Control of the Interferon Response in RNAi Experiments

Nejepinska, Jana; Flemr, Matyáš; Svoboda, Petr

doi:10.1007/978-1-61779-439-1_9

Cited by 5 publications

(5 citation statements)

References 63 publications

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“…This inhibition of RPA ubiquitination correlated precisely with decreases in His-ubiquitin levels rather than PRP19 depletion (Figures S4B, S4C). We suspect that this artifact is mediated by the interferon response, which normally detects long double-stranded RNAs but is also known to occur with siRNAs (Nejepinska et al, 2012). Interferon can inhibit expression from exogenous CMV promoters (Harms and Splitter, 1995).…”

Section: Resultsmentioning

confidence: 99%

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks

et al. 2015

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Summary We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings now reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells.

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Section: Resultsmentioning

confidence: 99%

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks

et al. 2015

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“…As the activation of the immune response has been described as a potential off-target effect induced after siRNA transfection, we questioned whether the four sequences were able to activate the innate immune signalling through Toll-like receptors [TLRs]. 28 We used the NFkB reporter cell line THP1-XBlue-MD2-CD14 [further referred to as THP1-XBlue cells], derived from the human monocytic THP1 cells. THP1-XBlue cells were exposed to siRNAs at a final concentration of 10 nM.…”

Section: Resultsmentioning

confidence: 99%

Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases

Clément

Nougarède

Combe

et al. 2021

Journal of Crohn's and Colitis

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Background and Aims Inflammatory bowel diseases are highly debilitating conditions that require constant monitoring and life-long medication. Current treatments are focused on systemic administration of immunomodulatory drugs, but they have a broad range of undesirable side-effects. The RNA interference is a highly specific endogenous mechanism that regulates the expression of the gene at the transcript level, which can be repurposed using exogenous short interfering RNA (siRNA) in order to repress the target gene’s expression. While siRNA therapeutics can offer an alternative to existing therapies, with a high specificity critical for chronically administrated drugs, evidence of their potency compared to chemical kinase inhibitors used in clinics is still lacking in alleviating an adverse inflammatory response. Methods We provide a framework to select highly specific siRNA, with a focus on two kinases strongly involved in pro-inflammatory diseases, namely JAK1 and JAK3. Using western-blot, RTqPCR and large scale analysis, we assessed the specificity profile of these siRNA drugs and compared their efficacy to the most recent and promising kinase inhibitors for Janus kinases (Jakinibs), Tofacitinib and Filgotinib. Results siRNA drugs can reach higher efficiency and selectivity at lower doses (5 pM versus 1 µM) than Jakinibs. Moreover, JAK silencing was lasting up to 11 days, even with 6h pulse transfection. Conclusions The siRNA-based drugs developed hold the potential to develop more potent therapeutics for chronic inflammatory diseases.

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“…a,b). It was reported that the IFN response is one of the most common non‐specific effects of RNAi experiments . To eliminate the possible confounding effects of a shRNA/shNC inducible IFN response, simvastatin, an inhibitor of HMGCR, was used.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase increases the expression of interferon-responsive genes

Ouyang

Chen

et al. 2014

Clin Exp Pharmacol Physiol

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The 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) pathway is an important metabolic route that is present in almost every organism. However, whether HMGCR affects the expression of interferon (IFN)-responsive genes is unclear. In the present study, expression levels of IFN-responsive genes were monitored by real time polymerase chain reaction and enzyme-linked immunosorbent assay. The results showed that expression levels of IFN-responsive genes were significantly increased in HMGCR-downregulated cells and HMGCR inhibitor-treated cells, indicating that inhibition of HMGCR activates the expression of IFN-responsive genes. The result in this study will provide new insight into the role of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in antiviral research.

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Control of the Interferon Response in RNAi Experiments

Cited by 5 publications

References 63 publications

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks

Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases

Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase increases the expression of interferon-responsive genes

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