The unusual stability of the IS10 anti‐sense RNA is critical for its function and is determined by the structure of its stem‐domain

IS10 transposition is regulated by an approximately 70 nt anti‐sense RNA, RNA‐OUT. RNA‐OUT folds into a duplex ‘stem‐domain’ topped by a loosely paired ‘loop‐domain’. The loop‐domain is critical for RNA‐RNA pairing per se; pairing initiates by interaction of the RNA‐OUT loop with the 5′ end of the target mRNA. We show here that RNA‐OUT is unusually stable in vivo (half‐life 60 min) and that this stability is conferred by specific features of the RNA‐OUT stem‐domain. One critical feature is stable base‐pairing: mutations that disrupt stem pairing destabilize RNA‐OUT in vivo and abolish anti‐sense control; combinations of mutations that restore pairing also restore both stability and control. We propose that the stem renders RNA‐OUT resistant to 3′ exoribonucleases. Other features of the stem‐domain prevent this essential duplex from being an effective substrate for double‐strand nucleases: two single base mutations disrupt antisense control by making RNA‐OUT susceptible to RNase III. Mutations in the loop region have little effect on RNA‐OUT stability. Implications for IS10 biology and the design of efficient anti‐sense RNAs are discussed.