Enhanced crosslinking and immunoprecipitation of SERBP1 in Hela cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE156015 | GEO | 2020/10/27
REPOSITORIES: GEO
Similar Datasets
Project description:Enhanced crosslinking and immunoprecipitation of SERBP1 in Hela cells
| PRJNA656424 | ENA
Project description:RNA-binding proteins (RBPs) have important roles in orchestrating posttranscriptional regulation and modulating many tumorigenesis events. SERBP1 has been recognized as an important regulator in multiple cancers, while it remains unclear whether SERBP1-regulated gene expression at the transcriptome-wide level is significantly correlated with tumorigenesis.We overexpressed SERBP1 in HeLa cells and explored whether SERBP1 overexpression (SERBP1-OE) affects the proliferation and apoptosis of HeLa cells. We analyzed the transcriptome-wide gene expression changes and alternative splicing changes mediated by SERBP1-OE using transcriptome sequencing method (RNA-seq). RT-qPCR was conducted to assay SERBP1-regulated alternative splicing.SERBP1-OE induced the apoptosis of HeLa cells. The downregulated genes were strongly enriched in the cell proliferation and apoptosis pathways according to the GO analysis, including FOS, FOSB, PAK6 and RAB26. The genes undergoing at least one SERBP1-regulated alternative splicing event were enriched in transcriptional regulation, suggesting a mechanism of the regulation of gene expression, and in pyruvate and fatty acid metabolic processes critical for tumorigenesis events. The SERBP1-regulated alternative splicing of ME3, LPIN3, CROT，PDP1, SLC27A1 and ALKBH7 was validated by RT-qPCR analysis.We for the first time demonstrated the cellular function and molecular targets of SERBP1 in HeLa cells at transcriptional and post-transcriptional levels. The SERBP1-regulated gene expression and alternative splicing networks revealed by this study provide important information for exploring the functional roles and regulatory mechanisms of SERBP1 in cancer development and progression.
2022-09-02 | GSE192950 | GEO
Project description:The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their S. cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1 mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA.
2009-12-10 | GSE19205 | GEO
Project description: Not available
| S-EPMC5991800 | biostudies-literature
Project description:The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their S. cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1 mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA. Cell culture conditions and media Human fibroblast cell strains (WS: AG05229, AG12795, AG12797; BS: GM02932, GM03402, GM16891; RTS: AG18371, AG18375, AG05013; Normal/Wild-type: AG04054, AG06310, AG09975) were obtained from the Coriell Repository (Camden, NJ), from donors matched for gender and of similar ages, and were at similar passage levels. Cells were cultured in MEM supplemented with Earle’s salts, 20% fetal bovine serum, 1x penicillin/streptomycin, and 1x fungizone in 3% O2 at 37oC and harvested for RNA extraction during active growth and at ~ 80% confluence. GeneChip microarray expression Total RNA from the 12 fibroblast cell strains was isolated by extraction with TRIzol (Invitrogen) and purified using the RNeasy system (Qiagen). Total RNA was amplified by in vitro transcription using the Ovation RNA Amplification System V2 (NuGen). The resultant cDNA was fragmented and labeled using the FL-Ovation cDNA Biotin Module V2 (NuGen), and then purified using QIAquick columns (Qiagen), as specified by the Ovation System manual. Labeled probe was hybridized to Affymetrix U133A 2.0 GeneChips, and ultimately scanned using an Axon GenePix array scanner. Statistical analysis of microarray expression experiment The output files were normalized by Robust Multiarray Average (RMA), using the R package GCRMA and gene expression levels were log2-transformed.
2009-12-09 | E-GEOD-19205 | biostudies-arrayexpress
Project description:Purpose: to identify the AGO1-bound miRNAs and mRNAs in human endothelial cells subject to hypoxic condition (2% oxygen) Methods: human microvascular endothelial cells was cultured under normoxia (20% oxygen) or hypoxia (2%) oxygen for 24 hours. Total RNA was extracted and RNA and small RNA libraries were prepared for Seq. Results: We found substantial changes in AGO1-bound mRNAs due to hypoxia. A portion of CLIP-seq reads also reveal direct binding to miRNA to mRNA. Conclusions: hypoxia induces profound changes in mRNAs associated with AGO1.
2019-09-06 | GSE136907 | GEO
Project description:Purpose: This study was performed to determine the RNA binding targets of the RNA binding protein CELF6 in the mouse brain. Method: 4 pools of CELF6-HA/YFP brains (cortices & cerebella removed) were snap frozen in liquid nitrogen, powdered, crosslinked (254 nm, 400 J/cm^2, 3 pulses), and homogenized and immunoprecipitated with anti-EGFP antibodies. Control samples consistent of input RNA from immunoprecipitates as well as anti-EGFP immunoprecipitates of wild-type (WT, HA/YFP negative) tissue. Sequencing libraries were prepared from harvested RNA with 9-mer unique molecular identifiers (UMIs). Procedure uses Trimmomatic to catalog UMIs as well as trim low quality bases, STAR to align to mm10 reference genome, Fulcrum Genomics FGBio to annotate BAM files with UMI information, Picard Tools to remove PCR duplicates, and Subread featureCounts to count reads mapping to features in a GTF in a strand specific manner. GTFs were prepared to contain annotated: 3'UTRs, 5'UTRs, CDS, as introns+/-10 bp overlap into surrounding exons. Additionally, BAMs were counted against features in a GTF corresponding to peaks identified by Piranha. To do this, an example BAM file (merge of all HA/YFP+ samples) was windowed using Bedtools, counted in a strand-specific manner using Subread featureCounts, and converted to BED format for peak calling with Piranha. The final called peaks were edited to a width of of 100 bp around significant maxima, and compiled into a GTF. This GTF was used for recounting individual sample files for immunoprecipitates and controls. Final counts were tested for significant enrichment in the HA/YFP-immunoprecipitated sample compared to (a) input samples, and (b) WT controls using edgeR. Samples were sequenced on 4 lanes of an 2xruns on an Illumina NextSeq, and BAMs were prepared on data for each lane separately and then merged across lanes by sample before counting. Results: Features showing nominal edgeR p values <0.05 in both YFP vs. Input and YFP vs. WT comparisons, as well as positive log2 fold change in YFP vs. Input and YFP vs. WT input comparison showed a predominance of significant comparisons falling in counts from 3'UTR regions. This was also true when restricting to only high confidence targets which showed FDR <0.05 by Benjamini-Hochberg. This was the case whether testing across sums under Piranha peaks in YFP vs. controls, as well as summing all counts across annotated 3'UTR, 5'UTR, introns, and CDS regardless of the presence of a called peak. Conclusions: CELF6 shows enriched binding in 3'UTR regions in YFP+ samples vs. controls. Nominally bound regions were used downstream to validate findings and explore the consequence of CELF6 associate in a massively parallel reporter assay. This is the first exploration of the binding targets of this RNA binding protein, linked to behavioral changes in knock-out mice (Dougherty et al. J. Neurosci 2013)
2018-08-17 | GSE118623 | GEO
Project description:Purpose: The goals of this study are to identify AID targets in DLBCL and investigate the overlapping of AID peaks and predicted G4 forming sequences (PQS) and the B-cell super enhancer (SE) U2932 DLBCL cells obtained from the American Type Culture Collection were grown in RPMI 1640 medium (Corning) supplemented with 10% FBS (Atlanta Biiologicals) and 1% Pen/Strep (Gibco). For each immunoprecipitation reaction, 4x106 chromatin cell equivalent were incubated overnight with either an AID antibody (Abcam, ab59361) or a rabbit IgG negative control antibody, subjected to protein A/G magnetic bead binding, IP elution and DNA recovery. The ChIP DNA was used to generate ChIPseq libraries with the ChIP-Seq sample preparation kit (Illumina) for sequencing at the Arkansas Children’s Research Institute Genomics Core. The sequence reads that passed quality filters were aligned to the human genome (hg19) using BWA-MEM and peakcalled using MACS2. Results: Using an optimized data analysis workflow, we mapped about 10-20 million sequence reads per sample to the human genome (build hg19) and identified 4,573 AID peaks which were filtered out if they overlap with the mock pull-down peaks and the blacklist (ENCODE blacklisted regions). We observed the intersection of AID peaks with PQS and SEs are significantly higher than expected by chance in all cases of comparisons supporting that the number of AID peaks sharing PQS and/or SEs is not a random event. Conclusions: Sequencing analyses reveal AID targets in both activated B-cells and lymphoma are highly enriched for G4 elements and validation studies confirm a representative set of these sequences form characteristic, stable G4 structures.
2020-04-08 | GSE146695 | GEO
Project description:Transcriptome-wide profiling of RNA-binding by IGF2BP1 and IGF2BP3 in the K562 cell line. Binding sites are characterized by T-to-C cross-linking mutations, depth and location on transcript
2020-02-10 | GSE138063 | GEO
Project description:Transcriptome-wide profiling of RNA-binding by four RBPs in v-Abl transformed 220-8 pro-B mouse cell line. Binding sites are characterized by T-to-C cross-linking mutations, depth, location on transcript, and overlap/exclusivity across different RBPs.
2019-06-27 | GSE98465 | GEO