Differential conformational dynamics in two type-A RNA-binding domains drive the double-stranded RNA recognition and binding

Abstract

TAR RNA binding protein (TRBP) has emerged as a key player in the RNA interference (RNAi) pathway. While facilitating Dicer-mediated pre-miRNA cleavage and recruitment of the RISC complex, TRBP binds to different pre-miRNAs & siRNAs, each varying in sequence, structure, or both. Our hypothesis posits that TRBP displays dynamic adaptability to accommodate this heterogeneity in target RNA structures. Thus, it is crucial to ascertain the role of intrinsic and RNA-induced protein dynamics in RNA recognition and binding. Prior investigations from the lab have elucidated the role of intrinsic and RNA- induced conformational exchange in the double-stranded RNA-binding domain 1 (dsRBD1) of TRBP in shape-dependent RNA recognition. Building further on this, the current study delves into the intrinsic and RNA-induced conformational dynamics of the dsRBD2 of TRBP, juxtaposed with dsRBD1. Remarkably, dsRBD2 exhibits a higher binding affinity to a short (12bp) dsRNA when compared with dsRBD1, which can be attributed to the presence of residues critical to RNA binding and structural plasticity in dsRBD2. Utilizing state-of- the-art NMR spin relaxation, relaxation dispersion experiments, and MD simulations, we report that dsRBD2 depicts constrained conformational plasticity when compared to dsRBD1, as evidenced by the fast (ps-ns) and slower (μs-ms) conformational dynamics measurements. Although, in the presence of RNA, dsRBD2 undergoes induced conformational exchange within the designated RNA-binding regions and the other residues, the amplitude of the motions remains modest when compared to those observed in dsRBD1. The culmination of our findings leads us to propose a dynamics-driven model of the two tandem domains of TRBP, substantiating their contributions to the versatility of dsRNA recognition and binding.