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. 2021 Sep 27;49(17):9978-9991.
doi: 10.1093/nar/gkab712.

Insights into the structure and RNA-binding specificity of Caenorhabditis elegans Dicer-related helicase 3 (DRH-3)

Kuohan Li  1   2   3 , Jie Zheng  4 , Melissa Wirawan  1   3 , Nguyen Mai Trinh  1   3 , Olga Fedorova  5   6 , Patrick R Griffin  4 , Anna M Pyle  5   6 , Dahai Luo  1   2   3
Affiliations

Insights into the structure and RNA-binding specificity of Caenorhabditis elegans Dicer-related helicase 3 (DRH-3)

Kuohan Li et al. Nucleic Acids Res. .

Abstract

DRH-3 is critically involved in germline development and RNA interference (RNAi) facilitated chromosome segregation via the 22G-siRNA pathway in Caenorhabditis elegans. DRH-3 has similar domain architecture to RIG-I-like receptors (RLRs) and belongs to the RIG-I-like RNA helicase family. The molecular understanding of DRH-3 and its function in endogenous RNAi pathways remains elusive. In this study, we solved the crystal structures of the DRH-3 N-terminal domain (NTD) and the C-terminal domains (CTDs) in complex with 5'-triphosphorylated RNAs. The NTD of DRH-3 adopts a distinct fold of tandem caspase activation and recruitment domains (CARDs) structurally similar to the CARDs of RIG-I and MDA5, suggesting a signaling function in the endogenous RNAi biogenesis. The CTD preferentially recognizes 5'-triphosphorylated double-stranded RNAs bearing the typical features of secondary siRNA transcripts. The full-length DRH-3 displays unique structural dynamics upon binding to RNA duplexes that differ from RIG-I or MDA5. These features of DRH-3 showcase the evolutionary divergence of the Dicer and RLR family of helicases.

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Figures

Figure 1.
Figure 1.
The overall structure of the DRH-3 N-terminal domain. (A) Schematic representation of the domain architecture of DRH-3. NTD, worm-specific N-terminal domain; HEL, the helicase domain; CTD, the C-terminal RNA-binding domain. The residues numbers corresponding to the predicted domain boundaries were labeled accordingly. (B) Crystal structure of dimeric DRH-3 NTD. Two DRH-3 NTD molecules in the asymmetric unit are shown as cartoons. Chain A and Chain B are indicated in different colors. (C) Cylinder depiction of DRH-3 NTD structure with helices labeled. DRH-3 NTD contains two CARD-like α-helical bundles, highlighted in red (NTD_C1) and pink (NTD_C2) colors, respectively. The topology of a 6-α-helical bundle is that of a Greek Key. Cylinders represent the helices, and the arrow represents the rotation direction of the polypeptide chain. (D) The surface of DRH-3 NTD colored according to the electrostatic surface charges where blue is for positive charges, red for negative charges, and white for the neutral surface. (E) Superposition of DRH-3 NTD1 with RIG-I CARD1. The relative position of the two CARD/CARD-like domain in DRH-3 and RIG-I are compared and illustrated in simplified cartoon models.
Figure 2.
Figure 2.
5′-ppp RNA recognition by DRH-3 CTD. (A) The overall structure of DRH-3 in complex with 5′-ppp 12-mer dsRNA. The crystallographic asymmetric unit contains a 2:1 complex between DRH-3 CTD and the dsRNA. Chain B and the 5′ stand are colored. The 5′-ppp is shown as orange spheres and the zinc molecule is shown as a purple sphere. (B) The schematic diagram of contacts between DRH-3 CTD and 5′-ppp 12-mer dsRNA. (C) The overall structure of DRH-3 in complex with 5′-ppp 8-mer ssRNA. The crystallographic asymmetric unit contains a 1:1 complex between DRH-3 CTD and the ssRNA. The symmetry mate structure is shown in gray. The 5′-ppp is shown as orange spheres and the zinc molecule is shown as a purple sphere. (D) The schematic diagram of contacts between DRH-3 CTD and 5′-ppp 8-mer ssRNA.
Figure 3.
Figure 3.
Comparison of C-terminal domain of DRH3 and RLRs. (A) Superposition of DRH-3 CTD structures bound to the 5′-ppp 12-mer dsRNA and the 5′-ppp 8-mer ssRNA. (B) Superposition of the RNA-bound DRH-3 CTD (yellow) and LGP2 CTD (orange, PDB code: 3EQT). (C) Superposition of the RNA-bound DRH-3 CTD (yellow) and MDA5 CTD (green, PDB code: 4GL2). (D) Superposition of the RNA-bound DRH-3 CTD (yellow) and RIG-I CTD (orange, PDB code: 3EQT). Polypeptide chains are shown as cartoons. Structural superimposition was done by pairwise alignment and represented by Pymol (98). (E) Electrostatic charge at solvent-accessible surfaces of dsRNA-bound DRH-3 CTD. Positively charged surfaces are colored blue and negatively charged surfaces are red. Key residues involved in the RNA binding were labeled. (F–H) Electrostatic charge at solvent-accessible surfaces of LGP2 CTD, MDA5 CTD and RIG-I CTD. Each CTD is shown in the same orientation as in Figure 3A–C. Positively charged surfaces are colored in blue and negatively charged surfaces are colored in red. The key charged residues are indicated. (I) Alignment of amino acid sequences of DRH and RLR CTDs. Sequences were aligned using the program ClustalW and visualized using Geneious software. The conservation of the amino acids is indicated by colors.
Figure 4.
Figure 4.
Conformational dynamics of DRH-3. Deuterium uptake profiles of the DRH-3 NTD (A) and DRH-3FL (B) are presented in a front and back view. The structure of DRH-3 NTD was obtained from this study and the DRH-3FL model was generated by assembling all three domains via fusing peptide bonds. The deuterium exchange data were mapped onto the structural models of DRH-3 NTD and DRH-3FL. The % deuterium exchange in the deuterium exchange of each peptide is colored according to the scale bar. Blue indicates the lowest and orange indicates the highest exchange rate. Black indicates that the region has no amide hydrogen exchange activities. (C) HDX-MS differential map for DRH-3FL with a short hairpin RNA in a front and back view. Differential single amino acid consolidation HDX data were mapped on to full-length RNA-bound DRH-3 structural model. The percentages of deuterium differences are color-coded according to the scale bar.
Figure 5.
Figure 5.
DRH-3 forms stable complexes with short dsRNAs with 5′-ppp. (A) Size exclusion chromatography (SEC) profile of DRH-3FL. Blue and red lines represent the absorbance at 280 nm (A280) and 260 nm (A260), respectively. The elution volume of the monomeric DRH-3FL in Superdex 200 Increase 10/300 GL is 11.4 ml. The A260/A280 ratio is typical for proteins. (B) SEC profile of DRH-3FL mixed with the 30-bp hairpin RNA. DRH-3FL forms a 1:1 complex with a 30-bp hairpin RNA. A higher A260/A280 ratio indicates that DRH-3FL forms a complex with the hairpin RNA. The elution volume of the DRH-3FL RNA complex is 11.1 ml. (C) SEC profile of DRH-3FL with a 30-bp dsRNA. DRH-3FL proteins form a dimer upon binding to the 30-bp dsRNA. The elution volume of the major DRH-3FL RNA complex is 10.2 ml, suggesting that more than one monomer is bound to the RNA.
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