The rate of pri-miRNA processing correlates with cooperative binding of DGCR8. (A) Schematic drawing of all the DGCR8 truncation constructs used in this study. The domain structure of the full-length DGCR8 is shown on the top as a reference. (B) Kinetic assays to measure the initial velocity of pri-mir-30a processing. The pri-mir-30a RNA uniformly labeled with [α-32P]UTP was annealed and incubated with recombinant Drosha (10–20 nM) and heme-bound NC1 (50 nM counting the number of protomers) for indicated time. (C) The accumulation of pre-mir-30a, as a processing product, was quantified, converted to the molar equivalent of pri-mir-30a using the ratios of U residues they contain, and was normalized to the amount of starting pri-mir-30a substrate. The initial rate of the reaction was the slope of the linear regression of the data. (D) The initial rates of pri-mir-30a processing were determined at a series of NC1 concentrations. The results are averages of two independent repeats, with bars representing the range of the rates. (E) The 45-min time points of the processing reactions in C were analyzed using a single denaturing polyacylamide gel. (F) The initial rates of pri-mir-30a processing were measured at a series of NC9 protein concentrations.
