Fig. 6: The single cleavage of DICER.

a The relative single cleavage (SC) of DICER was calculated as the ratio of SC to DC. The number of variants for each stem length: n = 1550 (19 nt); n = 2497 (20 nt); n = 2947 (21 nt); n = 6327 (22 nt); n = 3349 (23 nt); n = 1675 (24 nt); n = 953 (25 nt); n = 597 (26 nt); n = 132 (27 nt). The error bars were presented with 95% confidence intervals. b Human pre-miRNAs and their variants. The mutated nt are shown in red. The stem length of pre-miRNAs was the number of base pairs and mismatches from their 5'-end to their apical loop. c, d The in vitro DICER cleavage assays for human pre-miRNAs and their variants. The assays were repeated three times. e, f The two RNase III domains of human DICER (PDB: 5ZAM)¹⁰ were fitted with the RNA duplex. The RIIIDs of DICER from PDB: 5ZAM¹⁰ were superimposed on the RIIIDs of DROSHA from PDB: 6V5B³⁹ or PDB: 6LXD⁴⁰. The RNA duplex was a part of a pri-miRNA in PDB: 6V5B³⁹. The RIIIDa of DICER is in light pink, and the 3pCSH is in violet. The dashed line indicates the anticipated loop of the pre-miRNA. g The fractions of the shRNA variants sharing the same stem length and exhibiting 3p-strand or 5p-strand SC. h Superimposition of the RIIIDb of DROSHA and the RIIIDa or RIIIDb of DICER. DROSHA_RIIIDb is in cyan (PDB: 6V5B)³⁹, DICER_RIIIDa is in violet (PDB: 5ZAM)¹⁰, and DICER_RIIIDb is in blue (PDB: 5ZAM)¹⁰. The root-mean-square deviation (rmsd) and the number of aligned residues are shown. The superimposition and figures were drawn using PyMOL (http://www.pymol.org). i Close-up view of the possible interaction between 3pCSH and RNA. j, l The in vitro DICER cleavage assays. k, m Bar graphs showing ratios of the SC to the double-cut product (F1) band density (SC/DC ratios) from three repeated experiments as shown in (j, l). In (k, m), a two-tailed t-test calculated the p-values. The error bars were presented with 95% confidence intervals. Source data are provided as a Source Data file.