Fig. 5

Heme binding allows DGCR8 to recognize the terminal loop structure. a SHAPE analysis of pri-let-7d, showing hyperreactivity associated with MP^Heme binding. Secondary structure diagram (right) depicts the position of the SHAPE “hotspot” (red). Mature sequence is shown in light blue. Control reactions containing no SHAPE reagent (benzoyl cyanide) are shown in lanes 1–3. b SHAPE analysis of pri-let-7d with MP^Heme by capillary electrophoresis. c SHAPE analysis of pri-let-7d with DGCR8. Only the reactions containing the SHAPE reagent are shown, and (unshifted) nucleotide marker lanes are shown to the right. d Diagram summarizing SHAPE results for a series of pri-miRs, aligned according to the site of highest reactivity marked with red font on yellow background. The sequence of the apical fragment released by Dicer is shown for each miR. SHAPE gels for additional miRs are shown in Supplementary Fig. 5c–i. e, f EMSAs comparing the binding affinities of DGCR8^Heme and DGCR8^C352S for e pre-miR-9-1 and f pre-miR-9-1 containing a central break in the terminal loop. Diagrams of RNA sequences are shown to the left. Protein concentrations are (left to right): 33, 50, 67, 83, 100, 117, 133 , 150, 167, 183, 200, 217, 233, and 250 nM. All secondary structure diagrams were designed using mfold