m6A regulates ADAR1-mediated RNA editing during macrophage activation

Macrophages are a highly plastic innate immune cell subset that depends on environmental cues to activate, execute and resolve inflammatory responses. This plasticity of function is mirrored by the diversity of RNA modifications that dynamically decorate macrophage transcripts. Here, using the mouse macrophage line RAW 264.7 (RAW), we addressed the combinatorial effect of two major mRNA modifications: adenosine to inosine (A-to-I) deamination by ADAR1 and adenosine N6-methylation (m6A) by METTL3. Using both short-read and single molecule sequencing on RAW macrophages with genetic deletions of ADAR1 or METTL3, we identified transcripts that were modified by both enzymes, with specific functional outcomes on macrophage activation. While m6A levels remained relatively stable even in the absence of ADAR1, loss of METTL3 led to a global reduction in A-to-I editing levels. This interrelation was most apparent when m6A sites were distant from sites of deamination, suggesting a possible function of m6A in ADAR1-mediated editing. Using a dual reporter cell line where guided ADAR1 recruitment can be measured via eGFP reactivation, we observed that m6A modification of ADAR-engager guide RNAs substantially improved targeted RNA editing. Overall, we report the first example of an interdependence between modifications, which can also be therapeutically exploited.