DGCR8 recognizes primary transcripts of microRNAs through highly cooperative binding and formation of higher-order structures

Specific recognition of pri-miRNAs by DGCR8. (A) The secondary structure of the P4-P6ΔC209 RNA, which is used here to represent nonspecific RNAs. (B) Results of filter binding assays as a function of NC1 concentration. The data were fit using a cooperative dimer model, in which two NC1 molecules bind one RNA cooperatively. (C) Hill plot of the RNA binding data shown in B. Only the region in the binding transition is shown. (D) Competitive pri-mir-30a binding assays. Trace amount of ³²P-labeled pri-mir-30a RNA was mixed with unlabeled competitor RNA, either the pri-mir-30a or the P4-P6 RNA, at various concentrations prior to the addition of the NC1 protein. (E) Schematic drawing of a variety of pri-miRNA fragments used in additional competitive pri-mir-30a binding assays. (F) Results of the competitive binding assays using unlabeled RNA shown in E.