Identification of cellular double-stranded RNAs in mammalian embryonic stem cells

Antiviral defence mechanisms are typically activated upon sensing virus-derived nucleic acids. During replication, viruses generate double-stranded RNA (dsRNA) intermediates that the innate immune system can sense, triggering several defence pathways. Conversely, mammalian cells avoid accumulating their own endogenous dsRNA to prevent activating these defence mechanisms. However, we demonstrate that mammalian embryonic stem cells (ESCs) accumulate endogenous dsRNA without activating these responses, as they lack all classical dsRNA-mediated antiviral pathways. To identify these endogenous dsRNAs, we have developed a two-step purification method that includes an antibody-based immunoprecipitation followed by RNase I treatment to remove single-stranded RNA regions that do not contribute to dsRNA formation. RNase I treatment results in an enrichment of overlapping sense/antisense transcripts containing A-to-I editing sites, suggesting successful purification of cellular dsRNA. Our refined protocol reveals that transposable elements (TEs), including evolutionary young elements from the LINE and LTR classes, are the predominant source of dsRNA in ESCs. This approach will be useful for investigating the role of dsRNA in disease settings, such as autoimmunity or cancer, where endogenous dsRNA accumulation has also been observed.