Fig. 2

Heme dependence varies among miRs. a 5′ end-labeled in vitro processing assays comparing activities of MP^Heme (130 nM) and MP^C352S (130 nM) for a series of pri-miRs. Substrates are arranged from least heme dependent (left) to most heme dependent (right). b Cotransfection scheme for in vivo processing assays shown in c–h. Constructs containing pri-miR-9-1 and pri-miR-125a in tandem, or pri-miR-21 and pri-miR-125a in tandem, were cotransfected with either full-length FLAG-DGCR8^WT or FLAG-DGCR8^C352S into 293T cells. c Taqman quantitative PCR assay results from transfections in b. Mature miR levels were normalized to miR-125a expression levels. Data are represented as mean ± standard deviation from three biological replicates. Also see Supplementary Fig. 2a–d. d, e Representative Northern blots for pre-miRNAs indicated. U6 levels confirm normalized loading in each lane. f, g Quantitation of Northern blots as shown in d and e. Data are shown as mean ± standard deviation, for a total of three biological replicates. h Quantitative PCR of primary transcript levels for transfections in b. Levels were normalized to pri-miR-125a levels. i, j Representative splinted ligation assay results detecting miR-125a and miR-21 i or miR-9-1 j. U6 levels from Northern blots were used for normalization. k, l Statistical analyses of splinted ligation results, where miR levels with mutant DGCR8 were normalized to those with wild-type DGCR8. Data are represented as mean ± standard deviation, for a total of three biological replicates. *p < 0.05, **p < 0.005, n.s. (not significant), p > 0.05, Student’s t-test (two-sided, paired)