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.
Highlights
While all cellular RNA molecules are synthesized in singlestranded form, many can form into secondary structures that encompass segments of double stranded RNA
Certain dsRNA structures found in viruses activates protein kinase R (PKR), which in turn triggers the downstream antiviral immune pathways [6,7]; pri-microRNAs are recognized and cleaved by Drosha-DGCR8 to produce pre-microRNA in the nucleus; pre-microRNA is cleaved by Dicer-TRBP to form into mature microRNA [8,9]
We report on the dynamics motion involved in some protein–RNA interaction pairs probed by single molecule Protein Induced Fluorescence Enhancement [26]
Summary
While all cellular RNA molecules are synthesized in singlestranded (ss) form, many can form into secondary structures that encompass segments of double stranded (ds) RNA. DsRNA molecules are common in cells and are recognized as critical regulatory factors in many biological processes [1,2,3]. The family of proteins responsible for processing dsRNA is called double stranded RNA binding proteins (dsRBP). Various dsRNAs serve as cargoes, activators and substrates of dsRBPs in many biological pathways [4,5]. Certain dsRNA structures found in viruses activates protein kinase R (PKR), which in turn triggers the downstream antiviral immune pathways [6,7]; pri-microRNAs are recognized and cleaved by Drosha-DGCR8 to produce pre-microRNA in the nucleus; pre-microRNA is cleaved by Dicer-TRBP to form into mature microRNA [8,9]
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