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.
Highlights
Double-stranded RNA is a unique structure with important biological effects
Mos expression and function are restricted to oocytes [18,19]; effects observed in other cell types are sequenceindependent. pCAGEGFP-MosIR plasmid does not induce efficient RNA interference (RNAi) in cultured cells, presumably because of inefficient processing into small interfering RNAs (siRNAs) [8]
We show that expressed Double-stranded RNA (dsRNA) induces unique PKRdependent translational repression that is strongly biased towards transcripts from transiently transfected plasmids
Summary
Double-stranded RNA (dsRNA) is a unique structure with important biological effects. Viruses often give rise to dsRNA during their life cycle; dsRNA is recognized by a vertebrate cell as a hallmark of viral presence (reviewed in [1]). dsRNA can arise endogenously in a cell, being formed upon basepairing between complementary transcripts or by intramolecular pairing within a transcript, forming a hairpin. DsRNA can enter three pathways: RNA interference (RNAi), RNA editing, and the interferon response. RNAi mediates sequence-specific RNA degradation guided by ,22 nt small interfering RNAs (siRNAs) produced from long dsRNA by RNase III Dicer (reviewed in [2]). ADARs are nuclear and cytoplasmic enzymes activated by dsRNA that convert adenosines to inosines (which are recognized as guanosines during translation). The interferon response is a complex network of vertebrate pathways involved in the innate immune response against viruses (reviewed in [4]). One of the key factors in the interferon response is protein kinase R (PKR), which is activated upon binding of dsRNA to its dsRNA-binding domain. Activated PKR phosphorylates the a-subunit of the eukaryotic initiation factor 2 (eIF2a), which stabilizes the GEF-eIF2-GDP complex and, causes the inhibition of translation initiation (reviewed in [5]). Several groups observed restricted PKR effects and selective inhibition of specific mRNAs [6,7]
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