ADAR1 regulates dsRNA formation in nuclear and mitochondrial transcripts through editing-dependent and -independent mechanisms

Endogenous (self) double-stranded RNAs (dsRNAs) in human cells can activate innate immune responses. ADAR1, an A-to-I editing enzyme of dsRNAs, suppresses aberrant immune activation by self-dsRNAs. However, how ADAR1 influences the cellular dsRNA landscape remains unclear. We show that human ADAR1 downregulates self-dsRNA abundance through editing-dependent and editing-independent mechanisms. We further conducted quantitative dsRNA-sequencing on wild-type and ADAR1-deficient cells. dsRNAs are enriched in protein-coding mRNAs - especially those with repetitive elements and elongated 3'UTRs - and mitochondrial RNAs. ADAR1 loss markedly increased dsRNA quantity but minimally impacted dsRNA identity. ADAR1-regulated dsRNA transcripts consist of nuclear-encoded mRNAs and, unexpectedly, mitochondria-encoded RNAs rarely edited by ADAR1. Accordingly, dsRNAs accumulate to high levels within the mitochondria of ADAR1-deficient cells. Notably, ADAR1 loss sensitizes cells to inflammation under mitochondrial stress (e.g., mitochondrial herniation, X-ray radiation). Hence, we show that dsRNAs regulated by ADAR1 go beyond A-to-I edited transcripts, and that ADAR1 can control mitochondrial-dsRNAs. Overall design: Double-stranded RNA (dsRNA)-prone transcripts were enriched by immunoprecipitation with the J2 monoclonal antibody (SCICONS), which recognizes dsRNA structures. The enriched RNAs were then converted into a sequencing library, resulting in a dsRNA-seq library. For input control, total RNA was extracted from the cell lysate and converted into a total RNA-seq library. Experiments were performed using WT, ADAR1-E912A KI, and ADAR1 KO Hap1 cells.