RNAseq of hnRNPC and ADAR deficient THP-1
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA734583 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14711300_1.fastq.gz | Fastqsanger.gz | |||
| SRR14711300_2.fastq.gz | Fastqsanger.gz | |||
| SRR14711301_1.fastq.gz | Fastqsanger.gz | |||
| SRR14711301_2.fastq.gz | Fastqsanger.gz | |||
| SRR14711302_1.fastq.gz | Fastqsanger.gz |
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Project description:Endogenous double-stranded RNA is prevented from activating the cytosolic antiviral receptor MDA5 by adenosine-to-inosine editing by ADAR. Consequently, CRISPR/cas9 targeting ADAR in the human monocytic cell line THP-1 induces a spontaneous MDA5-dependent interferon response. This response is synergistically enhanced by concomitant CRISPR/cas9 targeting of hnRNPC. For this study, cas9 transgenic THP-1 were nucleofected with combinations of control gRNA, ADAR gRNA or hnRNPC gRNA to elucidate the basis for synergistic interferon-stmulated gene induction by combined ADAR and hnRNPC targeting. As a control for interferon-stimulated gene induction, IFN-alpha treated THP-1 were included, as well. To investigate potential interferon inducing mechanisms, we quantified gene expression, intron expression as well as the expression of regions rich in adenosine-to-inosine editing clusters. In a second part, nuclear vs cytosolic distribution of RNA rich in adenosine-to-inosine clusters was investigated by sequencing of total RNA or RNA from cytosolic and nuclear extracts of ADAR and/or hnRNPC targeted cells. In the first part of the study, STING-deficient, cas9 transgenic THP-1 were nucleofected with combinations of control, GFP-targeting, ADAR-targeting or hnRNPC-targeting gRNA and cells collected for total RNA extraction on days three, four and five after nucleofection. In addition, another control-nucleofected sample was treated on day 3 for 24 h. The experiment was repeated three times leading to three replicates per condition. In the second part, WT cas9-transgenic THP-1 were nucleofected with combinations of control, ADAR-targeting or hnRNPC-targeting gRNA and cells were either collected for total RNA extraction or separated into digitonin-soluble (cytosolic) and digitonin-resistant (nuclear and mitochondrial) compartments and then RNA extracted. The experiment was repeated twice leading to two replicates per condition. Differential gene expression analysis confirmed synergistic induction of interferon-stimulated genes. RNA-seq analysis demonstrated dysregulation of Alu-containing introns in hnRNPC-deficient cells via utilization of unmasked cryptic splice sites, including introns containing ADAR-dependent A-to-I editing clusters or regions enriched in editing clusters (ECRs) in general. These putative MDA5 ligands showed reduced editing in the absence of ADAR, providing a plausible mechanism for the combined effects of hnRNPC and ADAR. Part 2 of the study confirmed cytosolic access of a subset of ECRs to the cytosol.
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Project description:RNAseq transcriptome analysis of THP-1 macrophages activated by VHL-deficient kidney tubule cells
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Project description:The purpose of this experiment was to compare differences in Alu-exon inclusion in cells depleted of hnRNPC, Upf1, or both.
2016-04-01 | E-MTAB-4008 | biostudies-arrayexpress
Project description:HNRNPC plays an important role in HCC metastasis, HNRNPC knockdown by specific shRNA (HNRNPC-shRNA) significantly inhibited the migration and invasion of MHCC97H cells, while HNRNPC overexpression exerted the opposite effect. To elucidate the mechanisms by which HNRNPC facilitated HCC metastasis, we performed microarray analysis to compare the transcription profiling between the MHCC97H-shcontrol and MHCC97H-shHNRNPC cells.
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Project description:Cancer cells often co-opt post-transcriptional regulatory mechanisms to achieve pathologic expression of gene networks that drive metastasis. Translational control is a major regulatory hub in oncogenesis, however its effects on cancer progression remain poorly understood. To address this, we used ribosome profiling to compare genome-wide translation efficiencies of poorly and highly metastatic breast cancer cells and patient-derived xenografts. We developed novel regression-based methods to analyze ribosome profiling and alternative polyadenylation data, and identified HNRNPC as a translational controller of a specific mRNA regulon. Mechanistically, HNRNPC, in concert with PABPC4, binds near to poly(A) signals, thereby governing the alternative polyadenylation of a set of mRNAs. We found that HNRNPC and PABPC4 are downregulated in highly metastatic cells, which causes HNRNPC-bound mRNAs to undergo 3’ UTR lengthening and subsequently, translational repression. We showed that modulating HNRNPC expression impacts the metastatic capacity of breast cancer cells in xenograft mouse models. We also found that a small molecule, previously shown to induce a distal-to-proximal poly(A) site switching, counteracts the HNRNPC-PABPC4 driven deregulation of alternative polyadenylation and decreases the metastatic lung colonization by breast cancer cells in vivo.
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2013-09-20 | E-GEOD-38233 | biostudies-arrayexpress
Project description:To define and compare the interactomes of the RNA binding protein HNRNPC in poorly vs. efficiently metastatic breast adenocarcinoma cells, we carried out immunoprecipitation of endogenous HNRNPC from parental MDA-MB231 cells vs. its highly metastatic isogenic derivate, the MDA-MB231-LM2 cells. We used a non-specific MOUSE IgG IP from each line as control. Each IP was performed in triplicate, and analysed by LC-MS/MS, on a Thermo Q-Exactive-plus instrument.
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Project description:Elevated expression of RNA binding protein HNRNPC has been reported in cancer cells, while the essentialness and functions of HNRNPC in tumors were not clear. We showed that repression of HNRNPC in the breast cancer cells MCF7 and T47D inhibited cell proliferation and tumor growth. Our computational inference of the key pathways and extensive experimental investigations revealed that the cascade of interferon responses mediated by RIG-I was responsible for such tumor-inhibitory effect. Interestingly, repression of HNRNPC resulted in accumulation of endogenous double-stranded RNA (dsRNA), the binding ligand of RIG-I. These up-regulated dsRNA species were highly enriched by Alu sequences and mostly originated from pre-mRNA introns that harbor the known HNRNPC binding sites. Such source of dsRNA is different than the recently well-characterized endogenous retroviruses that encode dsRNA. In summary, essentialness of HNRNPC in the breast cancer cells was attributed to its function in controlling the endogenous dsRNA and the down-stream interferon response. This is a novel extension from the previous understandings about HNRNPC in binding with introns and regulating RNA splicing.
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Project description:Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery. HepG2 and K562 cell lines were stably transfected with plasmids containing siRNA designed to specifically knock down ADAR expression (ADAR KD). This in order to examine how ADAR affects alternative splicing globally.
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