Fig. 5: Discovery of the secondary YCR motif.

a Prediction of the secondary YCR motif. The secondary YCR motif, located at positions 22 and 23, presumably guides DICER to cleave shRNAs at DC21 and DC22, respectively. b Comparison of cleavage accuracy at DC21 and DC22 for between shRNA variants containing the YCR motif at position 22 and 23, respectively, with those that do not in high-throughput DICER-WT libraries. shRNA variants containing mismatches at position 22 were excluded from the analysis due to the violation of bipartite pairing rule for DC22. P-values were obtained from two-tailed Mann-Whitney U test. n indicated the sample size. The median value (center line), lower quartile and upper quartile (box edges), and whiskers extended to ±1.5 × IQR (interquartile range) were indicated in each boxplot. c Illustration of shRNA structures and sequences, with green and red arrowheads marking the DC21 and DC22 cleavage sites, respectively. d In vitro DICER cleavage assays for RNAs shown in (c) using DICER-WT. e Evaluation of cleavage accuracy at DC21 and DC22 for DICER-WT based on results from three repeated assays as depicted in (d). f Superimposition of human DICER RIIIDa (purple color) and RIIIDb (green color). g In vitro DICER cleavage assays on RNAs represented in c for different DICER mutants. (h and i) Analysis of cleavage accuracy at DC21 and DC22 for DICER-E1813A and DICER-E1813Q using data from three replicate assays as shown in (g). j A model illustrating the double YCR (dYCR) motif and the involvement of residues E1564 and E1813 in recognizing the major C-G or G-C pairs within the dYCR. In the bar plot representing quantitative data from the gel, data values are shown as dots, and the error bars are presented with 95% confidence intervals.