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. 2013 Jun;20(6):662-70.
doi: 10.1038/nsmb.2564. Epub 2013 Apr 28.

Substrate-specific Structural Rearrangements of Human Dicer

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Free PMC article

Substrate-specific Structural Rearrangements of Human Dicer

David W Taylor et al. Nat Struct Mol Biol. .
Free PMC article

Abstract

Dicer has a central role in RNA-interference pathways by cleaving double-stranded RNAs (dsRNAs) to produce small regulatory RNAs. Human Dicer can process long double-stranded and hairpin precursor RNAs to yield short interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Previous studies have shown that pre-miRNAs are cleaved more rapidly than pre-siRNAs in vitro and are the predominant natural Dicer substrates. We have used EM and single-particle analysis of Dicer-RNA complexes to gain insight into the structural basis for human Dicer's substrate preference. Our studies show that Dicer traps pre-siRNAs in a nonproductive conformation, whereas interactions of Dicer with pre-miRNAs and dsRNA-binding proteins induce structural changes in the enzyme that enable productive substrate recognition in the central catalytic channel. These findings implicate RNA structure and cofactors in determining substrate recognition and processing efficiency by human Dicer.

Figures

Figure 1
Figure 1. Domain architecture and cryo-EM structure of human Dicer
(a) Domain organization of human Dicer and color code used for labeling regions of the EM map (dashed lines) and docked atomic structures (solid boxes). (b) Reference-free 2D class averages of negatively stained human Dicer in complex with antibody mAb 77 (left) and mAb 83 (right), along with cartoon representations. The cap and branch of individual Dicer molecules are labeled. Scale bars represent 100 Å. (c) Cryo-EM reconstruction of human Dicer. Regions labeled by antibodies in (b) and the DExH/D domain assigned in our previous work are segmented and colored on the EM density: mAb 83-labeled region (orange), mAb 77-labled region (green), DExH/D domain (light blue). Crystal structures of homologous domains have been docked into the map based on the antibody localization and segmentation and are color coded and labeled as in (a): RNase IIIa (yellow), RNase IIIb (green), and PAZ domain (orange), from Giardia intestinalis Dicer (PDB 2QVW); ATP-binding (red) and helicase (light blue) domains of a homology model of Dicer’s helicase based on human RIG-I (PDB 2YKG). The cap and branch of the enzyme are labeled based on our previous nomenclature.
Figure 2
Figure 2. A pre-siRNA spans human Dicer between the cap and branch, while a pre-miRNA binds the platform of the enzyme
(a,b) Cryo-EM reconstructions of human Dicer–37ab (a) and human Dicer–pre-let7 (b) at ~29 Å and ~31 Å resolution, respectively. Segmented regions of the EM density and atomic structures are colored as in Fig. 1. The pre-siRNA, 37ab, is modeled as a 35-bp A-form RNA duplex within its segmented density colored purple (a). The pre-miRNA, pre-let7, is not modeled and the segmented density is colored red (b).
Figure 3
Figure 3. RNA substrates induce structural rearrangements in human Dicer
(a) Class averages corresponding to specific ranges of cap–branch distances (AC) and branch lengths (BC), mapped onto the graph used for plotting the raw data from negatively stained apo-Dicer and Dicer–RNA complexes. Scale bar indicates 100 Å. The inset shows a schematic of how domain positions and distance measurements were calculated. (b–d) Distribution of branch length (BC) vs. cap–branch distance (AC) for apo-Dicer (b), Dicer–pre-let7 (c), and Dicer–37ab (d) complexes, marked with open light blue, red, and purple circles, respectively. Normalized histograms of the distance distribution and Gaussian approximations are shown along each axis following the same coloring scheme.
Figure 4
Figure 4. A domain reorganization of human Dicer in response to pre-miRNAs correlates with increased dicing efficiency
(a) Distribution of branch length (BC) vs. cap–branch distance (AC) for negatively stained Dicer–pre-miR-430 complexes, marked with open orange circles. Normalized histograms of the distance distribution and Gaussian approximations are shown along each axis. (b) Cleavage activity (cleaved/total RNA %) vs. time of human Dicer on 37ab, pre-miR-430, and pre-let7. Error bars indicate the standard deviation from six independent experiments for 37ab and pre-let7 and three independent experiments for pre-miR-430, respectively.
Figure 5
Figure 5. dsRBPs promote conformational sampling of human Dicer for dsRNA substrate-loading
(a–c) Distribution of branch length (BC) vs. cap–branch distance (AC) for negatively stained Dicer–TRBP (a), Dicer–TRBP–37ab (b), and Dicer–PACT (c) complexes marked with open dark blue, grey, and green circles, respectively. Normalized histograms of the distance distribution and Gaussian approximations are shown along each axis following the same coloring scheme.
Figure 6
Figure 6. 3D reconstructions of major Dicer conformers show that branch position affects accessibility to the platform
(a–c) Reference-free 2D class averages used for RCT (left) and the final 3D conformers of the enzyme captured by multi-model refinement (right) of negatively stained Dicer–PACT particles against the three RCT models, showing the open (a), canonical (b), and closed (c) state of the enzyme in red, blue, and purple, respectively. The models are colored to reflect the similarity observed to conformations of Dicer–RNA complexes based on correlation with reference-free 2D class averages of unique subpopulations of Dicer–pre-let7 (red), apo-Dicer (light blue), and Dicer–37ab (purple) as shown in Fig. 3. Scale bar for 2D class averages corresponds to 100 Å. PACT is illustrated as a string of three yellow spheres with a flexible linker connecting it to the DExH/D domain in (b) based on our previous single particle analysis of Dicer–TRBP. The yellow arrow denotes its predicted range of motion.
Figure 7
Figure 7. Model for small RNA processing by human Dicer
RNA-binding by the helicase domain of human Dicer causes rearrangement of the enzyme in a substrate-specific manner. Binding of a pre-miRNA occurs along the platform and is accompanied by an outward bending of the helicase domain away from the platform, while pre-siRNAs are trapped between the PAZ and helicase domain away from the catalytic sites. dsRBPs may poise the enzyme for recognition of RNA precursors and/or productive loading of the enzyme. Dicer was drawn by tracing the contour of a class average from each state, respectively.

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