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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

KB Kyungmin Baeg
YT Yukihide Tomari
HI Hiro-oki Iwakawa

发布: 2018年01月05日第8卷第1期 DOI: 10.21769/BioProtoc.2673 浏览次数: 11204

评审: Tie LiuLaia ArmengotPooja Verma

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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) search Double-stranded RNA (双链RNA) search RNA silencing (RNA沉默) search Small RNA (小RNA) search siRNA (siRNA) search RNA-DEPENDENT RNA POLYMERASE6 (RNA依赖性RNA聚合酶6) search Arabidopsis thaliana (拟南芥) search

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

English
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文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

植物科学 > 植物生物化学 > 蛋白质 > 活性

生物化学 > 蛋白质 > 分离和纯化

植物科学 > 植物分子生物学 > RNA

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