The importance of internal loops within RNA substrates of ADAR1

Adenosine deaminases that act on RNA (ADARs) are a family of RNA editing en zymes that convert adenosines to inosines within double-stranded RNA (dsRNA ). Although ADARs deaminate perfectly base-paired dsRNA promiscuously, deam ination is limited to a few, selected adenosines within dsRNA containing mi smatches, bulges and internal loops. As a first step in understanding how R NA structural features promote selectivity, we investigated the role of int ernal loops within ADAR substrates. We observed that a dsRNA helix is deami nated 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. Sin ce ADAR1 deaminates short RNAs at fewer adenosines than long RNAs, loops de crease the number of deaminations within an RNA by dividing a long RNA into shorter substrates. For a series of symmetric internal loops related in se quence, larger loops (greater than or equal to six nucleotides) acted as he lix ends, whereas smaller loops (less than or equal to four nucleotides) di d not. Our work provides the first information about how secondary structur e within ADAR substrates dictates selectivity, and suggests a rational appr oach for delineating minimal substrates for RNAs deaminated by ADARs in viv o. (C) 1999 Academic Press.