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HomeNatureStructural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD

Structural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD

Information reporting

No statistical strategies had been used to predetermine pattern dimension. The experiments weren’t randomized and the investigators weren’t blinded to allocation throughout experiments and consequence evaluation.

Protein expression and purification

The gene encoding full-length Drosophila melanogaster Dcr-2 (UniProt: A1ZAW0) was cloned from the recombinant pFastBac-Dcr-2 plasmid (gifted by the Q. Liu laboratory). Full-length DmLoqs-PD (UniProt: M9MRT5) was PCR amplified from Drosophila cDNA and cloned right into a modified pET28a (with a 6×His-SUMO tag). The constructs of WT Dcr-2, Loqs-PD and different mutations had been generated utilizing a normal PCR-based cloning technique and cloned into the corresponding vectors, and their identities had been confirmed by sequencing evaluation.

Dcr-2 or its mutants was expressed utilizing the Bac-to-Bac baculovirus expression system (Invitrogen) in sf9 cells at 27 °C. One litre of cells (2 × 106 cells per ml, medium from Expression Techniques) was contaminated with 20 ml baculovirus at 27 °C. After development at 27 °C for 48 h, the cells had been collected, resuspended in buffer A (150 mM NaCl, 20 mM Tris-HCl pH  8.0, 10% glycerol, 20 mM imidazole) with 0.5 mM PMSF and protease inhibitors, and lysed by including 0.5% Triton X-100 and shaken gently for 30 min at 4 °C. Dcr-2 was purified to homogeneity utilizing Ni-NTA affinity, Hitrap Q column (Cytiva), 2nd Ni-NTA affinity and size-exclusion chromatography utilizing the Superdex 200 10/300 Improve column (Cytiva) (in that order).

Loqs-PD and its mutants had been expressed in Escherichia coli BL21 (DE3). Loqs-PD was first purified by Ni-NTA affinity chromatography. Utilizing protease ULP1 to take away the 6×His–SUMO tag, and dialysis was utilized to take away imidazole. The pattern was then utilized to a second Ni-NTA chromatography and the flow-through was collected for size-exclusion chromatography utilizing the Superdex 200 16/600 column (Cytiva). Fractions akin to the apo Loqs-PD had been collected and concentrated to about 10 mg ml−1.

Preparation of dsRNAs

The dsRNAs had been in vitro transcribed utilizing T7 RNA polymerase. The pUC19 plasmids containing goal sequences with 3′-HDV ribozyme sequences had been linearized by EcoRI, extracted with phenol–chloroform and precipitated with isopropanol. The in vitro transcription response was carried out at 37 °C for five h within the buffer containing 100 mM HEPES-Ok (pH 7.9), 10 mM MgCl2, 10 mM dithiothreitol (DTT), 6 mM NTP every, 2 mM spermidine, 200 μg ml−1 linearized plasmid and 100 μg ml−1 T7 RNA polymerase. For the 5′-monophosphate RNA, 40 mM GMP was added within the transcription reactions. EDTA at a closing focus of 20 mM was added to the samples containing palindromic transcripts. The samples had been heated to 95 °C for five min and then slowly cooled to room temperature. The annealed transcripts had been purified by 8% denaturing urea PAGE, eluted from gel slices and precipitated with isopropanol. After centrifugation, the RNA precipitant was collected, washed twice with 70% ethanol and air-dried, and the RNA was dissolved in ultrapure water. We subsequent used T4 PNK (NEB, M0201) to take away the two′,3′ cyclic phosphate on the 3′ finish of the RNA. The FAM-labelled dsRNA was produced by Silencer siRNA Labeling kit-FAM in accordance with the producer’s directions.

Pull-down assays

Pull-down assays had been carried out to detect Dcr-2–Loqs-PD interactions utilizing His-tagged proteins purified from bacterial or insect cells. First, 1.25 μM His-tagged Loqs-PD and 0.6 μM untagged Dcr-2 had been combined and incubated on ice for 30 min. The protein combination was then incubated with 15 μl Ni-NTA Agarose (Qiagen, 30210) in a complete quantity of 500 μl within the binding buffer (200 mM NaCl, 20 mM Tris pH 8.0, 5% glycerol, 20 mM imidazole) at 4 °C for 1 h with mild rotation. After centrifugation at 500i for 1 min, the supernatant was eliminated and the beads had been washed 5 occasions utilizing wash buffer (200 mM NaCl, 20 mM Tris pH 8.0, 5% glycerol, 20 mM imidazole, 0.1% NP-40) by centrifugation, adopted by SDS–PAGE evaluation.

In vitro dsRNA cleavage assays

Dicer-2–Loqs-PD cleavage assays had been carried out in cleavage buffer (50 mM HEPES pH 7.2, 100 mM NaCl, 1 mM DTT, 5 mM ATP) with dsRNA. Dcr-2–Loqs-PD and dsRNA had been preincubated at 25 °C for 15 min, then added with ATP and MgCl2 to a closing focus of 5 mM to begin the reactions. The reactions had been stopped with equal quantity of two× formamide loading buffer (95% formamide, 20 mM EDTA, 0.1% SDS, 0.005% xylene cyanol, 0.005% bromophenol blue). Samples had been separated by 12% denaturing PAGE, visualized on Storm FLA-9000 (GE Healthcare) system.

BS3/EDC-mediated cross-linking mass spectrometry

The purified complexes had been incubated with 0.25 mM bis(sulfosuccinimidyl)suberate (BS3; Thermo Fisher Scientific, 21580) within the response buffer containing 50 mM HEPES pH 7.5, 80 mM NaCl and 5% glycerol at 25 °C for two h or 5 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; Thermo Fisher Scientific, PG82073) within the response buffer containing 50 mM HEPES pH 7.2, 80 mM NaCl and 5% glycerol at 25 °C for two h. Cross-linked complexes had been additional purified to take away oligomer and glycerol by size-exclusion chromatography. The proteins (10 μg) had been precipitated and digested for 16 h at 37 °C by trypsin at an enzyme-to-substrate ratio of 1:50 (w/w). The tryptic digested peptides had been desalted and loaded on an in-house packed capillary reverse-phase C18 column (40 cm size, 100 µM ID × 360 µM OD, 1.9 µM particle dimension, 120 Å pore diameter) related to an Straightforward LC 1200 system. The samples had been analysed with a 120 min high-performance liquid chromatography gradient from 6% to 35% buffer B (buffer A: 0.1% formic acid in water; buffer B: 0.1% formic acid in 80% acetonitrile) at 300 nl min−1. The eluted peptides had been ionized and instantly launched right into a Q-Exactive mass spectrometer utilizing a nano-spray supply. Survey full-scan MS spectra (m/z = 300–1,800) had been acquired within the Orbitrap analyzer with decision r = 70,000 at m/z = 400. Cross-linked peptides had been recognized and evaluated utilizing pLink2 software program30.

Cryo-EM pattern preparation and information assortment

We used the identical specimen preparation and information assortment methodology for the entire cryo-EM datasets. An aliquot of 4 μl of purified or response pattern was utilized to a custom-made graphene grid31 (Quantifoil Au 1.2/1.3, 300 mesh), which had been glow-discharged (in a Harrick Plasma system) for 10 s at center stage after 2 min evacuation. The grids had been then blotted by a few 55 mm filter papers (Ted Pella) for 0.5 s at 22 °C and 100% humidity, then flash-frozen in liquid ethane utilizing the FEI Vitrobot Mark IV. Cryo-EM information had been collected on completely different Titan Krios electron microscopes, all of which had been operated at 300 kV, outfitted with a Gatan K3 direct electron detector and a Gatan Quantum power filter. All information had been robotically recorded utilizing AutoEMation32 or EPU (post-dicing state dataset) in counting mode and defocus values ranged from −1.5 μm to −2.0 μm. The opposite parameters of every dataset are supplied in Prolonged Information Desk 1.

Picture processing and 3D reconstruction

For the entire datasets, the picture processing was adopted in related steps. The entire uncooked dose-fractionated picture stacks had been 2× Fourier binned, aligned, dose-weighted and summed utilizing MotionCorr2 (ref. 33). The next steps had been then processed in RELION (v.3.1)34. The distinction switch operate parameters had been estimated utilizing CTFFIND4 (ref. 35). Roughly 2,000 particles had been manually picked and 2D-classified to generate preliminary templates for automated selecting. Numerous particles had been then robotically picked from uncooked micrographs on the premise of our templates. After one spherical of reference-free 2D classification and several other rounds of 3D classification, utilizing the preliminary 3D reference fashions obtained by ab initio calculation in RELION v.3.1, particles from good 3D courses, with higher total construction options, had been chosen for 3D refinement. The ultimate high-resolution homogeneous refinement was carried out in CryoSPARC36. The resolutions had been decided by gold-standard Fourier shell correlation. Native decision distribution was evaluated utilizing blocres command within the Bsoft software program bundle37. The detailed picture processing of every dataset is supplied in Prolonged Information Figs. 2 and 3.

Mannequin constructing and refinement

The very best decision EM density map of dimer standing was used for preliminary mannequin constructing, wherein the standard of density was enough for de novo mannequin constructing in COOT38. The preliminary mannequin was separated into three components (helicase-LoqsPD, DUF283 and different domains) and docked into EM 3D density maps of different states in Chimera39 after which adjusted manually in ISOLDE40 in Chimerax41 and COOT. Lastly, the entire fashions had been refined in opposition to the EM map by PHENIX42 in actual house with secondary construction and geometry restraints. The ultimate fashions had been validated in PHENIX software program bundle. The mannequin statistics are summarized in Prolonged Information Desk 1.

Statistics and reproducibility

For Prolonged Information Fig. 1a–c, experiments had been repeated at the least thrice. For Prolonged Information Figs. 1d, 2a,f, 3a,j, 9a and 10a–c, experiments had been repeated at the least twice.

Reporting abstract

Additional info on analysis design is out there within the Nature Analysis Reporting Abstract linked to this paper.



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