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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE149528 | 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 [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 [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 [RIP-Seq]

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

| PRJNA628993 | ENA

Effects of SOCS2-AS1 inhibition in prostate cancer cells

Project description:Prostate cancer is the most common cancer in men and AR downstream signalings promote prostate cancer cell proliferation. We identified a novel androgen-regulated long non-coding (lnc) RNA, SOCS2-AS1. In order to investigate the SOCS2-AS1 function in prostate cancer cells, we performed gene expression in AR-positive prostate cancer cell lines (LNCaP and LTAD) after siSOCS2-AS1 or siSOCS2 treatment. We also treated cells with vehicle or androgen to analyzed the effects of siSOCS2-AS1 on AR function. Observation of androgen dependent gene expression changes after treatmet with siSOCS2-AS1 with microarray.

2016-08-01 | E-GEOD-82224 | biostudies-arrayexpress

Expression profile analysis of long non-coding RNAs in condyloma acuminatum in a Chinese female population by RNA-sequencing and bioinformatics [clip-seq]

Project description:This study aimed to explore the differential lncRNAs in CA and their probable function on CA development.We used RNA-sequencing to assess the genome-wide expression levels of lncRNAs in CA and paired adjacent normal tissues. We further validated the candidate lncRNAs in larger-size of CA specimen using RT-qPCR. Furthermore, the functional enrichment analysis for these candidate lncRNAs and differential mRNAs were analyzed using GO terms, KEGG and STRING database. The coexpressed mRNAs for the candidate lncRNAs, calculated by Pearson?s correlation coefficient, were also analyzed using GO analysis.A total of 657 lncRNAs and 1486 mRNAs were found to dysregulated in CA compared to adjacent mucosal tissues. The microarray data of candidate lncRNAs, including HOXC13-AS, HOTAIR, PICSAR, FGF14-AS1, ADGRD1-AS1, TMEM108-AS1, TUSC7 and FGF10-AS1, were validated with a larger-size of specimen using RT-qPCR. The functional GO term and pathways analysis of DEGs are associated with the pathogenesis and development of CA, including cell proliferation and development, cellular adhesion, immune defense, cell migration and chemotaxis, angiogenesis, and maintaining skin hemostasis.LncRNAs that were differentially expressed between CA and normal tissues may be helpful to explore effective recurrence risk markers and potential intervention targets for CA.

2024-04-25 | GSE172139 | GEO

Expression profile analysis of long non-coding RNAs in condyloma acuminatum in a Chinese female population by RNA-sequencing and bioinformatics [RNA-seq]

2024-04-25 | GSE172137 | GEO

Transcriptome Analysis of Wild Type and FGD5-AS1 knockdown CCC-HEH-2 cells

Project description:Purpose: According to the previous analysis results of gene regulation in fetal heart tissues with tetralogy of Fallot (ToF), we constructed the ceRNA mediated network driven by lncRNAs using a causal inference framework based on the expression correlations and validated miRNA-lncRNA/mRNA evidences. Totally 4 lncRNAs were identified as hub lncRNAs in the network, and FGD5-AS1 was focused for further loss-of-function investigation. Methods: The specific shRNAs against FGD5-AS1 (sh-FGD5-AS1) as well as the corresponding negative control (sh-NC) were constructed along with lentiviral vector respectively. Then the CCC-HEH-2 human cardiac myocytes cell lines were infected with lentivirus and followed puromycin treatment for several days. The knockdown efficiencies of the FGD5-AS1 expression were validated by qPCR. Genome-wide RNA expression of control and knockdown CCC-HEH-2 cell lines were observed using RNA-Seq. Conclusions: Knockdown of this lncRNA interferes with glutamate receptor activity and metalloendopeptidase inhibitor activity. The expression of abundant CHD genes with |fold change| ≥ 2 was validated with qPCR. These results indicate that FGD5-AS1 plays an important role in CHD genes regulation networks.

2021-06-02 | GSE157626 | 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

| PRJNA628989 | ENA

Chromatin accessibility data in Wild Type and FGD5-AS1 knockdown CCC-HEH-2 cells (ATAC-Seq)

Project description:Purpose: According to the previous analysis results of gene regulation in fetal heart tissues with tetralogy of Fallot (ToF), we constructed the ceRNA mediated network driven by lncRNAs using a causal inference framework based on the expression correlations and validated miRNA-lncRNA/mRNA evidences. Totally 4 lncRNAs were identified as hub lncRNAs in the network, and FGD5-AS1 was focused for further loss-of-function investigation. Methods: The specific shRNAs against FGD5-AS1 (sh-FGD5-AS1) as well as the corresponding negative control (sh-NC) were constructed along with lentiviral vector respectively. Then the CCC-HEH-2 human cardiac myocytes cell lines were infected with lentivirus and followed puromycin treatment for several days. The knockdown efficiencies of the FGD5-AS1 expression were validated by qPCR. The landscape of accessible chromatin in control and knockdown CCC-HEH-2 cell lines were observed using ATAC-Seq. Conclusions: Our data reveal a unique different chromatin state between wild type and FGD5-AS1 knockdown CCC-HEH-2 cells.

2021-06-02 | GSE159464 | GEO

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