Mechanistic insights into editing-site specificity of ADARs

Abstract

Adenosine deaminases that act on RNA (ADARs) deaminate adenosines in dsRNA to produce inosines. ADARs are essential in mammals and are particularly important in the nervous system. Altered levels of adenosine-to-inosine (A-to-I) editing are observed in several diseases. The extent to which an adenosine is edited depends on sequence context. Human ADAR2 (hADAR2) has 5′and 3′ neighbor preferences, but which amino acids mediate thesepreferences, and by what mechanism, is unknown. We performeda screen in yeast to identify mutations in the hADAR2 catalyticdomain that allow editing of an adenosine within a disfavoredtriplet. Binding affinity, catalytic rate, base flipping, and preferenceswere monitored to understand the effects of the mutations onADAR reactivity. Our data provide information on the amino acidsthat affect preferences and point to a conserved loop as being ofkey importance. Unexpectedly, our data suggest that hADAR2'spreferences derive from differential base flipping rather than fromdirect recognition of neighboring bases. Our studies set the stagefor understanding the basis of altered editing levels in disease andfor developing therapeutic reagents.