Adar-mediated A-to-I editing is required for establishment of embryonic body axes in zebrafish
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE182714 | GEO | 2022/08/02
REPOSITORIES: GEO
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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.
2013-06-15 | E-GEOD-47945 | biostudies-arrayexpress
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.
2013-06-17 | E-GEOD-47997 | biostudies-arrayexpress
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.
2021-07-16 | GSE176012 | GEO
Project description:Identification of genes differentially regulated after treatment of zebrafish embryos from 50% epiboly to 24hpf with 6.5uM leflunomide
2011-03-30 | GSE24527 | GEO
Project description:Adenosine deaminases, RNA specific (ADAR) are proteins that deaminate adenosine to inosine which is then recognized in translation as guanosine. To study the roles of ADAR proteins in RNA editing and gene regulation, we carried out DNA and RNA sequencing, RNA interference and RNA-immunoprecipitation in human B-cells. We also characterized the ADAR protein complex by mass spectrometry. The results uncovered over 60,000 sites where the adenosines (A) are edited to guanosine (G) and several thousand genes whose expression levels are influenced by ADAR. We also identified more than 100 proteins in the ADAR protein complex; these include splicing factors, heterogeneous ribonucleoproteins and several members of the dynactin protein family. Our findings show that in human B-cells, ADAR proteins are involved in two independent functions: A-to-G editing and gene expression regulation. In addition, we showed that other types of RNA-DNA sequence differences are not mediated by ADAR proteins, and thus there are co- or post-transcriptional mechanisms yet to be determined. Here we studied human B-cells where ADAR proteins (ADAR1 and ADAR2) are expressed but APOBECs are not. We identified the sequence differences between DNA and the corresponding RNA in B-cells from two individuals. Then, we carried out RNA interference, RNA-immunoprecipitation and next generation sequencing to determine the contribution of ADAR proteins in mediating A-to-G editing and other types of RNA-DNA sequence differences.
2013-09-20 | E-GEOD-38233 | biostudies-arrayexpress
Project description:A recently proposed model of cell-cell communication, called “urocrine signaling”, hypothesizes that small (50-150 mm) extracellular vesicles (EVs) carry signals to “downstream” kidney tubule cells. We previously showed that the exocyst, a highly-conserved eight-protein (Exoc1-8) trafficking complex, is necessary for ciliogenesis, we show that the EV number released from MDCK cells was increased in EXOC5 OE, while it was decreased in Exoc5 KD, compared to control. To determine how Exoc5 changes the composition of EVs, we isolated EVs from Exoc5 control, KD, and OE MDCK cells, and subjected them mass spectrometry-based proteomic analysis to determine the protein composition of the EVs. By dendrogram, heat map, and principle component analysis, the protein content of the EVs was significantly different in Exoc5 KD, EXOC5 OE, and control MDCK cells. To confirm the proteomic results, we examined, by Western blot analysis, expression of the small guanosine triphosphate (GTP)-binding protein ADP-ribosylation factor (Arf6) and the epidermal growth factor receptor kinase substrate 8-like protein 2 (Eps8L2).
2019-11-06 | PXD013549 | Pride
Project description:Identification of genes differentially regulated after treatment of zebrafish embryos from 50% epiboly to 24hpf with 6.5uM leflunomide A six chip study comparing expression levels of zebrafish embryos treated with leflunomide 6.5uM
2011-03-30 | E-GEOD-24527 | biostudies-arrayexpress
Project description:Our observation suggest that ADAR may play important role to maintain GSC stemness. In order to address this hypothesis, we abrogated the expression of ADAR in GSCs using two distinct short and non-overlapping shot hairpin RNAs (shRNAs). A non-targeting control shRNA (shCONT) is used as control in these experiments. Both shRNAs significantly reduced ADAR protein expression. Depletion of ADAR impaired GSCs proliferation but did not affect matched DGCs. Similarly, lack of ADAR reduces GSC self-renewal capacity as assessed by in vitro limiting dilution assay . In order to identify the antitumor mechanisms triggered by ADAR depletion, we performed global transcriptional profiling through RNA-sequencing in GSCs upon ADAR knockdown. These analyses show that ADAR downregulation impairs the impairs the expression of genes involved in cancer proliferation, particularly those involved in cell cycle regulation
2022-07-19 | GSE153270 | GEO
Project description:Adenosine deaminases, RNA specific (ADAR) are proteins that deaminate adenosine to inosine which is then recognized in translation as guanosine. To study the roles of ADAR proteins in RNA editing and gene regulation, we carried out DNA and RNA sequencing, RNA interference and RNA-immunoprecipitation in human B-cells. We also characterized the ADAR protein complex by mass spectrometry. The results uncovered over 60,000 sites where the adenosines (A) are edited to guanosine (G) and several thousand genes whose expression levels are influenced by ADAR. We also identified more than 100 proteins in the ADAR protein complex; these include splicing factors, heterogeneous ribonucleoproteins and several members of the dynactin protein family. Our findings show that in human B-cells, ADAR proteins are involved in two independent functions: A-to-G editing and gene expression regulation. In addition, we showed that other types of RNA-DNA sequence differences are not mediated by ADAR proteins, and thus there are co- or post-transcriptional mechanisms yet to be determined.
2013-09-20 | GSE38233 | GEO
Project description:In order to test whether ADAR is responsible for mediating part of the transcriptional response to temperature changes in fly we determined the gene expression profile of fly heads from control and ADAR Hypomorph flies at 18 and 29°C by 3’ RNA-seq.
2017-07-20 | GSE95313 | GEO