Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules1. Although many editing sites have recently been discovered2–7, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood8–10. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.

SUMMARY Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs. In this study, we investigated the interaction of the RNA editing mechanism with the RNA interference (RNAi) machinery and found that ADAR1 forms a complex with Dicer through direct protein-protein interaction. Most importantly, ADAR1 increases the maximum rate (Vmax) of pre-microRNA (miRNA) cleavage by Dicer and facilitates loading of miRNA onto RNA-induced silencing complexes, identifying a new role of ADAR1 in miRNA processing and RNAi mechanisms. ADAR1 differentiates its functions in RNA editing and RNAi by formation of either ADAR1/ADAR1 homodimer or Dicer/ADAR1 heterodimer complexes, respectively. As expected, expression of miRNAs is globally inhibited in ADAR1−/− mouse embryos, which in turn alters expression of their target genes and might contribute to their embryonic lethal phenotype.

Exosome-like vesicles (ELV) are involved in mediating radiation-induced bystander effect (RIBE). Here, we used ELV from control cell conditioned medium (CCCM) and from 4 Gy of X-ray irradiated cell conditioned medium (ICCM), which has been used to culture normal human fibroblast cells to examine the possibility of ELV mediating RIBE signals. We investigated whether ELV from 4 Gy irradiated mouse serum mediate RIBE signals. Induction of DNA damage was observed in cells that were treated with ICCM ELV and ELV from 4 Gy irradiated mouse serum. In addition, we treated CCCM ELV and ICCM ELV with RNases, DNases, and proteinases to determine which component of ELV is responsible for RIBE. Induction of DNA damage by ICCM ELV was not observed after treatment with DNases. After treatment, DNA damages were not induced in CCCM ELV or ICCM ELV from mitochondria depleted (ρ0) normal human fibroblast cells. Further, we found significant increase in mitochondrial DNA (mtDNA) in ICCM ELV and ELV from 4 Gy irradiated mouse serum. ELV carrying amplified mtDNA (ND1, ND5) induced DNA damage in treated cells. These data suggest that the secretion of mtDNA through exosomes is involved in mediating RIBE signals.

ADAR1 is involved in adenosine-to-inosine RNA editing. The cytoplasmic ADAR1p150 edits 3’UTR double-stranded RNAs and thereby suppresses induction of interferons. Loss of this ADAR1p150 function underlies the embryonic lethality of Adar1 null mice, pathogenesis of the severe autoimmune disease Aicardi-Goutières syndrome, and the resistance developed in cancers to immune checkpoint blockade. In contrast, the biological functions of the nuclear-localized ADAR1p110 remain largely unknown. Here, we report that ADAR1p110 regulates R-loop formation and genome stability at telomeres in cancer cells carrying non-canonical variants of telomeric repeats. ADAR1p110 edits the A-C mismatches within RNA:DNA hybrids formed between canonical and non-canonical variant repeats. Editing of A-C mismatches to I:C matched pairs facilitates resolution of telomeric R-loops by RNase H2. This ADAR1p110-dependent control of telomeric R-loops is required for continued proliferation of telomerase-reactivated cancer cells, revealing the pro-oncogenic nature of ADAR1p110 and identifying ADAR1 as a promising therapeutic target of telomerase positive cancers.