Structural Insights into the Advances and Mechanistic Understanding of Human Dicer

Abstract

The RNase III endoribonuclease Dicer was discovered to be associated with cleavage of double-stranded RNA in 2001. Since then, many advances in our understanding of Dicer function have revealed that the enzyme plays a major role not only in microRNA biology but also in multiple RNA interference-related pathways. Yet, there is still much to be learned regarding Dicer structure-function in relation to how Dicer and Dicer-like enzymes initiate their cleavage reaction and release the desired RNA product. This Perspective describes the latest advances in Dicer structural studies, expands on what we have learned from this data, and outlines key gaps in knowledge that remain to be addressed. More specifically, we focus on human Dicer and highlight the intermediate processing steps where there is a lack of structural data to understand how the enzyme traverses from pre-cleavage to cleavage-competent states. Understanding these details is necessary to model Dicer's function as well as develop more specific microRNA-targeted therapeutics for the treatment of human diseases.

Figure 1.

Canonical miRNA biogenesis pathway highlighting the processing steps carried out by Drosha and Dicer. The 5P and 3P strands of microRNA are shown in dark blue and light blue, respectively, while the red arrows indicate the sites of cleavage. Purple and pink represent the 50S and 30S ribosome with additional proteins recruited for translation inhibition by RISC shown in orange, royal blue, and green. The 7-methlyguanosine 5′ mRNA cap is represented by an orange circle with the letter “G”, while the 3′ poly(A) tail is shown as a green circle with the letter “A”.

Figure 2.

Domain organization and distribution of the Class I, II, and III RNase III family of enzymes. Ec – E. coli; Aa – A. aeolicus; Sc – S. cerevisiae; Hs – Human; Dm – Drosophila; Gi – Giardia; Ce – C. elegans; At – Arabidopsis.

Figure 3.

Timeline of major findings that have expanded the understanding of the structural organization of Dicer. Significant advances that contributed to new information regarding full-length Dicer and Dicer-like structures are shown in red with their corresponding resolutions.

Figure 4.

Mechanistic insights into Drosophila and human Dicer through cryo-EM structural studies. Molecular models built from either EM density maps or predicted from AlphaFold* are organized into their proposed mechanistic sequence. The RNA substrates for human Dicer, dmDicer-2, and dmDicer-1 are colored blue, orange, and purple, respectively. Conformations yet to be structurally characterized are left as black outlines. Conformational states that are ATP dependent are only shown for dmDicer-2 (PDBs for Apo-hsDicer: 5ZAK; dmDicer-2: 7W0B; dmDicer-1: 8DG1; Early Translocation dmDicer-2: 7W0C; Midtranslocation dmDicer-2: 7W0D; Pre-Cleavage hsDicer: 5ZAL; Cleavage-Competent dmDicer-2: 7W0E; dmDicer-1: 8DG5; Post-cleavage dmDicer-2: 7W0F; dmDicer-1: 8DGA; Strand Release/Transfer dmDicer-2: 7V6C).

Figure 5.

Structural model of human Dicer. (A) Structural overview modeled from a 4.4 Å cryo-EM structure. Domain organization and color code used for labeling the domains from the N- to C-termini are also shown at the bottom. Dashed panels are zoomed-in views of the (B) RNase III processing center and (C) major binding pockets within the Platform-PAZ domains with key amino acids highlighted. A cartoon representation of RNA binding within the 3′ 2 nt overhang and 5′ phosphate binding pockets is depicted in the bottom right panel in (C). (PDB: 5ZAK).

Figure 6.

3D structural representation of disease-associated Dicer mutation and their locations relative to major binding and catalytic sites. Key residues are highlighted including metal binding residues (D1709, E1705, D1810, and E1813 within the RNase IIIB domain catalytic core), S1344 located within the RNase IIIA domain catalytic core, and Platform-PAZ domain mutants (S839, L881, and R944) (PDB: 5ZAK).

Figure 7.

Dicer cleavage counting rules. (A) 5′ and 3′ counting rules determined by RNA binding within the Platform (light blue) and PAZ (dark blue) domains, respectively. (B) Loop counting rule determined by binding within the helicase domain (green). Approximate sites of cleavage are represented by red arrows.

Figure 8.

Structural comparison of cryo-EM densities for the hsDicer pre-cleavage state and DCL3 cleavage-competent state. An overview of domain organization and color code for each protein is shown above. Region/domains missing density are shown in light gray in the domain organization and are represented by dashed lines in the EM structures. For both EM structures, the individual RNA strands are colored gray and dark gray (EMBD: hsDicer-6905; DCL3-31963).

Figure 9.

Overview of the major structural differences between the (A) hsDicer pre-cleavage complex and (B) DCL3 cleavage-competent complex cryo-EM structures. Outlined in black is the Dicer-specific α-helix which has a notable structural change from structured to unstructured within the transition between states. (C) Significant binding interactions occurring in the pre-cleavage structure with a red circle indicating the absence of complete binding by the pre-miRNA substrate in the Platform domain. (D) Zoomed in view of the catalytic core of DCL3 with Ca²⁺ bridging the contact between the RNase III domains and pre-siRNA substrate (PDB: hsDicer, 5ZAL; DCL3, 7VG2).