The molecular architecture of human Dicer

Abstract

Dicer is a multidomain enzyme that generates small RNAs for gene silencing in eukaryotes. Current understanding of Dicer structure is restricted to simple forms of the enzyme, whereas that of the large and complex Dicer in metazoans is unknown. Here we describe a new domain localization strategy developed to determine the structure of human Dicer by EM. A rearrangement of the nuclease core, compared to the archetypal Giardia lamblia Dicer, explains how metazoan Dicers generate products that are 21-23 nucleotides in length. The helicase domains form a clamp-like structure adjacent to the RNase III active site, facilitating recognition of pre-miRNA loops or translocation on long dsRNAs. Drosophila melanogaster Dicer-2 shows similar features, revealing that the three-dimensional architecture is conserved. These results illuminate the structural basis for small RNA production in eukaryotes and provide a versatile new tool for determining structures of large molecular machines.

Fig. 1. The conserved domain structure of metazoan Dicer

a, Schematic of the 2D domain structure of human Dicer with crystal structures homologous to each module. b, The EM map of Dicer (EMD-1646) shown in three orientations.

Fig. 2. Mapping the nuclease core of human Dicer

a–c, 2D class averages of Dicer labeled with streptavidin in the PAZ, platform or RNase IIIb domain (left) and corresponding RCT reconstruction (yellow) superimposed on the unlabeled Dicer map (gray). Streptavidin (red) is shown docked into the additional density. Estimated streptavidin attachment sites are indicated with a sphere. d–f, Estimated streptavidin attachment sites from 8 RCT reconstructions on the refined Dicer map g, Crystal structures of the PAZ-Platform and RNase III modules docked into the EM map of Dicer based on streptavidin labeling results. Blue spheres indicate positions of labeled sites in the crystal structures.

Fig. 3. Comparison of the human Dicer and Giardia Dicer nuclease cores

a, Modeled positions of the PAZ, platform and RNase III domains in human Dicer (left) compared to the Giardia Dicer crystal structure (right). The proposed Ruler domain in human Dicer, estimated by segmentation of the refined map, is shown as a wire mesh. b, The full-length crystal structure of Giardia Dicer docked onto the positions of the PAZ and platform domains identified in the human Dicer EM map (left). The RNase III domains modeled in to the human Dicer EM map, compared to the RNase III domains in the Giardia Dicer crystal structure (right). Double-headed arrow indicates the rearrangement required to align the RNase III domains in the two models.

Fig. 4. The helicase forms a clamp-like structure in the base

a, Schematic of full-length, ΔHEL1 and Δhelicase Dicers. b, Reconstruction of ΔHEL1 Dicer (blue) overlaid on the full length Dicer (transparent). HEL1 domain of the RIG-I helicase (red) is modeled into the major difference density. c, 3D reconstruction of Δhelicase Dicer (cyan) overlaid on the full length Dicer. The RIG-I helicase crystal structure is modeled into the base of the full-length map.

Fig. 5. Conformational states of the Dicer helicase

a, 2D class averages and corresponding RCT reconstructions of two distinct conformations of human Dicer observed when stained the presence of a dsRNA substrate. b, Alignment of the RCT maps showing the conformational differences between the two reconstructions. c, Docking RIG-I into the base of the L reveals that the two observed conformations of the Dicer helicase resemble RIG-I in its apo (PDB code: 4A2P) and dsRNA-bound bound forms (PDB code: 4A36). d, Overlay of the EM density maps of each helicase conformation shows a large scale rearrangement similar to that observed in the RIG-I crystal structures.

Fig. 6. Comparison of Human and Drosophila Dicer structures

a, Class averages of corresponding views of Drosophila (Dm) Dicer2 and Human (Hs) Dicer particles. b, Reconstructions of Dm Dicer2 and Hs Dicer reveal that the two proteins share a common overall shape and many 3D features.

Fig. 7. Architecture and Mechanism of Dicer

a, Segmented map of human Dicer with crystal structures of homologous domains docked. b, Model for pre-miRNA recognition. A pre-miRNA hairpin is modeled into the proposed binding channel of Dicer with the stem loop fit in the RNA-binding cleft of the helicase. c, Schematic for processive dicing: (1) The helicase translocates dsRNA into the nuclease core. (2) The PAZ domain (purple) recognizes the dsRNA end, positioning RNase III (orange) for cleavage. (3) The siRNA product is released while the dsRNA substrate remains bound to the helicase.