Cryo-EM Structures of Human Drosha and DGCR8 in Complex with Primary MicroRNA

Abstract

Metazoan microRNAs require specific maturation steps initiated by Microprocessor, comprising Drosha and DGCR8. Lack of structural information for the assembled complex has hindered an understanding of how Microprocessor recognizes primary microRNA transcripts (pri-miRNAs). Here we present a cryoelectron microscopy structure of human Microprocessor with a pri-miRNA docked in the active site, poised for cleavage. The basal junction is recognized by a four-way intramolecular junction in Drosha, triggered by the Belt and Wedge regions that clamp over the ssRNA. The belt is important for efficiency and accuracy of pri-miRNA processing. Two dsRBDs form a molecular ruler to measure the stem length between the two dsRNA-ssRNA junctions. The specific organization of the dsRBDs near the apical junction is independent of Drosha core domains, as observed in a second structure in the partially docked state. Collectively, we derive a molecular model to explain how Microprocessor recognizes a pri-miRNA and accurately identifies the cleavage site.

Keywords: DGCR8; Drosha; Microprocessor; RNA processing; RNase III; cryo-EM; dsRBD; microRNA; pri-miRNA; structure.

Figure 1.. Cryo-EM structure of a Drosha/DGCR8/pri-miRNA complex

(A) Domain organization of Drosha and DGCR8. CED, central domain of Drosha. RIIID, RNaseIII domain. dsRBD, double-stranded RNA-binding domain. HBR, heme-binding region. CTT, C-terminal tail. The sequences used for cryo-EM structure determination are marked underneath. (B) Cryo-EM maps of Drosha/DGCR8/pri-miR-16–2, segmented and colored by domain as shown in Figure 1A. The HBR regions of both DGCR8 monomers are in gray. (C) Model of the MP/RNA complex shown with three different views. Nucleotides on either side of cleavage sites are colored magenta. C-terminal tails (CTTs) of DGCR8 are labeled. (D) Secondary structure diagram of pri-miR-16–2 used for cryo-EM. Protein/RNA contacts (distance < 4.5 Å) are highlighted according to the protein domain or region, with the same color scheme used in Figures 1A–C. Wedge and Belt are subregions of the CED of Drosha. Nucleotides that were not modeled due to poor resolution or disorder are colored gray. Drosha cut sites are indicated with magenta arrowheads. Pri-miR-16–2 in the model is numbered from 1–105 from 5’ to 3’.

Figure 2.. Multiple rearrangements in Drosha enable recognition of the basal dsRNA-ssRNA junction.

(A-B) Views of Drosha and pri-miRNA with front (A) and side (B) views. Colored regions highlight major conformational changes of Drosha when compared to the isolated Drosha structure, except for RIIIDs (dark gray) which are colored for orientation. Nucleotides on either side of cleavage sites are colored magenta. (C) Cryo-EM density for Helix-1 of the Belt with model. (D-E) Cartoon illustration of MP/RNA showing the RNA-induced 4-way junction (D), and rotated to show the interaction with RIIIDb (E). Same color scheme as Figure 2A. 4-way junction is indicated by a red circle. (F) Close-up view of the interactions of the Belt with the rest of Drosha and RNA. Same color scheme as in Figure 2A. (G) Top-scoring intra-Drosha crosslinks in MP/RNA complex (top) and MP alone (bottom). Red lines represent hits identified in 3 out of 3 replicates. Gray lines represent hits found in 2 of 3 replicates. (H) Residues involved in red crosslinks from MP/RNA in Figure 2G are shown with distances.

Figure 3.. The Helical Belt is important for pri-miRNA processing

(A-B) Cartoon and surface representation of MP/RNA showing distances from cut site to apical and basal branch points with front (A) and back (B) views. CED and RIIIDs are shown in blue, Drosha dsRBD in cyan, Belt in green, DGCR8–1 in yellow-orange, and DGCR8–2 in purple. (C) Pri-miRNA structure compared to a model with an elongated stem. The Belt (green) clashes with the modeled dsRNA (right). Arg914, a residue within the “Bump Helix”, is labeled. (D) In vitro processing assays of pri-miR-16–2 and RNAs containing stem insertions. [MP] is 32 nM. (E) Quantified in vitro pri-miRNA processing assays with a series of Belt mutants. Data are from three replicates, and error bars represent ± S.D. [MP] is 8 nM. *p < 0.001 (unpaired t-test for WT v. each mutant). (F) In vitro processing assays using wild type Microprocessor or the ΔBelt mutant on various pri-miRNAs. [MP] is 65 nM. Solid or dashed lines indicate grouping of separate gel regions.

Figure 4.. The Wedge and Belt form a narrow tunnel for the ssRNA at the basal junction

(A) Vacuum electrostatic potential surface depicting basal side of the tunnel formed by the Belt and Wedge. RNA is shown in orange. (B) Cartoon representation of MP/RNA showing pathway of the basal junction between the Belt and Wedge. (C) Same as Figure 4B, but rotated 30° to show interactions between the Wedge and GHG motif (black). Nucleotides of the GHG motif (G88, U89, and G90) are indicated by white circles. (D) Overall view of Wedge and single-stranded nucleotides in flipped conformation near the basal junction. (E-F) Close-up views of interactions between Drosha and single-stranded nucleotides A99 (E) and U7 (F). (G-H) Quantification of in vitro pri-miRNA processing results on pri-miR-16–2 variants containing shortened 5’ (G) or 3’ (H) flanking segments. [MP] is 8 nM. Data are from three individual replicates, and error bars represent ± S.D. (I) RNA secondary structure diagram showing the essential ssRNA nucleotides in orange and the nonessential ones in gray, with the same number system as in Figure 1D.

Figure 5.. Cryo-EM structure of Drosha/DGCR8 with partially docked pri-miRNA

(A-B) Cryo-EM map (A) and model (B) of the partially docked Drosha/DGCR8/pri-miR-16–2 complex. The HBR regions of both DGCR8 monomers are gray in the map and not modeled. Colors are according to key in panel B. (C) Superimposition of partially docked (gray) and fully docked (colored according to Figure 3A) structures, showing shift in Drosha orientation. (D) Superimposition of inactive (gray) and active (marine, cyan and green) Drosha, showing the conformational rearrangements of the belt (green) and dsRBD (cyan) upon binding RNA. (E) Crosslink map of top-scoring intra-Drosha crosslinks for inactive (top) and active (bottom) MP/RNA conformations. Same color scheme as for Figure 2A. (F) Cartoon representation of apical RNA-binding modules observed in both partially and fully docked structures. Fully docked structure is shown, with same color scheme as Figure 5C. (G) Surface representation of RNA from fully docked structure. Colored nucleotides participate in interactions conserved between both MP structures, with cyan, yellow-orange and purple indicating interactions with Drosha, DGCR8–1, and DGCR8–2, respectively. (H-I) Electrophoretic mobility shift assay (EMSA) results showing the binding of DGCR8 (H) and Drosha^+CTT (I) to pri-miR-16–2. Protein concentrations are (left to right): 0, 0.13, 0.26, 0.52, 10.4, and 2.08 μM. Estimated dissociation constants (K_d) are listed below each gel. Spaces indicate grouping of separate gel regions.

Figure 6.. Model for assembly of Microprocessor/pri-miRNA complex

Proposed model for pri-miRNA recognition by Drosha. Different protein modules recognize specific structural features of pri-miRNAs, with the HBR recognizing the terminal loop, dsRBDs recognizing the stem, and the Belt and Wedge recognizing the basal junction. Flexibility between the modules enables recognition of diverse substrates that meet the structural requirements. The dsRBDs, the Belt and the Wedge, together form a molecular ruler that selects stem-loops with a length of approximately 35 bp. DGCR8 CTTs are removed for clarity.