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Effects of long dsRNA expression in HeLa and HEK293 cells

ABSTRACT: Double-stranded RNA (dsRNA) can enter different pathways in mammalian cells, including sequence-specific RNA interference, sequence-independent interferon response and editing by adenosine deaminases. To assess the potential of expressed dsRNA to induce interferon stimulated genes in somatic cells, we performed microarray analysis of HEK293 and HeLa cells transfected with a MosIR plasmid expressing an mRNA with a long inverted repeat structure in its 3’UTR (MosIR) or with a parental MosIR plasmid (without inverted repeat) as a control. Clustering analysis based on differentially expressed genes suggested that there was no common transcriptome signature in cells expressing dsRNA. Overall, the number of genes with altered expression upon transfection of the MosIR plasmid was rather small and only 19 probe sets, corresponding to 17 genes, were changed more than two-fold in both cell lines.

ORGANISM(S): Homo sapiens

PROVIDER: GSE27316 | GEO | 2011/10/11

SECONDARY ACCESSION(S): PRJNA137183

REPOSITORIES: GEO

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Effects of long dsRNA expression in HeLa and HEK293 cells

Project description:Double-stranded RNA (dsRNA) can enter different pathways in mammalian cells, including sequence-specific RNA interference, sequence-independent interferon response and editing by adenosine deaminases. To assess the potential of expressed dsRNA to induce interferon stimulated genes in somatic cells, we performed microarray analysis of HEK293 and HeLa cells transfected with a MosIR plasmid expressing an mRNA with a long inverted repeat structure in its 3’UTR (MosIR) or with a parental MosIR plasmid (without inverted repeat) as a control. Clustering analysis based on differentially expressed genes suggested that there was no common transcriptome signature in cells expressing dsRNA. Overall, the number of genes with altered expression upon transfection of the MosIR plasmid was rather small and only 19 probe sets, corresponding to 17 genes, were changed more than two-fold in both cell lines. Total RNA from cultured HEK293 or HeLa cells was used in each sample. Two independent biological replicates were analyzed for each condition.

2011-10-10 | E-GEOD-27316 | biostudies-arrayexpress

Ubiquitous expression of long dsRNA in mice causes mainly RNAi effects in the oocyte

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.

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2011-10-11 | GSE26577 | GEO

Expressed dsRNA provokes selective sequence-independent PKR response in transient co-transfection experiments

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.

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Cytosolic dsRNA-induced gene expression in mouse RAW24.7 macrophages and FIP200 knockout RAW cells

Project description:Our preliminary data showed that FIP200 is essential for dsRNA-induced interferon production. To corroborate the role of FIP200 in RIG-I signaling pathway, we are going to examine the cytosolic dsRNA-induced gene expression in mouse RAW24.7 macrophages and FIP200 knockout RAW cells.

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Pericentromeric hypomethylation elicits an interferon response in an animal model of ICF syndrome [dsRNA-seq]

Project description:The goal of this study was to investigate DNA methylation and gene expression changes in a zebrafish model of ICF Syndrome which were generated by mutation of ICF-gene zbtb24. Comparison of gene expression changes between wildtype and zbtb24 homozygous mutants revealed upregulation of interferon response genes following zbtb24 deletion. Upregulation of interferon response genes was blocked by mutation of the dsRNA helicase Mda5.

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