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

Abstract

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.

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

“…LacZ-expressing plasmid was co-transfected as a "neutral" co-expressed protein, which was later used as a control. LacZ yielded the same sequence-independent dsRNA effect as previously observed without a tested co-expressed protein (Nejepinska et al, 2014) and similar effect as expression of EGFP and RFP (Fig. S1B).…”

“…The long hairpin RNA organization is similar to some naturally occurring long dsRNA hairpins, which give rise to endogenous siRNAs in Caenorhabditis elegans (Morse and Bass, 1999) and mouse oocytes (Tam et al, 2008;Watanabe et al, 2008). Importantly, all three hairpin transcripts could be efficiently immunoprecipitated with an anti-dsRNA antibody (Nejepinska et al, 2014) and their expression induced robust RNAi in oocytes in vivo (Chalupnikova et al, 2014;Flemr et al, 2014;Stein et al, 2003b). In a control plasmid CAG-EGFP-MosMos ( Fig.…”

“…1C, S1B). We have shown previously that reporter expression from co-transfected plasmids is particularly sensitive in PKR-dependent manner to dsRNA expressed from a cotransfected plasmid (Nejepinska et al, 2014). The occurrence of this effect in raw data is thus a good indicator of dsRNA expression.…”

“…1A and 4A. Three dsRNA-expressing plasmids (MosIR, Lin28IR, and Elavl2IR), which efficiently induced RNAi in oocytes of transgenic mice (Chalupnikova et al, 2014;Flemr et al, 2014;Stein et al, 2003b) were modified by replacing the oocytes-specific ZP3 promoter with a strong ubiquitous CAG promoter as described previously (Nejepinska et al, 2014). pGL4-SV40 (Promega; for simplicity referred to as FL) and the parental plasmid for targeted Renilla reporter phRL-SV40 (Promega; for simplicity referred to as RL) are commercially available.…”

Resolving systematic errors in widely-used enhancer activity assays in human cells enables genome-wide functional enhancer characterization

“…Cytoplasmic DNA leads to IFN-I induction via the key signaling kinases TANK binding kinase 1 (TBK1) and IκB kinase ε (IKKε), which activate IRF3 28 . Similarly, double stranded RNA (dsRNA) that can arise at transfected plasmids is sensed by dsRNAactivated protein kinase (PKR) and can affect transgene expression [29][30][31] . The inhibition of these key signaling kinases should therefore ameliorate the changes of gene expression and enhancer activities described above.…”