Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA editing, additional functions have been proposed for ADAR1. To determine the specific role of RNA editing by ADAR1, we generated mice with an editing-deficient knock-in mutation (Adar1E861A, where E861A denotes Glu861→Ala861). Adar1E861A/E861A embryos died at ~E13.5 (embryonic day 13.5), with activated interferon and double-stranded RNA (dsRNA)–sensing pathways. Genome-wide analysis of the in vivo substrates of ADAR1 identified clustered hyperediting within long dsRNA stem loops within 3′ untranslated regions of endogenous transcripts. Finally, embryonic death and phenotypes of Adar1E861A/E861A were rescued by concurrent deletion of the cytosolic sensor of dsRNA, MDA5. A-to-I editing of endogenous dsRNA is the essential function of ADAR1, preventing the activation of the cytosolic dsRNA response by endogenous transcripts.

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.

Cancer stem cells (CSCs) have been hypothesized to represent the driving force behind tumour progression and metastasis, making them attractive cancer targets. However, conclusive experimental evidence for their functional relevance is still lacking for most malignancies. Here we show that the leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) identifies intestinal CSCs in mouse tumours engineered to recapitulate the clinical progression of human colorectal cancer. We demonstrate that selective Lgr5 cell ablation restricts primary tumour growth, but does not result in tumour regression. Instead, tumours are maintained by proliferative Lgr5 cells that continuously attempt to replenish the Lgr5 CSC pool, leading to rapid re-initiation of tumour growth upon treatment cessation. Notably, CSCs are critical for the formation and maintenance of liver metastasis derived from colorectal cancers. Together, our data highlight distinct CSC dependencies for primary versus metastasic tumour growth, and suggest that targeting CSCs may represent a therapeutic opportunity for managing metastatic disease.

We developed a computational framework to robustly identify RNA editing sites using transcriptome and genome deep-sequencing data from the same individual. As compared with previous methods, our approach identified a large number of RNA editing sites with high specificity in both Alu and non-Alu regions. We also found that the editing of non-Alu sites appears to be dependent on nearby edited Alu sites, possibly through the locally formed double-stranded RNA structure.

We show that RNA editing sites can be called with high confidence using RNA sequencing data from multiple samples across either individuals or species, without the need for matched genomic DNA sequence. We identified many previously unidentified editing sites in both humans and Drosophila; our results nearly double the known number of human protein recoding events. We also found that human genes harboring conserved editing sites within Alu repeats are enriched for neuronal functions.