In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site

The ADAR family of RNA-editing enzymes deaminates adenosines within RNA tha t is completely or largely double stranded. In mammals, most of the charact erized substrates encode receptors involved in neurotransmission, and these substrates are thought to be targeted by the mammalian enzymes ADAR1 and A DAR2. Although some ADAR substrates are deaminated very promiscuously, mamm alian glutamate receptor B (gluR-B) pre-mRNA is deaminated at a few specifi c adenosines. Like most double-stranded RNA (dsRNA) binding proteins, ADARs bind to many different sequences, but few studies have directly measured a nd compared binding affinities. We have attempted to determine if ADAR deam ination specificity occurs because the enzymes bind to targeted regions wit h higher affinities. To explore this question we studied binding of rat ADA R2 to a region of rat gluR-B pre-mRNA that contains the R/G editing site, a nd compared a wild-type molecule with one containing mutations that decreas ed R/G site editing. Although binding affinity to the two sequences was alm ost identical, footprinting studies indicate ADAR2 binds to the wild-type R NA at a discrete region surrounding the editing site, whereas binding to th e mutant appeared nonspecific.