The birth of long non-coding RNAs (lncRNAs) is closely associated with the presence and activation of repetitive elements in the genome. The transcription of endogenous retroviruses as well as long and short interspersed elements is not only essential for evolving lncRNAs but is also a significant source of double-stranded RNA (dsRNA). From an lncRNA-centric point of view, the latter is a minor source of bother in the context of the entire cell; however, dsRNA is an essential threat. A viral infection is associated with cytoplasmic dsRNA, and endogenous RNA hybrids only differ from viral dsRNA by the 5′ cap structure. Hence, a multi-layered defense network is in place to protect cells from viral infections but tolerates endogenous dsRNA structures. A first line of defense is established with compartmentalization; whereas endogenous dsRNA is found predominantly confined to the nucleus and the mitochondria, exogenous dsRNA reaches the cytoplasm. Here, various sensor proteins recognize features of dsRNA including the 5′ phosphate group of viral RNAs or hybrids with a particular length but not specific nucleotide sequences. The sensors trigger cellular stress pathways and innate immunity via interferon signaling but also induce apoptosis via caspase activation. Because of its central role in viral recognition and immune activation, dsRNA sensing is implicated in autoimmune diseases and used to treat cancer.

The testis transcriptome is highly complex and includes RNAs that potentially hybridize to form double-stranded RNA (dsRNA). We isolated dsRNA using the monoclonal J2 antibody and deep-sequenced the enriched samples from testes of juvenile Dicer1 knockout mice, age-matched controls, and adult animals. Comparison of our data set with recently published data from mouse liver revealed that the dsRNA transcriptome in testis is markedly different from liver: In testis, dsRNA-forming transcripts derive from mRNAs including promoters and immediate downstream regions, whereas in somatic cells they originate more often from introns and intergenic transcription. The genes that generate dsRNA are significantly expressed in isolated male germ cells with particular enrichment in pachytene spermatocytes. dsRNA formation is lower on the sex (X and Y) chromosomes. The dsRNA transcriptome is significantly less complex in juvenile mice as compared to adult controls and, possibly as a consequence, the knockout of Dicer1 has only a minor effect on the total number of transcript peaks associated with dsRNA. The comparison between dsRNA-associated genes in testis and liver with a reported set of genes that produce endogenous siRNAs reveals a significant overlap in testis but not in liver. Testis dsRNAs also significantly associate with natural antisense genes—again, this feature is not observed in liver. These findings point to a testis-specific mechanism involving natural antisense transcripts and the formation of dsRNAs that feed into the RNA interference pathway, possibly to mitigate the mutagenic impacts of recombination and transposon mobilization.

Recent studies have linked endogenous double-stranded RNA (dsRNA) to the development of inflammatory and autoimmune diseases and cancer. However, the scale of dsRNA formation and its biological consequences is still unclear. Malignant melanoma is the most lethal skin cancer, with an increasing prevalence worldwide. Despite the development of immunotherapy and targeted therapies for melanoma, treatment options are still limited for most patients. dsRNA derived from repetitive DNA elements has the potential to stimulate pattern recognition receptors (PRRs). These PRRs can trigger antiviral signalling cascades and induce an interferon response that renders resistant melanoma sensitive to immunotherapy. Consequently, dsRNA can add a new approach to treating melanoma. Tissue culture and molecular biology techniques were used to induce dsRNA formation in melanoma cell lines (with different mitogen-activated protein kinas-activating mutations) using PRRs agonist poly (I:C), azacitidine (Aza) and hydrogen peroxide (H2O2) as stressors. The impact was measured by quantifying cytosolic dsRNA sensors, also known as PRRs (pPKR, MDA5, RIG1 and ADAR1), using reverse transcriptase quantitative polymerase chain reaction and Western blotting. Immunofluorescence and high-resolution microscopy were then applied to investigate dsRNA and its sensors in skin biopsy samples from patients with melanoma. Moreover, the nature of dsRNA in melanoma was studied by isolating native dsRNA from multiple melanoma cell lines by immune enrichment followed by RNA sequencing. Analysis revealed that inducing endogenous dsRNA significantly activates PRR transcripts and protein levels. However, this activation showed different trends. A375 (BRAFV600E) showed robust MDA5 and RIG1 stimulation to poly (I:C) and a mild ADAR1 and PKR response to Aza and H2O2. Meanwhile, C8161 [BRAF wild type (WT)] was resistant to poly (I:C) with a favourable ADAR1 and RIG1 response to Aza. Immunofluorescence analysis of early stages I and II melanoma skin biopsies showed a significant reduction in ADAR1, which melts dsRNA, with increased dsRNA within BRAF-mutant biopsies. RNAseq analysis showed that A375 (BRAFV600E) produced significantly more dsRNA than C8161 (BRAF WT) and primary dermal fibroblasts. The increase was predominantly due to the enhanced production of dsRNA from nuclear DNA. However, about 11% of the reads mapped to the mitochondrial genome vs. 14% and 19% in C8161 and fibroblasts, respectively. In addition, compared to primary dermal fibroblasts, the dsRNA transcriptome of melanoma cell lines shows unique characteristics and altered dsRNA signalling pathways. To summarize, our data support the hypothesis that endogenous dsRNA activates dsRNA sensors and triggers innate immune signalling. Hence, dsRNA signalling may be explored as a potential therapeutic strategy to treat resistant melanoma with an attack from within.