Fig. 3: Molecular basis of the bipartite pairing rule.

a The arrangement shows RIIIDa in dark blue and RIIIDb in green, interacting with pre-miRNA (PDB: 7XW2). The designated cleavage site on the RNA is at DC22. “R-1” denotes the base pair at the −1 position relative to DC22. Two residues, D1709 and S1348, are positioned in potential interaction with the R-1 base pair. It is proposed that these residues might form hydrogen bonds with the 2-hydroxyl group of the ribose in the nucleotides at the 5p- and 3p-ends, respectively. b In the simplified model of the protein-RNA interaction shown in A, potential hydrogen bonds are depicted as dashed lines. c Anticipated comparison of cleavage accuracy and efficiency between DICER-WT and a DICER variant containing a mutation at the residue interacting with the −1 base pair. d In vitro cleavage assays conducted with DICER-S1348A to assess RNA substrates as detailed in Fig. 2d. e Analysis of cleavage accuracy at sites DC21 and DC22 for DICER-S1348A, using data from three repeated assays illustrated in (d). f Assessment of DICER-S1348A cleavage efficiency across various shRNA constructs, based on results from three replicate assays presented in (d). g In vitro cleavage assays conducted with DICER-D1709A to assess RNA substrates as detailed in Fig. 2d. h Analysis of cleavage accuracy at sites DC21 and DC22 for DICER-D1709A, using data from three repeated assays illustrated in (g). i Assessment of DICER-D1709A cleavage efficiency across various shRNA constructs, based on results from three replicate assays presented in (g). j A schematic representation explaining the influence of the D1709 residue on the bipartite pairing rule during cleavage by DICER. 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.