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Try out PMC Labs and tell us what you think. Learn More. The Dicer-specific insert is shown in red. B Comparison of sequences human, D. The platform, PAZ and connector helix are colored in yellow, blue and pink, respectively. E A view of the complex in the same orientation as in panel D, with the protein in an electrostatic surface representation.
The first insights into the mechanism of catalytic cleavage by Dicer emerged from biochemical Zhang et al. To precisely locate the cleavage site, Dicer utilizes multiple features of RNA substrates. Several additional factors modulate cleavage site choices, including the location of the loop Gu et al.
There are no x-ray crystallographic structures to date on either higher eukaryotic Dicer or its components bound to siRNAs. The structures define two different alignments of the siRNA along the hDicer PAZ cassette and more importantly, identify a phosphate-binding pocket positioned within the platform domain. Our initial attempts to express the isolated PAZ domain of hDicer did not yield soluble protein.
The structure of G. Therefore, we generated hDicer constructs that included these linkers and identified one construct, extending from positions to Figure 1Alower panelthat yielded soluble protein. However, during refinement it became apparent that it was difficult to as amino acids unambiguously for the platform domain, due to Rule 34 paz resolution and relatively poor electron density for this segment of the complex.
To improve diffraction quality, we introduced three separate pairs of surface mutations of large flexible residues to Ala to stabilize crystal contacts by reduction in surface entropy. The structure of this complex was solved by molecular replacement using the partially refined low-resolution structure of hDicer PAZ cassette with the mer self-complementary siRNA duplex presented above. Both the majority of the protein and the entire siRNA can be traced in the complex, with the omit map 1. When these mutants were introduced into Dicer knockout cells, the mutants were as competent as the wild type Dicer protein in miRNA production Figure 3.
Mutant residues are in red.
Western blot showed comparable expression of Dicer right panel. Of specific note are the hydrogen bonds formed between four tyrosine hydroxyls Tyr, Tyr, Tyr and Tyrtogether with the guanidinium of Arg, primarily to the non-bridging phosphate oxygens of the internucleotide phosphate Figure 2E. We have also grown 2. A striking feature of the hDicer PAZ cassette-siRNA mer complex involved the identification of a bound sulfate crystallization condition contained Li 2 SO 4 coordinated to the side chains of Arg, Arg, Arg and His cyan circle, Figure 4A ; x-ray statistics in Table S2which is suggestive of a basic phosphate-binding pocket.
The two pockets are located adjacent to each other on the same face of the hDicer PAZ cassette.
Note the positioning and hydrogen-bonding of a bound phosphate cyan circle in the basic phosphate pocket. The phosphate is anchored in a pocket composed of Arg, Arg, Arg and His A second basic patch is composed of Arg, Arg and His disordered in the structure and is positioned adjacent to the first patch.
The side chain atoms of Ser and Arg are disordered in this structure. We also observe an inorganic phosphate positioned inside the basic pocket in the 2. An additional complex with a mer siRNA was also successfully solved 3. Crystals of this complex diffracted to 2. Uned electron density is shown in a blue mesh representation, which could have originated from an additive used to facilitate crystallization. The G1 base of the terminal G-C base pair stacks on the indole ring of Trp Figure 5Bsimilar to what was observed in the complexes presented above. We solved the 2. We have also solved the 2.
While the bound mer siRNA Figure 6APAZ domain and connector helix can be traced with reasonable confidence, loop regions of the platform domain are poorly defined in this complex Figure 6B. In an earlier section we demonstrated that basic residues Arg, Arg and Arg and polar His lined the walls of the phosphate pocket. Dicer serves in two key steps during small RNA biogenesis. The model can be tested at the molecular level provided structural information becomes available.
To date, crystallographic structural information has been restricted to G. Our structural efforts have focused on human Dicer, and given its large size, been restricted to RNA complexes with constructs containing the PAZ domain and its adjacent structured linker platform and connector helix elements. The relative positioning of the platform, PAZ and connector helix domains are approximately the same in the structure of the hDicer PAZ cassette-siRNA mer complex reported in this study and in the structure of G.
In the current study, we have identified a common binding pocket for sulfate Figure 4A and phosphate Figure 4B in crystals grown from sulfate and phosphate-containing buffers, respectively. The duplex-single strand junctions also adopt distinct conformations in the two complexes.
In the absence of a structure of G. This model, where the bound dsRNA is aligned approximately in parallel to the long axis of the platform-PAZ-connector helix cassette, has been validated from biochemical experiments MacRae et al. Here we have identified such a phosphate-binding pocket within the platform domain Rule 34 paz 4. Indeed, the earlier research from the Kim laboratory established that mutations in the phosphate pocket reduced processing efficiency and altered cleavage sites in vitrowhile miRNA biogenesis was perturbed in vivo when Dicer-null embryonic stem cells are replenished with phosphate pocket mutants Park et al.
The cleavage-competent conformation where the Dicer-specific helix is disrupted and the siRNA is aligned parallel to the long axis of Dicer, may require additional intermolecular contacts with the pair of RNase III domains and perhaps the helicase domain present in intact Dicer, so as to stabilize the complex. Biochemical studies indicated that siRNA undergoes a ificant repositioning in Dicer complexes following the cleavage step, in the process biasing their orientation for guide strand selection based on the thermodynamic stability of the ends of the duplex Noland et al.
This model was further supported by a recent cryo-EM study, which showed that Dicer-RNA complexes can be in two different conformations, one representing cleavage-competent state and another indicative of loading intermediate or inactive state Taylor et al. However, we cannot at this time rule out the possibility that our rescue assay is not sensitive enough to detect the defect in miRNA release, transfer or Ago loading step.
Left panel The proposed features of the postulated model of the cleavage-competent complex are as follows: The Dicer-specific helix is disrupted, with the RNA duplex directed towards the protein. Please refer to the Supplemental Experimental Procedures for detailed methodology on protein expression and purification, RNA preparation, crystallization and Rule 34 paz collection, structure determination, and surface plasmon resonance SPR binding assay.
All clones were overexpressed in E. Proteins were further purified by gel filtration chromatography. The complex crystals were grown using the Rule 34 paz vapor-diffusion method by mixing the protein-RNA complex with equal volume of reservoir solution. RNA sequences, which gave diffraction quality crystals, are listed in Table S5. In this structure, we observed relatively poor electron density for segments of the platform domain compared to the PAZ domain and bound RNA. Because of limited resolution and relatively poor electron density, it was difficult to as amino acids unambiguously for the platform domain.
Careful analysis of the data suggested no of twinning.
Later, using the high resolution structure of hDicer PAZ cassette-siRNA mer complex, amino acid sequence asment was completed and the structure was further refined using Phenix. The structure refinement was completed with cycles of minimization, group B-factor refinement along with TLS parameters, leading to a final structure with R work of Three loop regions and a few side-chain atoms for which no electron density was observed were omitted from the final model. The final map 2Fo—Fc showed reasonably good quality electron density for the final model except for some segments in the platform domain, which has relatively poor density see Table S1 for crystallographic statistics.
The initial model was further improved by manual and automated model building as well as refinement using the PHENIX program package Adams et al.
This structure has clear and well interpretable electron density map for protein and RNA. All crystallographic data and refinement statistics are summarized in Tables S1, S2 and S3. For detailed information, please see Supplementary Experimental Procedures. For transfection, Dicer knockout mouse ES cells were separated from feeder cells and 1, cells were seeded on gelatin-coated 6-well plates one day before transfection. Protein and RNA was extracted at 48 h after transfection. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may Rule 34 paz discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
National Center for Biotechnology InformationU. Mol Cell. Author manuscript; available in PMC Feb Yuan Tian1, 4 Dhirendra K. Narry Kim3 and Dinshaw J. Dhirendra K. Narry Kim.Rule 34 paz
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