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In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6
拟南芥RDR6的体外RNA依赖性RNA聚合酶活性测定   

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Nature Plants
Mar 2017

Abstract

RNA-dependent RNA polymerases (RdRPs) in eukaryotes convert single-stranded RNAs into double-stranded RNAs, thereby amplifying small interfering RNAs that play crucial roles in the regulation of development, maintenance of genome integrity and antiviral immunity. Here, we describe a method of in vitro RdRP assay using recombinant Arabidopsis RDR6 prepared by an insect expression system. By using this classical biochemical assay, we revealed that RDR6 has a strong template preference for RNAs lacking a poly(A) tail. This simple method will be applicable to other RdRPs in Arabidopsis and different organisms.

Keywords: RNA-dependent RNA polymerase (RNA依赖性RNA聚合酶), Double-stranded RNA (双链RNA), RNA silencing (RNA沉默), Small RNA (小RNA), siRNA (siRNA), RNA-DEPENDENT RNA POLYMERASE6 (RNA依赖性RNA聚合酶6), Arabidopsis thaliana (拟南芥)

Background

RNA-dependent RNA polymerase (RdRP) genes have been found in all eukaryotic kingdoms–plants, fungi, protista and animals (Zong et al., 2009). They convert single-stranded RNAs (ssRNAs) into double-stranded RNAs (dsRNAs), thereby amplifying small interfering RNAs (siRNAs) that play crucial roles in various biological processes including regulation of development (Peragine et al., 2004; Li et al., 2005), maintenance of genome integrity (Volpe et al., 2002; Xie et al., 2004) and antiviral immunity (Mourrain et al., 2000; Yu et al., 2003; Garcia-Ruiz et al., 2010; Wang et al., 2010). In addition to this RdRP activity, RdRPs possess another enzymatic activity called terminal nucleotide transferase (TNTase) activity (Curaba and Chen, 2008; Aalto et al., 2010), which adds one or more nucleotides to the 3’ end of ssRNAs or dsRNAs in a template-independent manner.

Here, we describe a method for in vitro RdRP assay using recombinant Arabidopsis thaliana RDR6 prepared by an insect expression system. To accurately discriminate RdRP products from TNTase products, we designed two strategies: 1) elimination of single-stranded TNTase products by treating the reaction mixture with ssRNA-specific RNase I, and 2) electrophoresis of the reaction mixture on a native acrylamide gel, in which double-stranded RdRP products can be distinguished from single-stranded TNTase products based on their different mobility. By using this classical biochemical assay, we revealed that RDR6 has a strong template preference for the RNAs lacking a poly(A) tail (Baeg et al., 2017). This simple method should also be applied to other RdRPs in Arabidopsis and different organisms.

Materials and Reagents

  1. Preparation of recombinant RDR6
    1. 100 mm dish (Corning, catalog number: 430167 )
    2. 50 ml conical centrifuge tube (Nippon Genetics, catalog number: FG200 )
    3. Cell counter plate (Neubauer Improved) (FUKAEKASEI and WATSON, WATSON BIOLAB, catalog number: 177-112C )
    4. 1.5 ml tube (BM Equipment, BMBio, catalog number: NT-175 )
    5. Drosophila melanogaster Schneider 2 (S2) cells (Schneider, 1972)
    6. RDR6 expression plasmid for S2 cells (pAFW-SUMO-AtRDR6 [Baeg et al., 2017])
    7. Anti-FLAG antibody, M2 (Sigma-Aldrich, catalog number: F1804 )
    8. SUMOstar protease (LifeSensors, catalog number: 4110 )
    9. Schneider’s Drosophila medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21720001 )
    10. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 12483020 )
    11. Antibiotic-Antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240062 )
    12. X-tremeGENE HP transfection reagent (Sigma-Aldrich, Roche Diagnostics, catalog number: 06366546001 )
    13. Dynabeads proteins G (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10009D )
    14. Triton X-100 (Wako Chemical Pure Industries, catalog number: 169-21105 )
    15. Dithiothreitol (DTT) (NACALAI TESQUE, catalog number: 14128-04 )
    16. Glycerol (NACALAI TESQUE, catalog number: 17018-83 )
    17. Liquid nitrogen
    18. PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23227 )
    19. Sodium chloride (NaCl) (Wako Chemical Pure Industries, catalog number: 190-13921 )
    20. Potassium chloride (KCl) (NACALAI TESQUE, catalog number: 28514-75 )
    21. Sodium phosphate dibasic dodecahydrate (Na2HPO4·12H2O) (Sigma-Aldrich, catalog number: 28-3720 )
    22. Potassium dihydrogenphosphate (KH2PO4) (NACALAI TESQUE, catalog number: 28721-55 )
    23. HEPES (DOJINDO, catalog number: 340-01376 )
    24. Magnesium acetate tetrahydrate, Mg(OAc)2 (Wako Chemical Pure Industries, catalog number: 130-00095 )
    25. EDTA-free protease inhibitor cocktail (Sigma-Aldrich, catalog number: 11836170001 )
    26. Potassium acetate (KOAc) (NACALAI TESQUE, catalog number: 28405-05 )
    27. Phosphate-buffered saline (PBS) (see Recipes)
    28. Hypotonic lysis buffer (see Recipes)
    29. 1x lysis buffer (see Recipes)

  2. Preparation of template RNAs
    1. Fluorescent TLC plate
    2. 2 ml tube (BM Equipment, BMBio, catalog number: BM-20 )
    3. Sterilized pipette tips
    4. Plastic wrap (AsahiKASEI)
    5. T7-ScribeTM Standard RNA IVT Kit (CELLSCRIPT, catalog number: C-AS3107 )
    6. Distilled water
    7. Phenol:chloroform:isoamyl alcohol 25:24:1 mixed, pH 5.2 (NACALAI TESQUE, catalog number: 26058-96 )
    8. Chloroform (Wako Chemical Pure Industries, catalog number: 035-02616 )
    9. Isoamyl alcohol (Wako Chemical Pure Industries, catalog number: 135-12015 )
    10. Ammonium acetate (NACALAI TESQUE, catalog number: 02433-35 )
    11. 70% ethanol
    12. 2-Propanol (NACALAI TESQUE, catalog number: 29113-53 )
    13. Ethylenediaminetetraacetic acid (EDTA) (NACALAI TESQUE, catalog number: 15105-35 )
    14. Deionized formamide (NACALAI TESQUE, catalog number: 16229-95 )
    15. Xylene cyanol (Wako Chemical Pure Industries, catalog number: 240-00463 )
    16. Tris (Wako Chemical Pure Industries, catalog number: 207-06275 )
    17. Sodium chloride (NaCl) (Wako Chemical Pure Industries, catalog number: 190-13921 )
    18. Sodium dodecyl sulfate (SDS) (NACALAI TESQUE, catalog number: 02873-75 )
    19. Bromophenol blue (Wako Chemical Pure Industries, catalog number: 029-02912 )
    20. Urea (Wako Chemical Pure Industries, catalog number: 217-00171 )
    21. SequaGel-UreaGel Concentrate (National Diagnostics, catalog number: EC-830 )
    22. 2x formamide dye (see Recipes)
    23. PK buffer (see Recipes)

  3. RNA-dependent RNA polymerase activity assay
    1. RNasin® Plus RNase Inhibitor (Promega, catalog number: N2615 )
    2. α-32P-UTP (PerkinElmer, catalog number: NEG507H )
    3. 100% ethanol (NACALAI TESQUE, catalog number: 14713-53 )
    4. Ribonucleotide Solution set (New England Biolabs, catalog number: N0450L )
    5. Glycogen (NACALAI TESQUE, catalog number: 17110-11 )
    6. Proteinase K (20 mg/ml) (NACALAI TESQUE, catalog number: 29442-85 )
    7. Boric acid (H3BO3) (NACALAI TESQUE, catalog number: 05215-05 )
    8. Ethylenediaminetetraacetic acid (EDTA) (NACALAI TESQUE, catalog number: 15105-35 )
    9. HEPES (DOJINDO, catalog number: 340-01376 )
    10. Ammonium acetate (NH4OAc) (NACALAI TESQUE, catalog number: 02433-35 )
    11. Magnesium chloride hexahydrate (MgCl2·6H2O) (Wako Chemical Pure Industries, catalog number: 135-00165 )
    12. RNase I (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2294 )
    13. PEG 4000 (Sigma-Aldrich, catalog number: 95904-250G-F )
    14. Glycerol (NACALAI TESQUE, catalog number: 17018-83 )
    15. Tartrazine (NACALAI TESQUE, catalog number: 32706-22 )
    16. 40(w/v)%-Acrylamide/Bis Mixed Solution(19:1) (NACALAI TESQUE, catalog number: 06140-45 )
    17. 1 mM NTP mixture (see Recipes)
    18. PK buffer (EDTA-) (see Recipes)
    19. PK mixture (see Recipes)
    20. PK mixture (EDTA-) (see Recipes)
    21. 5x TBE (see Recipes)
    22. 10x RdRP buffer (see Recipes)
    23. 2x RNase I buffer (see Recipes)
    24. 2x loading buffer (see Recipes)

Equipment

  1. Pipettes (Gilson)
  2. Cell incubator (SANYO, catalog number: MIR-153 )
  3. Centrifuge (TOMY SEKIO, catalog number: MX-301 )
  4. Vortex mixer (Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0286)
  5. Rotator (TAITEC, model: RT-30mini, catalog number: 0057154-000 )
  6. Magnetic stand (Thermo Fisher Scientific, catalog number: 120.20 )
  7. Block incubator (TAITEC)
  8. UV lamp (UVP, catalog number: 95-0004-11 )
  9. Gel dryer (Bio-Rad Laboratories, catalog number: 165-1745 )
  10. Imaging plate cassette (GE Healthcare, catalog number: 63-0035-45 )
  11. Imaging plate (GE Healthcare, catalog number: 28-9564-74 )
  12. Typhoon FLA 7000 IP (GE Healthcare, model: Typhoon FLA 7000 IP )

Procedure

  1. Preparation of recombinant RDR6
    The recombinant RDR6 protein with N-terminal 3xFLAG-tags followed by a SUMOstar protease cleavage site is overexpressed by using Drosophila S2 cell expression system. This system produces higher levels of soluble recombinant RDR6 compared to plant cell-free translation systems or Escherichia coli expression systems. After lysis of the cells, the recombinant protein is purified by anti-FLAG antibody conjugated magnetic beads, and eluted with SUMOstar protease. Although we use the tag-less recombinant RDR6 protein, the N- or C-terminally tagged RDR6 can also be used for RdRP activity assay (Devert et al., 2015; Baeg et al., 2017).
    1. When S2 cells reach between 1.0 x 107 and 1.5 x 107 cells/ml in a 100-mm dish, collect them into a 50-ml tube(s). Centrifuge at 1,500 x g for 3 min, remove supernatant, and resuspend the cell pellet gently with Schneider’s Drosophila medium containing 10% FBS. Count the cell numbers using a cell counter plate. Seed 1.5 x 107 cells (1.5 x 106 cells/ml) in a 100-mm dish in 10 ml of Schneider’s Drosophila medium containing 10% FBS (see Notes 1 and 2).
    2. Dilute 10 μl of 1 μg/μl pAFW-SUMO-AtRDR6 plasmid with 500 μl of Schneider’s Drosophila medium in a 1.5 ml tube. Add 20 μl of X-tremeGENE HP transfection reagent to the diluent, mix by tapping, and incubate it at room temperature (~25 °C) for 10 min.
    3. Add the transfection mixture into S2 cells in a dropwise manner, gently agitate the dish in a circular motion to evenly disperse the transfection mixture, and statically incubate at 27 °C for 72 h.
    4. Collect the cells into a 50-ml conical centrifuge tube(s). Centrifuge in a swinging bucket rotor at 1,500 x g for 3 min at 25 °C. Remove the supernatant with a pipette.
    5. Resuspend the cell pellet gently in 300 μl of PBS (see Recipes). Transfer the cell suspension into a 1.5 ml tube. Centrifuge at 1,500 x g for 3 min at 4 °C. Remove the supernatant with a pipette completely.
    6. Weigh the cells and add an equal amount of pre-chilled hypotonic lysis buffer (see Recipes). Resuspend the cell pellet gently by tapping the bottom of the tube.
    7. Incubate the cell suspension on ice for 15 min, then vortex the cell suspension for 30 sec to disrupt the cell membrane.
    8. Centrifuge at 17,000 x g for 20 min at 4 °C.
    9. Transfer the supernatant into the 1.5 ml tube containing Dynabeads protein G coated with anti-FLAG antibody (see Note 3). Mix by tapping, and incubate on a rotator at 4 °C for 1 h.
    10. Transfer the tube to a magnetic stand. Wash the beads twice with an equal volume of 1x lysis buffer (see Recipes) containing 800 mM NaCl and 1% Triton X-100, and once with an equal volume of 1x lysis buffer.
    11. Resuspend the beads with 100 μl of 1x lysis buffer containing 1 mM DTT, 20% glycerol and 0.05 U per μl SUMOstar protease. Incubate the slurry on a rotator at 4 °C for 20 min.
    12. Recover the supernatant in a pre-chilled tube, aliquot it into several tubes on ice, freeze with liquid nitrogen, and store at -80 °C. Measure the concentration of purified RDR6 by SDS-PAGE along with the defined dilutions of bovine serum albumin standard (PierceTM BCA Protein Assay Kit) after Coomassie Brilliant Blue (CBB) staining (Figure 1).


      Figure 1. Recombinant RDR6. CBB staining of purified recombinant protein. The arrow indicates purified recombinant RDR6.

  2. Preparation of template RNAs
    Because in vitro transcribed RNAs often contain unwanted shorter or longer byproducts, polyacrylamide gel electrophoresis (PAGE) purification of RNAs is essential for accurate determination of the template specificity of RdRPs. When synthetic oligo RNAs are used as the template for RdRPs, the use of HPLC purification is recommended.
    1. Transcribe reporter RNAs from DNA templates at 37 °C for 2 h using T7-Scribe Standard RNA IVT Kit following manufacturer’s instructions.
    2. Add 1 μl of DNase I, mix thoroughly and incubate at 37 °C for 20 min.
    3. Adjust the volume of the reaction mixture to 100 μl with distilled water on ice.
    4. Add an equal volume of phenol:chloroform:isoamyl alcohol into the reaction mixture, and mix by vortexing at room temperature (~25 °C). Centrifuge at 17,000 x g for 2 min at 4 °C.
    5. Recover the aqueous phase into a new tube with an equal volume of chloroform:isoamyl alcohol, and mix by vortexing at room temperature (~25 °C). Centrifuge at 17,000 x g for 1 min at 4 °C.
    6. Recover the aqueous phase into a new chilled tube, add an equal volume of pre-chilled 5 M ammonium acetate, mix the solution by tapping the tube, and incubate on ice for 15 min.
    7. Centrifuge at 21,500 x g for 20 min at 4 °C.
    8. Remove the supernatant and add 700 μl of 70% ethanol.
    9. Centrifuge at 17,000 x g at 4 °C for 3 min and remove the supernatant completely.
    10. Place the open tube on ice to air dry.
    11. Resuspend the pellet in 40 μl of 1x formamide dye (see Recipes). Incubate the sample at 95 °C for 5 min, mix by vortexing and load onto a urea gel containing 0.5x TBE and appropriate concentration of polyacrylamide (see Notes 4 and 5).
    12. Perform electrophoresis (see Note 6).
    13. Transfer the gel onto a plastic wrap, cover the gel with another wrap, and place it on a fluorescent TLC plate. Briefly expose the gel to UV light and excise a gel slice containing the RNA of interest (see Note 7). Transfer the gel slice into a 2 ml tube (Figures 2A-2C).


      Figure 2. Workflow for RNA purification. A. The gel is transferred onto a plastic wrap after denaturing PAGE. B. Gel excision under UV light; C. A gel slice containing the RNA of interest is transferred into a tube with sterilized pipette tips.

    14. Add 700 μl of PK buffer (see Recipes) and incubate the tube at room temperature (~25 °C) for 24 h on a rotator.
    15. Transfer the supernatant into a new 1.5 ml tube and add 700 μl of 2-propanol and mix by vortexing.
    16. Centrifuge at 17,000 x g at 4 °C for 20 min and remove the supernatant carefully.
    17. Add 700 μl of 70% ethanol.
    18. Centrifuge at 17,000 x g at 4 °C for 3 min and remove the supernatant.
    19. Repeat Steps B17 and B18.
    20. Air dry the pellet for 10 min and dissolve it in distilled water.
    21. Quantify the concentration of the RNA with a spectrophotometer.

  3. RNA-dependent RNA polymerase (RdRP) activity assay
    The flow chart of this assay is shown in Figure 3. After the reaction, the samples are electrophoresed on a urea gel or native gel. In urea PAGE, dsRNAs are denatured into two ssRNAs and migrate based on their molecular weights. Although RNase I treatment is essential for discrimination between RdRP products and TNTase products, this method allows the accurate estimation of the size of newly synthesized complementary strand. In contrast, because dsRNAs retain their double-helical structure in native PAGE, double-stranded RdRP products can be distinguished from single-stranded TNTase products based on their different mobilities without RNase I treatment. A drawback of native PAGE is that the mobility does not reflect the actual size of RNAs. The simultaneous use of both PAGE methods provides more accurate information for the enzymatic properties of RdRPs.


    Figure 3. Flow chart of the procedure for in vitro RdRP assay

    1. Combine the following reagents on ice in the order given: 20 μl of distilled water, 4 μl of 10x RdRP buffer (see Recipes), 4 μl of 5x diluted RNasin, 4 μl of 1 mM NTP mixture, 2 μl of 1 μM template RNA, 2 μl of α-32P-UTP (~3,000 Ci/mmol) and 4 μl of 100-200 nM RDR6.
    2. Incubate the tube at 25 °C for 2 h.
    3. Split the mixture into 4 tubes (tube A, B, A’ and B’) containing 10 μl each. Place them on ice.
    4. Add 111 μl of PK mixture (see Recipes) and 111 μl of PK mixture (Mg2+) (see Recipes) into tube A and tube A’, respectively (see Note 8). Vortex, and incubate at 25 °C until Step C6 is completed.
    5. Add 10 μl of RNase I mixture (see Recipes) into tube B and tube B’ (see Note 9) and incubate at 37 °C for 20 min.
    6. Add 111 μl of PK mixture and 111 μl of PK mixture (Mg2+) into tube B and tube B’, respectively, and vortex.
    7. Incubate all tubes at 65 °C for 20 min (see Note 10).
    8. Add 350 μl of 100% ethanol to each tube, vortex and centrifuge at 17,000 x g for 10 min at 4 °C. Remove the supernatant.
    9. Add 700 μl of 70% ethanol to each tube and centrifuge at 17,000 x g for 3 min at 4 °C. Remove the supernatant completely.
    10. Air dry the pellet for 10 min.
    11. For urea PAGE, dissolve the pellet (tube A and B) in 10 μl of 2x formamide dye and incubate at 95 °C for 5 min. For native PAGE, dissolve the pellet (tube A’ and B’) in 10 μl of 1x loading buffer (see Recipes) and keep the samples on ice.
    12. Prepare a urea gel containing 0.5x TBE and appropriate concentration of polyacrylamide or a native gel containing 0.5x TBE, 2 mM MgCl2 and appropriate concentration of polyacrylamide (see Note 11).
    13. Perform electrophoresis (see Notes 12, 13 and 14).
    14. Dry the gel using gel dryer under vacuum at 80 °C for 2 h.
    15. Expose the dried gel onto an imaging plate for ~12 h. Scan the plate using a laser scanner.

Data analysis

Here, we explain typical results of in vitro RdRP assay with the 100-nt RNA without the poly(A) tail. When total RNAs after the RdRP reaction are electrophoresed in a urea gel, multiple smeared bands are generally observed (Figure 4A RNase I-). At this time, these bands cannot be assigned to either TNTase or RdRP products. After ssRNA-specific RNase I treatment, some bands disappear and only a sharp band remains (Figure 4A RNase I+). This RNase I-resistant band is the RdRP product. In contrast, all the other bands erased by the RNase I treatment are single-stranded TNTase products (Figure 4A). When total RNAs after the RdRP reaction are electrophoresed in a native gel, double-stranded RdRP products migrate more slowly than single-stranded TNTase products (Figure 4B RNase I-), which can be validated by digestion of ssRNAs by RNase I treatment (Figure 4B RNase I+). Note that although the optimal concentration of RNase I is determined, RNase I treatment decreases dsRNA signals to some extent (Figure 4B). The signal of dsRNA products in the absence of RNase I treatment in native PAGE reflects the actual RdRP activity of RDR6.


Figure 4. Representative results of in vitro RdRP assay. A. A typical banding pattern of RdRP- and TNTase-products from the 100-nt template RNA without the poly(A) tail on an 8% acrylamide-urea gel. B. A typical banding pattern of RdRP- and TNTase-products from the 100-nt template RNA without the poly(A) tail on a 4% acrylamide-native gel.

Notes

  1. S2 cells are statically cultured in a 100-mm dish with 10 ml of Schneider’s Drosophila medium containing 10% FBS and 1x antibiotic-antimycotic in an incubator at 27 °C. For maintenance of cells, passage S2 cells when the cell density reaches between 1.0 x 107 and 1.5 x 107 per ml, and split at a 1:10 dilution into a new 100-mm dish.
  2. To obtain high a concentration of recombinant RDR6, we usually prepare 500 ml of S2 cells (1.5 x 106 cells per ml). We aliquot it into 50 dishes (10 ml/dish) for transfection of pAFW-SUMO-AtRDR6. After expression of RDR6, S2 cells are collected into twelve 50-ml tubes (~40 ml each). After centrifugation, the cell pellet in each tube is resuspended with 300 μl of PBS, and transferred into a 1.5 μl tube. At Step A10, the washed magnetic beads in the 12 tubes are collected in a new tube. Then, 100 μl of 1x lysis buffer containing 1 mM DTT, 20% glycerol and 0.05 U per μl SUMOstar protease are added for elution of RDR6.
  3. To prepare the anti-FLAG antibody conjugated Dynabeads for immunopurification from e.g., 100 μl of S2 cell extract, 2 μl of anti-FLAG antibody and 100 μl slurry of Dynabeads are mixed in a tube by tapping, and incubated on a rotator at 4 °C for 1 h. Then, the tube is placed on a magnetic stand, and the supernatant is removed. The beads are washed once with 200 μl of 1x lysis buffer, and resuspended in 200 μl of 1x lysis buffer. The supernatant is removed just before incubation with S2 cell extract.
  4. We use SequaGel-UreaGel Concentrate to prepare urea gel.
  5. We usually use 5%, 8% and 15% acrylamide-urea gels for migration of 400-700 nt, 80-200 nt and < 50 nt RNAs, respectively.
  6. We resolve the RNAs at a constant voltage of 500 V in 0.5x TBE as running buffer.
  7. If the template RNAs are undetectable by UV shadowing, stain the gel with a nucleic acid staining dye for gel excision.
  8. For native PAGE samples (tube A’ and B’), we use ‘PK buffer (Mg2+)’ which contains magnesium ions but not EDTA to stabilize double-stranded RNAs (see Recipes).
  9. Although RNase I treatment allows discrimination of double-stranded RdRP products from single-stranded TNTase products, the use of a too high concentration of RNase I leads to degradation of dsRNAs. Therefore, the optimal concentration of RNase I should be carefully determined prior to the RdRP activity assay by using 32P body-labeled ss and dsRNA markers as substrates for RNase I.
  10. To avoid unwinding of dsRNAs, if the lengths of template RNAs are < 25 nt, it is better to incubate the RNA samples for native PAGE at 25 °C for 60 min or at 37 °C for 30 min in deproteinization step.
  11. We use 40(w/v)%-Acrylamide/Bis Mixed Solution(19:1) to prepare native gel.
  12. The running time should be optimized. The RdRP- and TNTase-products from the 100-nt template RNAs are well separated on an 8% acrylamide-urea gel (100 mm (H) x 150 mm (W) x 1 mm (D)) at a constant voltage of 500 V for 40 min or 4% acrylamide-native gel (100 mm (H) x 150 mm (W) x 1 mm (D)) at a constant voltage of 500 V for 40 min.
  13. To avoid spontaneous unwinding of double-stranded RNAs, keep the running buffer cold using plastic bars filled with coolant and perform electrophoresis in a cold room (Figure 5).
  14. To determine the relative size of the products, 5’ radiolabeled template RNAs can be used as the size markers.


    Figure 5. Representative image of native PAGE. The white arrow indicates a plastic bar filled with coolant.

Recipes

  1. Phosphate-buffered saline (PBS), pH 7.4
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4
  2. Hypotonic lysis buffer
    10 mM HEPES-KOH, pH 7.4
    10 mM KCl
    1.5 mM Mg(OAc)2
    5 mM DTT
    1x EDTA-free protease inhibitor cocktail
  3. 1x lysis buffer
    30 mM HEPES-KOH, pH 7.4
    100 mM KOAc
    2 mM Mg(OAc)2
  4. 2x formamide dye
    10 mM EDTA, pH 8.0
    98% (w/v) deionized formamide
    0.025% (w/v) xylene cyanol
    0.025% (w/v) bromophenol blue
  5. PK buffer
    100 mM Tris-HCl, pH 7.5
    200 mM NaCl
    2 mM EDTA, pH 8.0
    1% (w/v) SDS
  6. 1 mM NTP mixture
    1 μl of 100 mM ATP in Ribonucleotide Solution set
    1 μl of 100 mM UTP in Ribonucleotide Solution set
    1 μl of 100 mM GTP in Ribonucleotide Solution set
    1 μl of 100 mM CTP in Ribonucleotide Solution set
    96 μl of distilled water
  7. PK buffer (Mg2+)
    100 mM Tris-HCl, pH 7.5
    200 mM NaCl
    1 mM MgCl2
    1% (w/v) SDS
  8. PK mixture
    2 μl of glycogen
    20 μl of Proteinase K
    200 μl of PK buffer
  9. PK mixture (Mg2+)
    2 μl of glycogen
    20 μl of Proteinase K
    200 μl of PK buffer (Mg2+)
  10. 5x TBE
    0.446 M Tris
    0.445 M H3BO3
    0.01 M EDTA, pH 8.0
  11. 10x RdRP buffer
    500 mM HEPES-KOH, pH 7.6
    200 mM NH4OAc
    80 mM MgCl2
    1 mM EDTA, pH 8.0
    20% (w/v) PEG 4000
  12. 2x RNase I buffer
    100 mM Tris-HCl pH 8.0
    600 mM NaCl
    30 mM MgCl2
  13. RNase mixture
    10 μl of 2x RNase I buffer
    1 μl of RNase I
    9 μl of distilled water
  14. 2x loading buffer
    4 mM MgCl2
    0.5x TBE
    50% (w/v) glycerol
    0.04% (w/v) tartrazine
    0.02% (w/v) bromophenol blue
    0.02% (w/v) xylene cyanol

Acknowledgments

This protocol has been adapted and modified from Baeg et al., 2017 and Makeyev and Bamford, 2002. We are grateful to members of Tomari laboratory for comments on the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research on Innovative Areas (‘Nascent-chain Biology’) 26116003 (to H.I.), (‘Non-coding RNA neo-taxonomy’) 26113007 (to Y.T.), Grant-in-Aid for Young Scientists (A) 16H06159 (to H.I.), Grant-in-Aid for Challenging Exploratory Research 15K14444 (to H.I.) and Grant-in-Aid for JSPS Fellows 16J07290 (to K.B.). We declare no conflicting or competing interests.

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  8. Mourrain, P., Beclin, C., Elmayan, T., Feuerbach, F., Godon, C., Morel, J. B., Jouette, D., Lacombe, A. M., Nikic, S., Picault, N., Remoue, K., Sanial, M., Vo, T. A. and Vaucheret, H. (2000). Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101(5): 533-542.
  9. Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L. and Poethig, R. S. (2004). SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev 18(19): 2368-2379.
  10. Schneider, I. (1972). Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol 27(2): 353-365
  11. Volpe, T. A., Kidner, C., Hall, I. M., Teng, G., Grewal, S. I. and Martienssen, R. A. (2002). Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297(5588): 1833-1837.
  12. Wang, X. B., Wu, Q., Ito, T., Cillo, F., Li, W. X., Chen, X., Yu, J. L. and Ding, S. W. (2010). RNAi-mediated viral immunity requires amplification of virus-derived siRNAs in Arabidopsis thaliana. Proc Natl Acad Sci U S A 107(1): 484-489.
  13. Xie, Z., Johansen, L. K., Gustafson, A. M., Kasschau, K. D., Lellis, A. D., Zilberman, D., Jacobsen, S. E. and Carrington, J. C. (2004). Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2(5): E104.
  14. Yu, D., Fan, B., MacFarlane, S. A. and Chen, Z. (2003). Analysis of the involvement of an inducible Arabidopsis RNA-dependent RNA polymerase in antiviral defense. Mol Plant Microbe Interact 16(3): 206-216.
  15. Zong, J., Yao, X., Yin, J., Zhang, D. and Ma, H. (2009). Evolution of the RNA-dependent RNA polymerase (RdRP) genes: duplications and possible losses before and after the divergence of major eukaryotic groups. Gene 447(1): 29-39.

简介

真核生物中的RNA依赖性RNA聚合酶(RdRP)将单链RNA转化为双链RNA,从而扩增在调节发育,维持基因组完整性和抗病毒免疫方面起关键作用的小干扰RNA。 在此,我们描述了使用通过昆虫表达系统制备的重组拟南芥RDR6的体外RdRP测定的方法。 通过使用这种经典的生物化学分析,我们发现RDR6有一个强大的模板偏好RNAs缺乏poly(A)尾巴。 这个简单的方法将适用于拟南芥属和其他生物体中的其他RdRPs。

【背景】已经在所有真核生物王国 - 植物,真菌,原生动物和动物中发现RNA依赖性RNA聚合酶(RdRP)基因(Zong等人,2009)。它们将单链RNA(ssRNA)转化为双链RNA(dsRNA),从而扩增在各种生物过程中发挥关键作用的小干扰RNA(siRNA),包括调节发育(Peragine等人, ,2004; Li等人,2005),维持基因组完整性(Volpe等人,2002; Xie等人,2004年, )和抗病毒免疫性(Mourrain等人,2000; Yu等人,2003; Garcia-Ruiz等人,2010; Wang ,2010)。除了这种RdRP活性之外,RdRP还具有称为末端核苷酸转移酶(TNTase)活性的另一种酶活性(Curaba和Chen,2008; Aalto等,2010),其将一个或多个核苷酸添加到ssRNA或dsRNA的3'末端以模板独立的方式。

在这里,我们描述了使用通过昆虫表达系统制备的重组拟南芥RDR6进行体外RdRP测定的方法。为了准确区分RdRP产物与TNT酶产物,我们设计了两种策略:1)通过用ssRNA特异性RNA酶I处理反应混合物消除单链TNT酶产物,和2)在天然丙烯酰胺凝胶上电泳反应混合物,其中双链RdRP产物可以根据其不同的迁移率与单链TNTase产物区分开来。通过使用这种经典的生物化学测定,我们发现RDR6对缺乏poly(A)尾巴的RNA具有强烈的模板偏好(Baeg等人,2017)。这个简单的方法也应该适用于拟南芥属和其他生物体中的其他RdRP。

关键字:RNA依赖性RNA聚合酶, 双链RNA, RNA沉默, 小RNA, siRNA, RNA依赖性RNA聚合酶6, 拟南芥

材料和试剂

  1. 制备重组RDR6
    1. 100毫米培养皿(康宁,目录号:430167)
    2. 50ml锥形离心管(Nippon Genetics,目录编号:FG200)
    3. 细胞计数板(Neubauer Improved)(FUKAEKASEI和WATSON,WATSON BIOLAB,目录号:177-112C)
    4. 1.5毫升管(BM设备,BMBio,目录号:NT-175)
    5. Schneider 2(S2)细胞(Schneider,1972)
    6. 用于S2细胞的RDR6表达质粒(pAFW-SUMO-AtRDR6 [Baeg em et al。 ,2017))
    7. 抗FLAG抗体M2(Sigma-Aldrich,目录号:F1804)
    8. SUMOstar蛋白酶(LifeSensors,目录号:4110)
    9. 施奈德氏果蝇培养基(Thermo Fisher Scientific,Gibco TM,目录号:21720001)
    10. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:12483020)
    11. 抗生素 - 抗真菌药(100x)(Thermo Fisher Scientific,Gibco TM,目录号:15240062)
    12. X-tremeGENE HP转染试剂(Sigma-Aldrich,Roche Diagnostics,目录号:06366546001)
    13. Dynabeads蛋白G(Thermo Fisher Scientific,Invitrogen TM,目录号:10009D)
    14. Triton X-100(Wako Chemical Pure Industries,目录编号:169-21105)
    15. 二硫苏糖醇(DTT)(NACALAI TESQUE,目录号:14128-04)
    16. 甘油(NACALAI TESQUE,目录号:17018-83)
    17. 液氮
    18. Pierce TM BCA蛋白质分析试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:23227)
    19. 氯化钠(NaCl)(Wako Chemical Pure Industries,目录号:190-13921)
    20. 氯化钾(KCl)(NACALAI TESQUE,目录号:28514-75)
    21. 磷酸氢二钠十二水合物(Na 2 HPO 4•12H 2 O)(Sigma-Aldrich,目录号:28-3720) >
    22. 磷酸二氢钾(KH 2 PO 4)(NACALAI TESQUE,目录号:28721-55)
    23. HEPES(DOJINDO,目录号:340-01376)
    24. 醋酸镁四水合物,Mg(OAc)2(Wako Chemical Pure Industries,目录号:130-00095)
    25. 无EDTA的蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:11836170001)
    26. 醋酸钾(KOAc)(NACALAI TESQUE,目录号:28405-05)
    27. 磷酸盐缓冲盐水(PBS)(见食谱)
    28. 低渗裂解缓冲液(见食谱)
    29. 1x裂解缓冲液(见食谱)

  2. 模板RNA的制备
    1. 荧光TLC板
    2. 2毫升管(BM Equipment,BMBio,目录号:BM-20)
    3. 消毒的移液枪头
    4. 保鲜膜(AsahiKASEI)
    5. T7-Scribe TM标准RNA IVT试剂盒(CELLSCRIPT,目录号:C-AS3107)
    6. 蒸馏水
    7. 苯酚:氯仿:异戊醇25:24:1混合,pH5.2(Nacalai TESQUE,目录号:26058-96)
    8. 氯仿(Wako Chemical Pure Industries,目录号:035-02616)
    9. 异戊醇(Wako Chemical Pure Industries,目录号:135-12015)
    10. 醋酸铵(NACALAI TESQUE,目录号:02433-35)
    11. 70%乙醇
    12. 2-丙醇(NACALAI TESQUE,目录号:29113-53)
    13. 乙二胺四乙酸(EDTA)(NACALAI TESQUE,目录号:15105-35)
    14. 去离子甲酰胺(NACALAI TESQUE,目录号:16229-95)
    15. 二甲苯蓝(Wako Chemical Pure Industries,目录号:240-00463)
    16. Tris(Wako Chemical Pure Industries,目录号:207-06275)
    17. 氯化钠(NaCl)(Wako Chemical Pure Industries,目录号:190-13921)
    18. 十二烷基硫酸钠(SDS)(NACALAI TESQUE,目录号:02873-75)
    19. 溴酚蓝(Wako Chemical Pure Industries,目录号:029-02912)
    20. 尿素(Wako Chemical Pure Industries,目录号:217-00171)
    21. SequaGel-UreaGel Concentrate(National Diagnostics,目录号:EC-830)
    22. 2x甲酰胺染料(见食谱)
    23. PK缓冲(见食谱)

  3. RNA依赖性RNA聚合酶活性测定
    1. RNasin Plus RNA酶抑制剂(Promega,目录号:N2615)
    2. α-32 P-UTP(PerkinElmer,目录号:NEG507H)
    3. 100%乙醇(NACALAI TESQUE,目录号:14713-53)
    4. 核糖核苷酸溶液组(New England Biolabs,目录号:N0450L)
    5. 糖原(NACALAI TESQUE,目录号:17110-11)
    6. 蛋白酶K(20mg / ml)(NACALAI TESQUE,目录号:29442-85)
    7. 硼酸(H 3 BO 3)(NACALAI TESQUE,目录号:05215-05)
    8. 乙二胺四乙酸(EDTA)(NACALAI TESQUE,目录号:15105-35)
    9. HEPES(DOJINDO,目录号:340-01376)
    10. 乙酸铵(NH 4 OAc)(NACALAI TESQUE,目录号:02433-35)
    11. 氯化镁六水合物(MgCl 2•6H 2 O)(Wako Chemical Pure Industries,目录号:135-00165)
    12. RNA酶I(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2294)。
    13. PEG 4000(Sigma-Aldrich,目录号:95904-250G-F)
    14. 甘油(NACALAI TESQUE,目录号:17018-83)
    15. 酒石黄(NACALAI TESQUE,目录号:32706-22)
    16. 40(w / v)% - 丙烯酰胺/双混合溶液(19:1)(NACALAI TESQUE,目录号:06140-45)
    17. 1毫米NTP混合物(见食谱)
    18. PK缓冲液(EDTA-)(见食谱)
    19. PK混合物(见食谱)
    20. PK混合物(EDTA-)(见食谱)
    21. 5倍TBE(见食谱)
    22. 10倍RdRP缓冲区(见食谱)
    23. 2个RNase I缓冲液(见食谱)
    24. 2x加载缓冲区(请参阅食谱)

设备

  1. 移液器(吉尔森)
  2. 细胞培养箱(SANYO,目录号:MIR-153)
  3. 离心机(TOMY SEKIO,目录号:MX-301)
  4. 涡旋混合器(Scientific Industries,型号:Vortex-Genie 2,产品目录号:SI-0286)
  5. 旋转器(TAITEC,型号:RT-30mini,目录号:0057154-000)
  6. 磁力架(Thermo Fisher Scientific,目录号:120.20)
  7. 块孵化器(TAITEC)
  8. 紫外灯(UVP,目录号:95-0004-11)
  9. 凝胶干燥器(Bio-Rad Laboratories,目录号:165-1745)
  10. 成像板盒(GE Healthcare,目录号:63-0035-45)
  11. 成像板(GE Healthcare,目录号:28-9564-74)
  12. 台风FLA 7000 IP(GE Healthcare,型号:台风FLA 7000 IP)

程序

  1. 制备重组RDR6
    使用果蝇S2细胞表达系统过表达具有N-末端3xFLAG-标签,然后是SUMOstar蛋白酶切割位点的重组RDR6蛋白。与无植物细胞翻译系统或大肠杆菌表达系统相比,该系统产生更高水平的可溶性重组RDR6。细胞裂解后,用抗FLAG抗体偶联磁珠纯化重组蛋白,用SUMOstar蛋白酶洗脱。虽然我们使用无标签重组RDR6蛋白,但是N-或C-末端标记的RDR6也可以用于RdRP活性测定(Devert等人,2015; Baeg等人, ,2017)。
    1. 当S2细胞在100-mm培养皿中达到1.0×10 7〜1.5×10 7细胞/ ml时,将其收集到50ml管中。 1,500×g离心3分钟,去除上清液,用含10%FBS的Schneider's果蝇培养基轻轻悬浮细胞沉淀。用细胞计数板计数细胞数量。在10毫升Schneider's果蝇介质中的100-mm培养皿中播种1.5×10 7个细胞(1.5×10 6细胞/ ml) 10%FBS(见注1和2)。
    2. 在1.5ml试管中,用500μlSchneider's 果蝇培养基稀释10μl1μg/μlemA pAFW-SUMO-AtRDR6质粒。在稀释液中加入20μlX-tremeGENE HP转染试剂,轻敲混匀,在室温(〜25℃)下孵育10分钟。
    3. 将转染混合物以逐滴方式加入到S2细胞中,以圆周运动轻轻搅动培养皿以均匀分散转染混合物,并在27℃静置培养72小时。
    4. 将细胞收集到50毫升圆锥形离心管中。在25°C下,在摇动的转子中以1,500×g 离心3分钟。用吸管取出上清液。
    5. 在300μl的PBS中轻轻地重悬细胞沉淀(参见食谱)。将细胞悬液转移到1.5ml管中。在4℃下在1,500gxg离心3分钟。
      用移液器完全除去上清液
    6. 称量细胞并加入等量的预冷的低渗裂解缓冲液(参见食谱)。
      轻轻地重新悬浮细胞颗粒
    7. 在冰上孵育细胞悬液15分钟,然后涡旋细胞悬液30秒,以破坏细胞膜。
    8. 在4℃下以17,000×g克离心20分钟。
    9. 将上清转移至含有抗FLAG抗体包被的Dynabeads蛋白G的1.5ml管中(参见注释3)。轻敲混合,并在4°C的旋转器孵育1小时。
    10. 将试管转移到磁力架上。用等体积的含有800mM NaCl和1%Triton X-100的1倍溶解缓冲液(见配方)洗两次珠,用等体积的1倍裂解缓冲液洗一次。
    11. 用100μl含1mM DTT,20%甘油和0.05U /μlSUMOstar蛋白酶的1x裂解缓冲液重悬珠。
      在4°C的旋转器上孵育浆液20分钟
    12. 在预先冷却的管中回收上清液,在冰上将其分成几个管,用液氮冷冻,并保存在-80°C。在考马斯亮蓝(CBB)染色后(图1),通过SDS-PAGE连同确定的牛血清白蛋白标准稀释液(Pierce TM BCA蛋白质分析试剂盒)一起测量纯化的RDR6的浓度。 />

      图1.重组RDR6。纯化的重组蛋白的CBB染色。箭头指示纯化的重组RDR6。

  2. 模板RNA的制备
    由于体外转录RNA通常含有不想要的更短或更长的副产物,所以RNA的聚丙烯酰胺凝胶电泳(PAGE)纯化对准确测定RdRP的模板特异性是必不可少的。当使用合成的寡聚RNA作为RdRP的模板时,建议使用HPLC纯化。
    1. 使用T7-Scribe标准RNA IVT试剂盒,按照制造商的说明书,在37°C下从DNA模板转录报告RNAs 2小时。
    2. 加入1微升DNase I,充分混匀,37°C孵育20分钟。

    3. 用蒸馏水将反应混合物的体积调整到100μl
    4. 加入等体积的酚:氯仿:异戊醇到反应混合物中,并在室温(〜25℃)下涡旋混合。
      在17,000 xg g离心2分钟
    5. 用等体积的氯仿:异戊醇将水相回收到新管中,并在室温(〜25℃)下涡旋混合。在4℃下以17,000×g g离心1分钟。
    6. 将水相回收到一个新的冷冻管中,加入等量的预先冷却的5 M醋酸铵,通过轻敲管混合溶液,并在冰上孵育15分钟。

    7. 在21,500×g×4℃下离心20分钟
    8. 去除上清液并加入700μl的70%乙醇。
    9. 在4℃下以17,000×g克离心3分钟,完全除去上清液。
    10. 将开放的管放在冰上风干。
    11. 重悬在40微升的1x甲酰胺染料颗粒(见食谱)。将样品在95℃孵育5分钟,通过涡旋混合并加载到含有0.5×TBE和适当浓度的聚丙烯酰胺的尿素凝胶上(见注4和5)。
    12. 进行电泳(见注6)。
    13. 将凝胶转移到保鲜膜上,用另一层保鲜膜覆盖凝胶,并将其放在荧光TLC板上。简单地将凝胶暴露于紫外光下,切下含有感兴趣RNA的凝胶片(参见注释7)。将凝胶片转移到2毫升试管中(图2A-2C)。


      图2.纯化RNA的工作流程:一种。变性PAGE后将凝胶转移到保鲜膜上。 B.紫外线下凝胶切除; C.将含有感兴趣的RNA的凝胶片转移到带有无菌吸头的管中。

    14. 加入700μl的PK缓冲液(见食谱),并在室温(〜25℃)下在旋转器上孵育24小时。
    15. 转移上清到一个新的1.5毫升管,并添加700μL的2-丙醇和涡流搅拌。
    16. 在4℃下以17,000×g克离心20分钟,小心地除去上清液。
    17. 加700μl的70%乙醇。
    18. 在4℃下以17,000×g克离心3分钟,除去上清液。
    19. 重复步骤B17和B18。
    20. 将颗粒风干10分钟,溶于蒸馏水。
    21. 用分光光度计量化RNA的浓度。

  3. RNA依赖性RNA聚合酶(RdRP)活性检测
    该测定的流程图显示在图3中。反应后,将样品在尿素凝胶或天然凝胶上电泳。在尿素PAGE中,dsRNA被变性为两个ssRNA,并基于其分子量迁移。尽管RNA酶I处理对于RdRP产物和TNTase产物之间的区分是必需的,但是该方法允许准确估计新合成的互补链的大小。相比之下,因为dsRNA在天然PAGE中保留其双螺旋结构,因此根据不具有RNA酶I处理的不同流动性,双链RdRP产物可以与单链TNT酶产物区分开来。原生PAGE的缺点是移动性不能反映RNA的实际大小。同时使用两种PAGE方法为RdRP的酶特性提供更准确的信息。


    图3.体外RdRP测定的流程图

    1. 按照给定的顺序将以下试剂组合在一起:20μl蒸馏水,4μl10x RdRP缓冲液(参见食谱),4μl5x稀释的RNasin,4μl1mM NTP混合物,2μl1μM模板RNA ,2μlα-32P-UTP(〜3,000Ci / mmol)和4μl100-200nM RDR6。

    2. 在25°C孵育管2小时。
    3. 将混合物分成4个管(A,B,A'和B'管),每个含10μl。把它们放在冰上。
    4. 将111μlPK混合物(参见食谱)和111μlPK混合物(Mg2 + +)(参见食谱)分别加入管A和管A'中(参见注释8)。涡旋,并在25°C孵育,直到步骤C6完成。
    5. 将10μlRNase I混合物(见配方)加入B管和B'管中(见注9),37°C孵育20分钟。
    6. 将111μlPK混合物和111μlPK混合物(Mg2 + +)分别加入管B和管B'中,涡旋。

    7. 在65°C孵育20分钟(见注10)
    8. 向每个管中加入350μl100%乙醇,涡旋并在4℃下以17,000×g g离心10分钟。去除上清液。
    9. 向每个试管中加入700μl70%乙醇,并在4℃下以17,000×gg离心3分钟。
      完全去除上清液
    10. 将颗粒风干10分钟。
    11. 对于尿素PAGE,将沉淀(试管A和B)溶解在10μl2x甲酰胺染料中,并在95℃孵育5分钟。对于非变性PAGE,将沉淀(管A'和B')溶解在10μl1x上样缓冲液中(参见食谱),并将样品保存在冰上。
    12. 准备含有0.5×TBE和适当浓度的聚丙烯酰胺或含有0.5×TBE,2mM MgCl 2和适当浓度的聚丙烯酰胺的天然凝胶的尿素凝胶(参见注释11)。
    13. 进行电泳(见注12,13和14)。
    14. 使用凝胶干燥器在80℃真空下干燥凝胶2小时。
    15. 将干燥的凝胶暴露于成像板上约12小时。使用激光扫描仪扫描平板。

数据分析

在这里,我们解释使用不含poly(A)尾部的100-nt RNA体外 RdRP测定的典型结果。当RdRP反应之后的总RNA在尿素凝胶中电泳时,通常观察到多条模糊条带(图4A RNA酶I-)。目前,这些频段不能分配到TNTase或RdRP产品。 ssRNA特异性RNase I处理后,一些条带消失,只剩下一条清晰的条带(图4A RNase I +)。这个RNase I抗性带是RdRP产品。相反,RNase I处理消除的所有其他带是单链TNTase产物(图4A)。当RdRP反应后的总RNA在天然凝胶中电泳时,双链RdRP产物比单链TNT酶产物(图4B RNase I-)迁移得更慢,这可以通过RNA酶I处理消化ssRNA来验证(图4B RNase I +)。请注意,尽管确定了RNase I的最佳浓度,但RNase I处理在一定程度上降低了dsRNA信号(图4B)。 dsRNA产物在天然PAGE中不存在RNA酶I处理的信号反映了RDR6的实际RdRP活性。


图4.体外 RdRP测定的代表性结果。 :一种。在8%丙烯酰胺 - 尿素凝胶上没有聚(A)尾的100-nt模板RNA的RdRP-和TNTase-产物的典型带型。 B.来自在4%丙烯酰胺 - 天然凝胶上没有聚(A)尾的100-nt模板RNA的RdRP-和TNTase-产物的典型带型。

笔记

  1. S2细胞在含有10%FBS和1x抗生素 - 抗真菌剂的10ml Schneider's果蝇培养基中于27℃的培养箱中在100-mm培养皿中静态培养。为了维持细胞,当细胞密度达到1.0×10 7至1.5×10 7 / ml时,通过S2细胞,并以1:10稀释成1新的100毫米碟。
  2. 为了获得高浓度的重组RDR6,我们通常制备500ml的S2细胞(1.5×10 6个细胞/ ml)。我们将其分成50个培养皿(10ml /培养皿),用于转染pAFW-SUMO-AtRDR6 。在RDR6表达后,将S2细胞收集到12个50ml管(各40ml)中。离心后,将每管中的细胞沉淀用300μlPBS重悬,并转移到1.5μl管中。在步骤A10中,将12个管中洗过的磁珠收集在一个新管中。然后,加入100μl含有1mM DTT,20%甘油和0.05U每μlSUMOstar蛋白酶的1x裂解缓冲液用于RDR6的洗脱。
  3. 为了制备偶联抗FLAG抗体的Dynabeads用于免疫纯化,将100μlS2细胞提取物,2μl抗FLAG抗体和100μlDynabeads浆液在管中通过轻拍混合,在旋转器上在4℃下孵育1小时。然后,将管置于磁力架上,除去上清液。珠子用200μl1x裂解缓冲液洗涤一次,并重悬于200μl1x裂解缓冲液中。就在与S2细胞提取物温育之前除去上清液。
  4. 我们使用SequaGel-UreaGel浓缩液来制备尿素凝胶。
  5. 我们通常使用5%,8%和15%丙烯酰胺 - 尿素凝胶进行400-700nt,80-200nt和< 50 nt的RNA分别。
  6. 我们在0.5x TBE中以500V的恒定电压作为运行缓冲液来解析RNA。
  7. 如果模板RNA不能通过UV阴影检测到,则用核酸染色染料染色凝胶以进行凝胶切除。
  8. 对于天然PAGE样品(管A'和B'),我们使用含有镁离子而不是EDTA的“PK缓冲液(Mg2 +)”来稳定双链RNA(参见食谱)。
  9. 尽管RNA酶I处理允许区分来自单链TNT酶产物的双链RdRP产物,但使用过高浓度的RNA酶I会导致dsRNA的降解。因此,在使用32 P体标记的ss和dsRNA标记物作为RNase I底物之前,应该在RdRP活性测定之前仔细确定RNase I的最佳浓度。
  10. 为了避免dsRNA的解旋,如果模板RNA的长度< 25 nt,最好将RNA样品在25°C温育60分钟或在脱蛋白步骤中于37°C温育30分钟。
  11. 我们使用40(w / v)% - 丙烯酰胺/ Bis混合溶液(19:1)来制备天然凝胶。
  12. 运行时间应该优化。来自100-nt模板RNA的RdRP-和TNTase-产物在8%丙烯酰胺 - 尿素凝胶(100mm(H)×150mm(W)×1mm(D))上以500的恒定电压(V)40分钟或4%丙烯酰胺 - 天然凝胶(100mm(H)×150mm(W)×1mm(D))在500V的恒定电压下处理40分钟。
  13. 为了避免双链RNA的自发退绕,使用装有冷却剂的塑料棒保持冷却缓冲液,并在冷藏室中进行电泳(图5)。
  14. 为了确定产物的相对大小,可以使用5'放射性标记的模板RNA作为大小标记。


    图5.原生PAGE的代表性图像。白色箭头指示填充冷却剂的塑料条。

食谱

  1. 磷酸盐缓冲盐水(PBS),pH 7.4
    137mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4 4/2 1.8mM KH 2 PO 4 4/2
  2. 低渗裂解缓冲液
    10mM HEPES-KOH,pH7.4
    10 mM KCl
    1.5mM Mg(OAc)2•/ 2 5 mM DTT
    1x无EDTA蛋白酶抑制剂鸡尾酒
  3. 1x裂解缓冲液
    30mM HEPES-KOH,pH7.4
    100 mM KOAc
    2mM Mg(OAc)2•/ 2
  4. 2x甲酰胺染料
    10mM EDTA,pH8.0
    98%(w / v)去离子甲酰胺
    0.025%(w / v)二甲苯氰醇
    0.025%(w / v)溴酚蓝
  5. PK缓冲区
    100mM Tris-HCl,pH 7.5
    200 mM NaCl
    2mM EDTA,pH8.0
    1%(w / v)SDS
  6. 1 mM NTP混合物
    在核糖核苷酸溶液组中1μl100mM ATP
    1μl100 mM UTP Ribonucleotide Solution set
    在核糖核苷酸溶液组中1μl100mM GTP
    在核糖核苷酸溶液组中1μl100mM CTP
    96μl蒸馏水
  7. PK缓冲液(Mg <2 +)
    100mM Tris-HCl,pH 7.5
    200 mM NaCl
    1mM MgCl 2
    1%(w / v)SDS
  8. PK混合物
    2微升的糖原
    20μl蛋白酶K
    200微升PK缓冲液
  9. PK混合物(Mg2 +)
    2微升的糖原
    20μl蛋白酶K
    200μl的PK缓冲液(Mg 2 +)。
  10. 5倍TBE
    0.446 M Tris
    0.445 M H 3 BO 3 3 0.01M EDTA,pH8.0
  11. 10倍RdRP缓冲区
    500mM HEPES-KOH,pH 7.6
    200mM NH 4 OAc
    80mM MgCl 2•/ 2 1mM EDTA,pH8.0
    20%(w / v)PEG 4000
  12. 2x RNase I缓冲液
    100 mM Tris-HCl pH 8.0
    600 mM NaCl
    30mM MgCl 2•/ 2
  13. 核糖核酸酶混合物
    10μl2×RNase I缓冲液
    1μlRNase I
    9μl蒸馏水
  14. 2个加载缓冲区
    4mM MgCl 2•/ 2 0.5x TBE
    50%(w / v)甘油
    0.04%(w / v)酒石黄
    0.02%(w / v)溴酚蓝
    0.02%(w / v)二甲苯氰醇

致谢

该协议已经由Baeg等人于2017年和马克叶夫和Bamford于2002年进行了修改和修改。我们感谢Tomari实验室的成员对该手稿提出意见。这项工作得到了部分创新领域科学研究资助(“新生链生物学”)26116003(对于HI)(“非编码RNA新分类学”)26113007(YT),Grant (A)16H06159(对于HI),对于挑战性探索性研究15K14444(对于HI)和对于JSPS研究员的援助16J07290(对KB)的援助。我们宣布没有冲突或竞争的利益。

参考

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Baeg, K., Tomari, Y. and Iwakawa, H. (2018). In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6. Bio-protocol 8(1): e2673. DOI: 10.21769/BioProtoc.2673.
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