In vitro binding of single-stranded RNA by human Dicer.
While Dicer alone has been shown to form stable complexes with double-stranded RNAs and short interfering RNAs, its interactions with single-stranded RNAs (ssRNAs) have not been characterized. Here, we show that recombinant human Dicer alone can bind 21-nt ssRNAs in vitro, independent of their sequence and structure. We also demonstrate that Dicer binds ssRNAs having a 5'-phosphate with greater affinity versus those with a 5'-hydroxyl. In addition, 3'-biotinylated ssRNAs are bound by Dicer with lower affinity than 3'-hydroxyl ssRNAs. The stability of ssRNA-Dicer complexes was found to depend on divalent cations. Together, our results suggest a role for the PAZ domain of Dicer in binding ssRNAs and may indicate roles for Dicer in cellular function beyond those currently known.
Kini
Hemant K
HK
Applied Biomolecular Engineering Laboratory of the Cellular and Biomolecular Laboratory, Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824-1226, USA.
Walton
S Patrick
SP
D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D006801:Humans; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III
D002413:Cations, Divalent; D011994:Recombinant Proteins; D012313:RNA; D004722:Endoribonucleases; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases