title: Dynamic profiling of double-stranded RNA binding proteins.
abstract: Double-stranded (ds) RNA is a key player in numerous biological activities in cells, including RNA interference, anti-viral immunity and mRNA transport. The class of proteins responsible for recognizing dsRNA is termed double-stranded RNA binding proteins (dsRBP). However, little is known about the molecular mechanisms underlying the interaction between dsRBPs and dsRNA. Here we examined four human dsRBPs, ADAD2, TRBP, Staufen 1 and ADAR1 on six dsRNA substrates that vary in length and secondary structure. We combined single molecule pull-down (SiMPull), single molecule protein-induced fluorescence enhancement (smPIFE) and molecular dynamics (MD) simulations to investigate the dsRNA-dsRBP interactions. Our results demonstrate that despite the highly conserved dsRNA binding domains, the dsRBPs exhibit diverse substrate specificities and dynamic properties when in contact with different RNA substrates. While TRBP and ADAR1 have a preference for binding simple duplex RNA, ADAD2 and Staufen1 display higher affinity to highly structured RNA substrates. Upon interaction with RNA substrates, TRBP and Staufen1 exhibit dynamic sliding whereas two deaminases ADAR1 and ADAD2 mostly remain immobile when bound. MD simulations provide a detailed atomic interaction map that is largely consistent with the affinity differences observed experimentally. Collectively, our study highlights the diverse nature of substrate specificity and mobility exhibited by dsRBPs that may be critical for their cellular function.
authors.affiliation: Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Institute for Genomic Biology, University of Illinois, 1206 W. Gregory St,. Urbana, IL 61801, USA.
authors.forename: Xinlei
authors.initials: X
authors.lastname: Wang
authors.affiliation: Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Lela
authors.initials: L
authors.lastname: Vukovic
authors.affiliation: Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Institute for Genomic Biology, University of Illinois, 1206 W. Gregory St,. Urbana, IL 61801, USA Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Hye Ran
authors.initials: HR
authors.lastname: Koh
authors.affiliation: Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Biophysics and Computational Biology, University of Illinois, 1110 W. Green St., Urbana, IL 61801, USA.
authors.forename: Klaus
authors.initials: K
authors.lastname: Schulten
authors.affiliation: Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Institute for Genomic Biology, University of Illinois, 1206 W. Gregory St,. Urbana, IL 61801, USA Biophysics and Computational Biology, University of Illinois, 1110 W. Green St., Urbana, IL 61801, USA smyong@illinois.edu.
authors.forename: Sua
authors.initials: S
authors.lastname: Myong
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D012313:RNA