The double-stranded RNA-binding domains of Xenopus laevis ADAR1 exhibit different RNA-binding behaviors

Abstract

We have cloned cDNAs encoding two versions of Xenopus double-stranded RNA adenosine deaminase (ADAR1). Like ADAR1 proteins from other species Xenopus ADAR1 contains three double-stranded RNA-binding domains (dsRBDs) which are most likely required for substrate binding and recognition of this RNA-editing enzyme. Analysis of mammalian ADAR1 identified the third dsRBD in this enzyme as most important for RNA binding. Here we analyzed the three dsRBDs of Xenopus ADAR1 for their in vitro RNA-binding behavior using two different assays. Northwestern assays identified the second dsRBD in the Xenopus protein as most important for RNA binding while in-solution assays demonstrated the importance of the third dsRBD for RNA binding. The differences between these two assays are discussed and we suggest that both the second and third dsRBD of Xenopus ADAR1 are important for RNA binding in vivo. We show further that all three dsRBDs can contribute to a cooperative binding effect.