The importance of internal loops within RNA substrates of ADAR1

Abstract

Adenosine deaminases that act on RNA (ADARs) are a family of RNA editing enzymes that convert adenosines to inosines within double-stranded RNA (dsRNA). Although ADARs deaminate perfectly base-paired dsRNA promiscuously, deamination is limited to a few, selected adenosines within dsRNA containing mismatches, bulges and internal loops. As a first step in understanding how RNA structural features promote selectivity, we investigated the role of internal loops within ADAR substrates. We observed that a dsRNA helix is deaminated at the same sites whether it exists as a free molecule or is flanked by internal loops. Thus, internal loops delineate helix ends for ADAR1. Since ADAR1 deaminates short RNAs at fewer adenosines than long RNAs, loops decrease the number of deaminations within an RNA by dividing a long RNA into shorter substrates. For a series of symmetric internal loops related in sequence, larger loops (⩾six nucleotides) acted as helix ends, whereas smaller loops (⩽four nucleotides) did not. Our work provides the first information about how secondary structure within ADAR substrates dictates selectivity, and suggests a rational approach for delineating minimal substrates for RNAs deaminated by ADARs in vivo.