Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Nejepinska, Jana; Malı́k, Radek; Moravec, Martin; Svoboda, Petr

doi:10.1371/journal.pone.0043283

Cited by 34 publications

(33 citation statements)

References 28 publications

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“…3G). To confirm that pCAGEGFP-MosIR produces dsRNA, we analyzed adenosine deamination of short SOLiD sequence tags as previously described [8], [9]. Consistently with previous results, RNA editing of adenosine manifested as adenosine/guanosine (A/G) conversion in sequence tags clustering to the MosIR region (Fig.…”

Section: Resultssupporting

confidence: 81%

“…Our previous results suggested that dsRNA originating from a plasmid suppresses the expression of co-transfected reporters in a sequence-independent manner [9]. To examine the phenomenon, we used dsRNA-expressing pCAGEGFP-MosIR plasmid, in which the inverted repeat of the Mos gene sequence (MosIR) is inserted into the 3′UTR of an EGFP reporter controlled by a strong chimeric (CMV/β-actin) promoter (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Reporters Transiently Transfected into Mammalian Cells Are Highly Sensitive to Translational Repression Induced by dsRNA Expression

et al. 2014

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In mammals, double-stranded RNA (dsRNA) can mediate sequence-specific RNA interference, activate sequence-independent interferon response, or undergo RNA editing by adenosine deaminases. We showed that long hairpin dsRNA expression had negligible effects on mammalian somatic cells—expressed dsRNA was slightly edited, poorly processed into siRNAs, and it did not activate the interferon response. At the same time, we noticed reduced reporter expression in transient co-transfections, which was presumably induced by expressed dsRNA. Since transient co-transfections are frequently used for studying gene function, we systematically explored the role of expressed dsRNA in this silencing phenomenon. We demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits the expression of co-transfected reporter plasmids but not the expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent, it is found in different cell types, and it is independent of transfection method and dsRNA sequence. The inhibition occurs at the level of translation and involves protein kinase R, which binds the expressed dsRNA. Thus, dsRNA expression represents a hidden danger in transient transfection experiments and must be taken into account during interpretation of experimental results.

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Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Reporters Transiently Transfected into Mammalian Cells Are Highly Sensitive to Translational Repression Induced by dsRNA Expression

et al. 2014

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“…To investigate whether MERS-CoV accessory proteins can suppress the stress response pathway, we expressed them individually as EGFP fusion proteins and monitored SG formation in transfected cells. This approach is based on the observation that transfection of plasmid DNA, and in particular the pEGFP plasmids, can activate PKR, most likely due to the production of dsRNA formed from positive and negative sense mRNA transcription from cryptic promoters in these plasmids [25]. Indeed, we observed that transfection of pEGFP plasmid DNA in HeLa cells triggered SG formation in a PKR-dependent manner, as no SGs were observed in PKR knockout cells (HeLa-PKR KO ), which we generated using the CRISPR-Cas9 system (S1 Fig) (Fig 2A and 2B).…”

Section: Mers-cov P4a Suppresses Dsrna-and Pkr-dependent Formation Ofmentioning

confidence: 99%

Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

et al. 2016

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Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infections that can be life-threatening. To establish an infection and spread, MERS-CoV, like most other viruses, must navigate through an intricate network of antiviral host responses. Besides the well-known type I interferon (IFN-α/β) response, the protein kinase R (PKR)-mediated stress response is being recognized as an important innate response pathway. Upon detecting viral dsRNA, PKR phosphorylates eIF2α, leading to the inhibition of cellular and viral translation and the formation of stress granules (SGs), which are increasingly recognized as platforms for antiviral signaling pathways. It is unknown whether cellular infection by MERS-CoV activates the stress response pathway or whether the virus has evolved strategies to suppress this infection-limiting pathway. Here, we show that cellular infection with MERS-CoV does not lead to the formation of SGs. By transiently expressing the MERS-CoV accessory proteins individually, we identified a role of protein 4a (p4a) in preventing activation of the stress response pathway. Expression of MERS-CoV p4a impeded dsRNA-mediated PKR activation, thereby rescuing translation inhibition and preventing SG formation. In contrast, p4a failed to suppress stress response pathway activation that is independent of PKR and dsRNA. MERS-CoV p4a is a dsRNA binding protein. Mutation of the dsRNA binding motif in p4a disrupted its PKR antagonistic activity. By inserting p4a in a picornavirus lacking its natural PKR antagonist, we showed that p4a exerts PKR antagonistic activity also under infection conditions. However, a recombinant MERS-CoV deficient in p4a expression still suppressed SG formation, indicating the expression of at least one other stress response antagonist. This virus also suppressed the dsRNA-independent stress response pathway. Thus, MERS-CoV interferes with antiviral stress responses using at least two different mechanisms, with p4a suppressing the PKR-dependent stress response pathway, probably by sequestering dsRNA. MERS-CoV p4a represents the first coronavirus stress response antagonist described.

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“…3A, lower part). While MosMos transcript should not fold into dsRNA, it is possible that cells expressing high PKR levels were sensitized to dsRNA such that some cryptic transcription from the plasmid backbone could cause the effect (Nejepinska et al 2012c).…”

Section: Effects Of Expression Dsrna Binding Proteins On Rnaimentioning

confidence: 99%

Main constraints for RNAi induced by expressed long dsRNA in mouse cells

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Vaškovičová

Malı́k

et al. 2018

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RNA interference (RNAi) is sequence-specific mRNA degradation guided by small RNAs (siRNAs) produced from long double-stranded RNA (dsRNA) by RNase Dicer. Proteins executing RNAi are present in mammalian cells but sustain a gene-regulating microRNA pathway while dsRNA-induced innate immunity relies on a sequence-independent interferon response. While striving to benchmark mammalian RNAi analysis, we report that the main RNAi constraint is siRNA production, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of dsRNA-binding Dicer co-factors

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Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Cited by 34 publications

References 28 publications

Reporters Transiently Transfected into Mammalian Cells Are Highly Sensitive to Translational Repression Induced by dsRNA Expression

Reporters Transiently Transfected into Mammalian Cells Are Highly Sensitive to Translational Repression Induced by dsRNA Expression

Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

Main constraints for RNAi induced by expressed long dsRNA in mouse cells

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