Double-Strand RNA Binding Protein Profiling

Abstract

The double-strand RNA-binding proteins (DRBP), which are featured by the existence of evolutionarily conserved dsRNA-binding domain (DRBD), have been reported to function in a variety of significant cellular activities, involving RNA processing, RNA cleavage, RNA interference and anti-viral immunity pathways.However, it still remains unclear about the molecular mechanism and dynamic properties underlying the interaction between DRBP and dsRNA. Recently single-molecule experiments have shown an ATP-independent diffusion/sliding activity of purified TRBP as well as its two homologs, PKR activator and R3D1-L, on double strand RNA molecules (Koh et al., 2013), indicating a potential generalizability of diffusion/sliding activity in dsRNA-binding proteins family. To test this hypothesis and to further characterize DRBPs in terms of substrate specificity, binding affinity, features of motion upon binding and multiple DRBD collaboration or redundancy, we examined in vivo expressed proteins with one or more DRBDs at single-molecule level.In this research, DRBP genes have been cloned from Human Open Reading Frame Library and overexpressed in human A549 cells. Then we immobilized the protein products on quartz slide surface by Single Molecule Pull Down (Jain et al. 2011) and investigated their dynamic property upon biding various dsRNA substrates using Protein Induced Fluorescence Enhancement (PIFE) (Hwang et al, 2011). PIFE data from one of our candidates, TRBP, showed the intensity fluctuation of Cy3, the fluorescent label of dsRNA substrates, indicating that TRBP is sliding back and forth along dsRNA strand upon binding to it (Fig. 1). This result is highly consistent with the experimental observation in vitro (Koh et al., 2013).1. Koh H. R et al. (2013) Proc. Natl. Acad. Sci USA 110:151-15.2. Jain A et al. (2012) Nat Protoc 7:445-452.3. Hwang H et al. (2011) Proc Natl Acad Sci USA 108: 7414-7418.View Large Image | View Hi-Res Image | Download PowerPoint Slide