1-124K5.4 dominantly distributed in the cytoplasm, whereas lnc-CCTT, specifically and almost exclusively, localized to the nucleus. Thus, lnc-CCTT appears to be a prime interacting RNA of CENP-C at centromere.

2022-04-20 | GSE149528 | GEO

Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA [ChIRP-Seq]

Project description:To investigate the exact locations of CCTT-chromatin interaction on a genome-wide scale, we modified previously reported ChIRP-seq (chromatin isolation by RNA purification followed by deep-sequencing) with a crosslinker 4’- aminomethyltrioxalen (AMT, a psoralen derivative), which allows fixation of nucleic acid interaction by ultraviolet light without crosslinking proteins. We designed 8 complementary DNA oligonucleotides that tiled the Alu-depleted part of CCTT. Affinity-purified CCTT-binding DNAs were sequenced and mapped to the HuRef genome hg38, which contains human α-satellite sequence models in each centromeric region. In contrast with 5.38% of input reads mapping to α-satellite sequence, 16.89% of CCTT-binding reads were enriched at α-satellites. Peaks bound by lnc-CCTT were identified by MACS2 algorithm, which compared the reads in CCTT-captured samples with that in input ones. CCTT-binding peaks mapped across the centromeric regions of all 23 reference centromeres. Next, we validated the enrichment of centromeric peaks located at all chromosomes via ChIRP-qPCR. To validate our ChIRP-seq results, we selected representatives of three major subpopulations: multimapping peaks in one specific chromosome and several chromosomes, as well as single-copy peaks. We performed ChIRP-qPCR and found abundant CCTT-binding centromeric peaks throughout all 23 chromosomes. Importantly, CCTT ChIRP-seq profile was highly correlated with the previously reported ChIP-seq profiles of CENP-C (Pearson correlation R = 0.82), both of which showed very strong, extensive signals across entire centromeric regions in HeLa cells.

2022-04-20 | GSE149532 | GEO

secondary structure based on icSHAPE of pre-let-7 and hDicer-TRBP bound pre-let-7

Project description:we resolved precusor let7 (pre-let-7) and human Dicer-TRBP complex bound precusor let7 (hDicer-TRBP-pre-let-7) secondary structure based on in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE). We found the free pre-let-7 RNA is more flexible comparing with hDicer-TRBP-pre-7 in the double strand region [10-20nt and 50- 60 nt].

2018-02-14 | GSE110516 | GEO

Dynamic landscapes and regulome of mRNA structure during zebrafish early embryogenesis

Project description:We resolved the RNA secondary structure during zebrafish early embryogenesis based on in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE). We analyzed the RNA structure dynamics among different development stages. Also, we studied which factors regulate maternal gene decay by RNA structure switch.

2020-04-14 | GSE120724 | GEO

Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

Project description:New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2â²-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems. We sequenced in-vitro transcribed RNaseP in wild type and barcoded variants and probed them with SHAPE-Seq. Comparison to existing crystalography data and previous SHAPE experiments verified the accuracy of the technique and the absence of bias due to our multiplexiing strategy.

2011-08-26 | E-GEOD-31573 | biostudies-arrayexpress

lncRNA SLERT controls phase separation of FC/DFCs to facilitate Pol I transcription

Project description:Probe SLERT secondary structure by SHAPE-MaP in PA1 cell line

2021-05-11 | GSE174140 | GEO

ANKRD52_SHAPE-MaP

Project description:Probe ANKRD52 secondary structure by SHAPE-MaP in PA1 cell line.

2023-12-18 | GSE183927 | GEO

RNA secondary structome of different cellular compartments in human and mouse

Project description:We resolved the RNA secondary structure of three cellular compartments including chromatin (ch), nucleoplasm(np) and cytoplasm(cy) in HEK293 and mES cells based on in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE). We analyze the RNA structure dynamics among cellular compartments and different RNA processings. Also, we study which factors perturb the RNA structures.

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Project description:we resolved the RNA secondary structure of human (HEK293, HeLa, K562, HepG2, H9) and mouse (mES) cells based on optimized in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE). we describe a deep discriminative neural network (PrismNet) that integrates big data of RBP binding and RNA secondary structure from matched living cells and accurately models the RNA sequence and structural preferences of protein-RNA interactions in vivo, enabling precise prediction of the perturbation effects of genetic variants.

2021-02-05 | GSE145805 | GEO

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