In mammals, a single Dicer participates in biogenesis of small RNAs in microRNA (miRNA) and RNAi pathways. In mice, endogenous RNAi is highly active in oocytes, but not in somatic cells, which we ascribe here to an oocyte-specific Dicer isoform (Dicer(O)). Dicer(O) lacks the N-terminal DExD helicase domain and has higher cleavage activity than the full-length Dicer in somatic cells (Dicer(S)). Unlike Dicer(S), Dicer(O) efficiently produces small RNAs from long double-stranded (dsRNA) substrates. Expression of the Dicer(O) isoform is driven by an intronic MT-C retrotransposon promoter, deletion of which causes loss of Dicer(O) and female sterility. Oocytes from females lacking the MT-C element show meiotic spindle defects and increased levels of endogenous small interfering RNA (endo-siRNA) targets, phenocopying the maternal Dicer null phenotype. The alternative Dicer isoform, whose phylogenetic origin demonstrates evolutionary plasticity of RNA-silencing pathways, is the main determinant of endogenous RNAi activity in the mouse female germline.

Double-stranded RNA (dsRNA) can enter different pathways in mammalian cells, including sequence-specific RNA interference (RNAi), sequence-independent interferon (IFN) response and editing by adenosine deaminases. To study the routing of dsRNA to these pathways in vivo, we used transgenic mice ubiquitously expressing from a strong promoter, an mRNA with a long hairpin in its 3′-UTR. The expressed dsRNA neither caused any developmental defects nor activated the IFN response, which was inducible only at high expression levels in cultured cells. The dsRNA was poorly processed into siRNAs in somatic cells, whereas, robust RNAi effects were found in oocytes, suggesting that somatic cells lack some factor(s) facilitating siRNA biogenesis. Expressed dsRNA did not cause transcriptional silencing in trans. Analysis of RNA editing revealed that a small fraction of long dsRNA is edited. RNA editing neither prevented the cytoplasmic localization nor processing into siRNAs. Thus, a long dsRNA structure is well tolerated in mammalian cells and is mainly causing a robust RNAi response in oocytes.

Transient plasmid transfection is a common approach in studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We have found that the entire plasmid sequence is transcribed at different levels. Spurious transcription may have undesirable effects as some plasmids, when co-transfected, inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to a Kan/Neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression from transiently transfected plasmids underscores the importance of appropriate experimental controls.

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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Double-stranded RNA (dsRNA) is involved in different biological processes. At least three different pathways can respond to dsRNA in mammals. One of these pathways is RNA interference (RNAi) where long dsRNA induces sequence-specific degradation of transcripts carrying sequences complementary to dsRNA. Long dsRNA is also a potent trigger of the interferon pathway, a sequence-independent response that leads to global suppression of translation and global RNA degradation. In addition, dsRNA can be edited by adenosine deamination, which may result in nuclear retention and degradation of dsRNA or in alteration of RNA coding potential. Here, we provide a technical review summarizing different strategies of long dsRNA usage. While the review is largely focused on long dsRNA-induced RNAi in mammalian cells, it also provides helpful information on both the in vitro production and in vivo expression of dsRNAs. We present an overview of currently available vectors for dsRNA expression and provide the latest update on oocyte-specific transgenic RNAi approaches.

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The RNA interference (RNAi) and interferons have been an uneasy marriage. Ever since the discovery of RNAi in mammals, the interferon response has been a feared problem. While RNAi became an efficient and widespread method for gene silencing in mammals, numerous studies recognized several obstacles, including undesirable activation of the interferon response, which need to be overcome to achieve a specific and robust RNAi effect. The aim of this text is to provide theoretical and practical information for scientists who want to control interferon response and other adverse effects in their RNAi experiments.