Enhanced crosslinking and immunoprecipitation of SERBP1 in Hela cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE156015 | GEO | 2020/10/27
REPOSITORIES: GEO
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Project description:Enhanced crosslinking and immunoprecipitation of SERBP1 in Hela cells
| PRJNA656424 | ENA
Project description:Background
RNA binding proteins (RBPs) containing intrinsically disordered regions (IDRs) are present in diverse molecular complexes where they function as dynamic regulators. Their characteristics allow liquid-liquid phase separation (LLPS) and formation of membraneless organelles or molecular condensates such as stress granules and nucleoli. IDR-RBPs are particularly relevant in the nervous system, regulating brain function, neurogenesis and synapse plasticity. Their dysfunction is associated with neurodegenerative diseases and brain tumor development. SERBP1 is a unique member of this group, being mostly disordered and lacking canonical RNA-binding domains. We have previously defined SERBP1 as an oncogenic factor in glioblastoma. Here, we investigated the SERBP1 interactome using a proteomics approach.
Results
We defined novel roles for SERBP1 in splicing, cell division, DNA repair and ribosomal biogenesis based on the protein's interactome and functional analyses. SERBP1 preferentially interacts with other G-quadruplex (G4) binders, implicated in different stages of gene expression, suggesting that G4 binding is a critical component of SERBP1 function in different settings. Similarly, we identified important associations between SERBP1 and PARP1/polyADP-ribosylation (PARylation). SERBP1 interacts with PARP1 and its associated factors and influences PARylation. Moreover, SERBP1 and most of its associated proteins get PARylated and/or bind to PAR, indicating that SERBP1 is present in regulatory complexes that are built on PAR binding. Finally, we determined that SERBP1 is potentially implicated in Alzheimer's where it is highly expressed and present in pathological stress granules and Tau aggregates.
Conclusion
Our study established SERBP1 as a multi-functional protein that participates in distinct regulatory complexes assembled via G4- and PAR-binding.
2023-11-10 | MSV000093355 | MassIVE
Project description:Background
RNA binding proteins (RBPs) containing intrinsically disordered regions (IDRs) are present in diverse molecular complexes where they function as dynamic regulators. Their characteristics allow liquid-liquid phase separation (LLPS) and formation of membraneless organelles or molecular condensates such as stress granules and nucleoli. IDR-RBPs are particularly relevant in the nervous system, regulating brain function, neurogenesis and synapse plasticity. Their dysfunction is associated with neurodegenerative diseases and brain tumor development. SERBP1 is a unique member of this group, being mostly disordered and lacking canonical RNA-binding domains. We have previously defined SERBP1 as an oncogenic factor in glioblastoma. Here, we investigated the SERBP1 interactome using a proteomics approach.
Results
We defined novel roles for SERBP1 in splicing, cell division, DNA repair and ribosomal biogenesis based on the protein's interactome and functional analyses. SERBP1 preferentially interacts with other G-quadruplex (G4) binders, implicated in different stages of gene expression, suggesting that G4 binding is a critical component of SERBP1 function in different settings. Similarly, we identified important associations between SERBP1 and PARP1/polyADP-ribosylation (PARylation). SERBP1 interacts with PARP1 and its associated factors and influences PARylation. Moreover, SERBP1 and most of its associated proteins get PARylated and/or bind to PAR, indicating that SERBP1 is present in regulatory complexes that are built on PAR binding. Finally, we determined that SERBP1 is potentially implicated in Alzheimer's where it is highly expressed and present in pathological stress granules and Tau aggregates.
Conclusion
Our study established SERBP1 as a multi-functional protein that participates in distinct regulatory complexes assembled via G4- and PAR-binding.
2023-04-19 | MSV000091749 | MassIVE
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:N6-methyladenosine (m6A) is the most abundant chemical modification in mRNA, and plays important roles in human embryonic stem cell pluripotency, maintenance, and differentiation. However, the role of m6A and the precise mechanisms involved during the development of β-cells are unexplored. Here, we performed enhanced crosslinking and immunoprecipitation (eCLIP) assays to identify m6A sites regulated by METTL14 in EndoC-βh1 human fetal β-cells.
2024-06-13 | GSE236324 | GEO
Project description:Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.
| S-EPMC5991800 | biostudies-literature
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: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: 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