Extended Data Fig. 3: Dicing of 26S shRNAs and characteristics of DICER–26S complexes.
From: DICER cleavage fidelity is governed by 5′-end binding pockets
a, In vitro dicing assay of 26S-UG and 26S-GU shRNAs by DICER. F1 and F3 are 22 nt, while F2 is 20 nt. b, Partial density of the helicase domain observed in the cryo-EM maps of DICER–shRNA complexes. The helicase (Hel1) is outlined in dashed circles, with adjacent domains (PAZ, platform, RIIIDa, RIIIDb, and dsRBD) labelled in distinct colours. c, Detailed view of the cleavage site (DC22) in the DICER–shRNA complexes. Calcium ions (green spheres) and the four catalytic acidic residues (sticks). Black arrowheads indicate hydrolysed phosphates at the cleavage sites. The cleavage sites align with the hydrolysis products observed from the in vitro dicing assay. d, Movement of the helix and β-sheet in the PAZ domain of DICER–shRNA complexes relative to DICER (PDB:7XW3), illustrated by RMSD values. e, Comparison of RNA expansion in pre-let-7a-1GYM (PDB: 7XW2) versus shRNAs (26S-UG and 26S-GU). The RNA duplex in pre-let-7a-1GYM expands near the catalytic centre, whereas the shRNAs remain compact. Alignment of AlphaFold3-predicted RNA (AF3-shRNAs) with experimental structures highlights structural differences. f, RNA conformation change analysis in PDB: 7XW2. The resolved RNA (blue) is compared with the AlphaFold3-predicted RNA (salmon). Calcium ions (red spheres) are positioned at the 3′ and 5′ catalytic centres. Measured distances from the calcium ions to the hydrolysed phosphates are displayed, showing structural differences in RNA alignment.
