Distinguishing self from non-self RNA by editing-specific inosine patterns

The cytoplasmic antiviral sensor MDA5 is activated by double-stranded RNAs. Endogenous double-stranded RNAs are modified by the A to I RNA-editing enzymes of the ADAR family to prevent activation of MDA5. In vivo, the cytoplasmic ADAR1p150 version seems critically required to suppress MDA5 activation in vivo, while the editing signature of all ADAR isoforms seems surprisingly overlapping in mice. Further, it is not clear, how A to I modifications in dsRNA prevent MDA5 activation. Here we show that 3' UTRs harboring inverted repeat elements when not edited are able to activate MDA5 in vitro and in cells. Interestingly, in vitro editing by either ADAR isoform leads to editing at overlapping hotspot regions and prevents MDA5 activation in vitro and in cells. Remarkably, only inosines introduced by RNA-editing are capable to suppress MDA5 activation while substitution of guanosines by inosines during in vitro transcription has no impact on MDA5 activation. A comparison of inosines introduced by ADAR1p150 in vitro, in cells, and in vivo, suggests that a small number of A to I conversions may be critically required to suppress MDA5 activation. As those critical editing events are predominantly altering A:U basepairs into I:U wobble basepairs, we suggest that the helical distortion introduced by those wobble pairs may prevent MDA5 polymerization and thus downstream activation of interferon stimulated genes.