Production and Application of Long dsRNA in Mammalian Cells

Abstract

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.

Antiviral RNA Interference in Mammalian Cells

“…First, in many infected somatic cells, viral double-stranded RNA (dsRNA) triggers the potent and non-sequence-specific interferon (IFN) response (3) that may have largely supplanted antiviral RNAi functions (4). Second, several mammalian viral proteins display viral suppressor of RNAi (VSR)–like activities still awaiting validation in authentic virus expression contexts (1).…”

“…Second, virus-derived siRNA levels were at least one order of magnitude higher in undifferentiated than in differentiated mESCs or BHK-21 cells (23). This probably relates to the distinctive efficacy of long dsRNA-triggered RNAi in undifferentiated cells derived from embryonic or adult tissues, which is possibly underpinned by their generally reduced ability to mount non–sequence specific immune responses, including the IFN response, against long dsRNA (4). Alternatively, or coincidently, DCR siRNA-processing activity might decrease during cell differentiation, perhaps via modification of its internal autoinhibitory helicase domain (20).…”

Suppressing PAR ylation by 2′,5′‐oligoadenylate synthetase 1 inhibits DNA damage‐induced cell death

“…OASs are latent enzymes that require activation by dsRNA species that satisfy specific structural criteria (Donovan et al, 2013). Viral dsRNAs are typical OAS activators (Player & Torrence, 1998;Lin et al, 2009;Kristiansen et al, 2011), but dsRNA species that are present naturally in the nuclei of normal cells (Gantier & Williams, 2007;Chalupnikova et al, 2013) may possibly serve as endogenous activators of OASs in that compartment. Indeed, a recent study estimated the numbers of potential host dsRNA OAS activators as > 1,000 for microRNAs, 10-100 for lncRNAs, and 10-100 for coding RNAs per cell (Donovan et al, 2013).…”

Production of functional double‐stranded RNA using a prokaryotic expression system in Escherichia coli

“…PCR, in vitro transcription based on double‐strand annealing, is a typical method to synthesize dsRNA molecules (Chalupnikova, Nejepinska, & Svoboda, ), although false amplification leads to poor quality dsRNA products. Transcription based on a prokaryotic expression system is another way to obtain high‐quality dsRNA.…”

“…The terminal stem-loop of hpRNA is cut by spliceosomes after entering the cells and forms a complete dsRNA to perform PTGS as dsRNA, although the mechanism of terminal stemloop cutting remains unclear (Okamura et al, 2008;Xiang, Fruehauf, & Li, 2006). PCR, in vitro transcription based on double-strand annealing, is a typical method to synthesize dsRNA molecules (Chalupnikova, Nejepinska, & Svoboda, 2013), although false amplification leads to poor quality dsRNA products. Transcription based on a prokaryotic expression system is another way to obtain high-quality dsRNA.…”