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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE149530 | 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 [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 [SHAPE-Map]

Project description: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.

2022-04-20 | GSE149529 | 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

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

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

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Rbm15-irCLIP-seq

Project description:we performed infrared crosslinking immunoprecipitation followed by sequencing (irCLIP-seq) (Zarnegar et al., 2016) for Rbm15 to directly map its binding sites on RNA. As a principle of concept, the cells were engineered to simultaneously express emGFP-Rbm15 and Xist RNA together, as Rbm15 strongly interacts with Xist A-repeat to deposit the m6A methylation downstream. Cross-linking induced truncation site (CITS or RT stops) is the main signature occurring at our irCLIP-seq datasets. RNA meta-profile plot against normalized transcripts shows that these CITSs reside across the transcript, with 2 main peaks in transcript starts and near the stop-codon regions, in agreement with the RBM15/15B binding profile in human cells (Patil et al., 2016).Motif analysis against CITSs revealed that Rbm15 binding sites prefer U-rich stretches, namely 3 or 4 consecutive Us. This is also true for crosslinking induced mutations (CIMS).

2020-09-15 | GSE154708 | GEO

Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA

Project description:Long noncoding RNA CCTT recruits CENP-C to centromeres by directly binding to centromeric DNA

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

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

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

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

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

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