Fig. 4: YCR recognizing mechanism.

a Enrichment analysis of C-G and G-C pairs within identified YCR motifs. YCR motifs previously identified were subjected to enrichment analysis using the Logomaker package³⁶ to evaluate the prevalence of nucleotides at specific positions. The graphical representation employs stacks, where the height of each stack signifies the information content of the sequence position. b Representation of shRNA structures and sequences, with green and red arrowheads indicating the DC21 and DC22 cleavage sites, respectively. c In vitro DICER cleavage assays performed on RNAs depicted in (b) using DICER-WT. d In C-G and G-C pairs, G contains an -NH2 group indicated with the red arrow that acts as a hydrogen bond donor toward the minor groove, a feature does not present in C, U, and A. e In vitro DICER cleavage assays on RNAs illustrated in (b) for various DICER mutants. f Assessment of cleavage accuracy at DC21 and DC22 for both DICER-WT and its mutants based on data from three replicate assays as shown in (c and e). “SC” denotes the single cleavage on the 5p-strand of shRNAs. g In vitro DICER cleavage assays conducted on RNAs displayed in (b) for DICER variants with different amino acids at position 1564. h, Assessment of cleavage accuracy at DC21 and DC22 for DICER-E1564Q based on data from three replicate assays as shown in (g). “SC” denotes the single cleavage on the 5p-strand of shRNAs. i A schematic representation illustrates the DICER-RNA complex during the dicing state (PDB: 7XW2). E1564 within RIIIDa and the C-G base pair in the pre-miRNA are depicted as sticks. The residue E1564 identifies the C-G pair situated within the minor groove by establishing a hydrogen bond with the -NH2 group on the guanine base. j Representation of pre-mir-30a structures and sequences, with green and red arrowheads indicating the DC21 and DC22 cleavage sites, respectively. (k, l) In vitro DICER cleavage assays performed on RNAs depicted in (j) using DICER-TRBP (k) or DICER-E1564A-TRBP and DICER-E1564Q-TRBP (l). m Analysis of cleavage accuracy at sites DC21 and DC22 for DICER-TRBP from three replicate assays as shown in (k) using the F2 fragments as indicators for the cleavage site because of the overlapping between F1 and F3 derived from DC21 and DC22. n Analysis of cleavage accuracy at sites DC21 and DC22 for DICER-E1564A-TRBP and DICER-E1564Q-TRBP from three replicate assays as shown in (l) using the SC fragments (F1) as indicators for the cleavage site because the mutant DICER disrupts 3p cleavage generating F1 and F23 fragments. 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.