Extended Data Fig. 6: Massively parallel dicing assay of Dcr-1 on pre-mir-324.
From: DICER cleavage fidelity is governed by 5′-end binding pockets
a, Sequence alignment of DICERs highlighting conserved residues in the 5′- and 3′-end binding pockets. 5′-pocket: R821, D991, H992, R1003; 3′-pocket: Y936, R937, Y956, Y971, Y972, K975, Y976 (shaded red). b, Number of recovered variants. The assay recovered all 256/256 expected variants, with equal contributions from each subgroup (A, U, G, C). c, Reproducibility of three independent dicing assays. Orange dots represent individual variants, and the Pearson correlation coefficient (r) reflects consistency between replicates. d, Cleavage sites (DC20–DC23) identified from sequencing results. e, Cleavage accuracy at DC21 and DC22 for pre-mir-324 groups, based on sequencing data. Left: RNAs grouped by 5′-nt; Right: RNAs grouped by 3′-nt. f, Similar effect of the 5′-nt on cleavage-site selection between DICER and Dcr-1. Variants were grouped by 5′-nt, and DC21 was calculated for each variant. g, Dicer cleavage across species for pre-miRNAs with different 5′-nt. Left: Workflow for cross-species analysis (34 species; MirGeneDB), assigning 5′-nt identity (A, U, G, C) to pre-miRNAs, predicting secondary structures, annotating major cleavage sites (DC21 or DC22), and comparing cleavage patterns. Right: Box-and-whisker plots show the proportion of pre-miRNAs cleaved at DC21 (top) or DC22 (bottom) by 5′-nt. Statistics: two-tailed, two-sample t-tests; **** p < 0.0001; n.s., not significant. h, Per-species distributions of cleavage outcomes. Top, proportion of pre-miRNAs cleaved at DC21 across species; 5′-G (green) versus others (grey). Bottom, proportion cleaved at DC22; 5′-U (red) versus others (grey).
