DsRNA immunoprecipitation sequencing on a murine model of endotoxin-induced acute kidney injury
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE244941 | GEO | 2023/11/10
REPOSITORIES: GEO
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Project description:In mammals, double-stranded RNA (dsRNA) plays roles in sequence-specific RNA interference, sequence-independent interferon response, and RNA editing by adenosine deaminases. We have previously shown that long hairpin dsRNA expression in cultured cells does not activate the interferon response, it is poorly processed into siRNAs, and it is partially edited. Here, we demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits expression of co-transfected reporter plasmids but not expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent and independent of a cell type, transfection method, or dsRNA sequence. The inhibition occurs at the level of translation and is mediated by protein kinase R (PKR). PKR binds the expressed dsRNA, becomes phosphorylated and changes its distribution along polysome fractions. In conclusion, we demonstrate that expression from plasmids is selectively repressed if one of co transfected plasmids produces dsRNA. Our results highlight the importance of proper controls and careful interpretation of co-transfection experiments. HEK293 cells (human origin) were transiently transfected with hairpin dsRNA-expressing (MosIR) and control (pCag) plasmids.
2013-12-27 | E-GEOD-46959 | biostudies-arrayexpress
Project description:In mammals, double-stranded RNA (dsRNA) plays roles in sequence-specific RNA interference, sequence-independent interferon response, and RNA editing by adenosine deaminases. We have previously shown that long hairpin dsRNA expression in cultured cells does not activate the interferon response, it is poorly processed into siRNAs, and it is partially edited. Here, we demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits expression of co-transfected reporter plasmids but not expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent and independent of a cell type, transfection method, or dsRNA sequence. The inhibition occurs at the level of translation and is mediated by protein kinase R (PKR). PKR binds the expressed dsRNA, becomes phosphorylated and changes its distribution along polysome fractions. In conclusion, we demonstrate that expression from plasmids is selectively repressed if one of co transfected plasmids produces dsRNA. Our results highlight the importance of proper controls and careful interpretation of co-transfection experiments.
2013-12-27 | GSE46959 | GEO
Project description:8 week-old male C57BL6J mice were given Gram-negative endotoxin (LPS O111:B4, 10 mg/kg) intraperitoneally at time 0. 18 hrs thereafter, they were administered 10 ml/kg 0.9% saline. Mice were sacrificed at 0, 18, or 42 hrs after LPS challenge. Kidneys were immediately collected into TRIzol for RNA preparation. Renal function was measured on blood collected at the time of tissue harvest At t=0hr, mice had normal baseline renal function. At t=18hr, mice exhibited early renal injury, At t=42hr, mice had either recovered normal renal function or had persistent renal injury. We collected kidneys from 3 mice per time point. For the 42 hr time point, we collected kidneys from 3 mice with recovered renal function and kidneys from 3 mice with persistent renal injury.
2011-07-12 | GSE30576 | GEO
Project description:8 week-old male C57BL6J mice were given Gram-negative endotoxin (LPS O111:B4, 10 mg/kg) intraperitoneally at time 0. 18 hrs thereafter, they were administered 10 ml/kg 0.9% saline. Mice were sacrificed at 0, 18, or 42 hrs after LPS challenge. Kidneys were immediately collected into TRIzol for RNA preparation. Renal function was measured on blood collected at the time of tissue harvest At t=0hr, mice had normal baseline renal function. At t=18hr, mice exhibited early renal injury, At t=42hr, mice had either recovered normal renal function or had persistent renal injury. We collected kidneys from 3 mice per time point. For the 42 hr time point, we collected kidneys from 3 mice with recovered renal function and kidneys from 3 mice with persistent renal injury. Mouse kidneys selected at successive stages of renal injury and recovery following systemic LPS challenge and volume resuscitation following LPS challenge.
2011-07-12 | E-GEOD-30576 | biostudies-arrayexpress
Project description:Small RNA produced by Dicer (Dcr1) are used to map dsRNA in wild-type strain and a xrn1-delta mutant of S. cerevisiae, inactivated for the cytoplasmic 5'-3' RNA decay pathway.
2015-08-11 | GSE64090 | GEO
Project description:The ADAR RNA editing enzymes deaminate adenosine bases to inosines in cellular RNAs, recoding open reading frames. Human ADAR1 mutations cause Aicardi-Goutieres Syndrome (AGS) and Adar1 mutant mice showing an aberrant interferon response and death by embryonic day E12.5 model the human disease. Searches have not identified key ADAR1 RNA editing sites recoding immune/haematopoietic proteins but editing is widespread in Alu sequences. We show that Adar1 embryonic lethality is rescued in Adar1; Mavs double mutant mice in which general antiviral responses to cytoplasmic dsRNA are prevented. We propose that inosine bases are epigenetic marks identifying cellular RNA as innate immune ÒselfÓ. Consistent with this idea we show that an editing-active cytoplasmic ADAR is required to prevent aberrant immune responses in Adar1 mutant mouse embryo fibroblasts. No dramatic increase in repetitive transcripts is observed. AGS mutations in ADAR1 affect editing by the interferon-inducible cytoplasmic ADAR1 isoform. RNA-seq expression profiling in Adar1 and Adar1/Mavs knockout mice embryos.
2014-12-19 | E-GEOD-62917 | biostudies-arrayexpress
Project description:Small RNA produced by Dicer (Dcr1) are used to map dsRNA in wild-type strain and a xrn1-delta mutant of S. cerevisiae, inactivated for the cytoplasmic 5'-3' RNA decay pathway. Small RNA sequencing in wild-type and xrn1-delta strains of S. cerevisiae, with or without reconstituted RNAi pathway.
2015-08-11 | E-GEOD-64090 | biostudies-arrayexpress
Project description:Purpose: The purpose of this experiment is to expand the repertoire of C. elegans edited transcripts and identify the roles of ADR-1 as indirect regulator of editing and ADR-2 as the only active deaminase in vivo. Methods: Strand-specific RNA sequencing of wild-type and adr mutant worms, followed by a novel RNA variant calling and comparative analysis pipeline. Results: Despite lacking deaminase function, ADR-1 affects editing of over 60 adenosines within the 3’ UTRs of 16 different mRNAs. Furthermore, ADR-1 interacts directly with ADR-2 substrates, even in the absence of ADR-2; and mutations within its dsRNA binding domains abolished both binding and editing regulation. Conclusions: ADR-1 acts as a major regulator of editing by binding ADR-2 substrates in vivo and raises the possibility that other dsRNA binding proteins, including the inactive human ADARs, regulate RNA editing by deaminase-independent mechanisms. Strand-specific RNA sequencing of wild-type and adr mutant worms, followed by a novel RNA variant calling and comparative analysis pipeline.
2014-01-20 | E-GEOD-51556 | biostudies-arrayexpress
Project description:Double-stranded RNA (dsRNA) can enter different pathways in mammalian cells, such as the sequence-specific RNA interference (RNAi) pathway, the sequence-independent interferon response, and RNA editing by adenosine deaminases. To study routing of long dsRNA into different pathways in different tissues, we generated transgenic mice carrying an inverted repeat transcribed by a strong, ubiquitously active polII promoter. Here, we provide the first report of effects caused by ubiquitous long dsRNA expression in the adult mouse. Long dsRNA is poorly processed into siRNAs in somatic cells but readily induces the RNAi effects in the oocyte, suggesting that somatic cells are missing a component of RNAi that facilitates siRNA biogenesis. Expression of dsRNA is not sufficient to activate the interferon pathway and has a minimal effect on the transcriptome in somatic cells. The interferon response in somatic cells could be induced with high doses of dsRNA expression, suggesting that somatic cells can tolerate endogenous dsRNA expression to a large extent. Our data demonstrate that the association between long dsRNA and the interferon pathway in somatic cells cannot be generalized and that cells recognize other features of dsRNA molecules, which increase or reduce the likelihood activation of a particular dsRNA-induced pathway. Brain or kidney tissue (transgenic mice), or HEK293 or HeLa cell lines (human), expressing long dsRNA within the 3'-UTR of the EGFP reporter gene.
2011-10-10 | E-GEOD-26577 | biostudies-arrayexpress
Project description:Eukaryotic cells recognize intracellular pathogens through pattern recognition receptors, including sensors of aberrant nucleic acid structures. Sensors of double-stranded RNA (dsRNA) are known to detect replication intermediates of RNA viruses. It has been suggested that annealing of mRNA from symmetrical transcription of both top and bottom strands of DNA virus genomes can produce dsRNA during infection. Supporting this hypothesis, nearly all DNA viruses encode inhibitors of dsRNA-recognition pathways. However, direct evidence that DNA viruses produce dsRNA is lacking. Contrary to dogma, we show that the nuclear-replicating DNA virus adenovirus (AdV) does not produce detectable levels of dsRNA during infection. In contrast, abundant dsRNA is detected within the nucleus of cells infected with AdV mutants that lack the virus-directed ubiquitin ligase required for efficient processing of viral RNA. In the presence of nuclear dsRNA, the cytoplasmic dsRNA sensor PKR is relocalized and activated within the nucleus. Accumulation of viral dsRNA occurs in the late phase of infection, when unspliced viral transcripts form intron/exon base pairs between top and bottom strand transcripts. We propose that DNA viruses actively promote efficient splicing and mRNA processing to limit dsRNA formation, thus avoiding detection and restriction by host nucleic acid sensors.
2021-07-15 | GSE179388 | GEO