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Measurement of RNA-induced PKR Activation in vitro
RNA诱导的PKR活化的体外测定   

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Abstract

Protein kinase R (PKR) is one of the key RNA-activated sensors for innate immunity. PKR is activated by pathogenic or aberrant RNAs such as short double-stranded RNAs or those with imperfect secondary structures, as well as a reduction in the amount and number of RNA modifications. Activation of PKR may be an underlying mechanism for the pathogenesis of human diseases. In this protocol, I describe a method for studying levels of RNA-induced PKR activation in vitro.

Keywords: PKR(PKR), Stress response(应激反应), Innate immunity(先天免疫), Aberrant RNA(异常RNA)

Background

PKR is one of four mammalian kinases that phosphorylate eukaryotic initiation factor 2-α subunit (eIF2α) in response to stress signals. PKR is activated mainly in response to viral infection (Holcik and Sonenberg, 2005). PKR is a key component of innate immunity that recognizes and binds to pathogenic RNAs. The interaction of RNAs with PKR promotes and stabilizes its dimerization. PKR then undergoes auto-phosphorylation and subsequently phosphorylates eIF2α to shut off general translation, while activating the downstream signaling cascade including the increased translation of the ATF4 stress response transcription factor (Hinnebusch, 2005).

PKR is known to be activated by short double-stranded RNAs (Manche et al., 1992; Zheng and Bevilacqua, 2004) as well as RNAs with some imperfect secondary structures such as hairpin loops (Bevilacqua et al., 1998). In addition, defects in RNA biogenesis, including lower levels of m6A modification, lead to a stress response via the activation of PKR (Nallagatla and Bevilacqua, 2008). Reduced levels of m6A modification followed by the activation of the PKR-mediated stress response may serve as an underlying molecular etiology of human diseases such as Cornelia de Lange syndrome (Yuen et al., 2016). The method described in this protocol allows us to study the stress response triggered by foreign or aberrant RNAs by examining their effect on the activation of PKR in vitro.

Materials and Reagents

  1. 35 mm TC-treated culture dish (Corning, catalog number: 430165 )
  2. Razor blades
  3. Protein purification column (Kimble Chase Life Science and Research Products, catalog number: 420400-1010 )
  4. Pipette tips (VWR, catalog number: 53508-794 )
  5. 50 ml tube (Corning, catalog number: 430290 )
  6. VWR 10 ml serological pipet (VWR, catalog number: 89130-888 )
  7. Parafilm M (Bemis, catalog number: PM996 )
  8. Immobilon-P polyvinylidene difluoride membrane (EMD Millipore, catalog number: IPVH00010 )
  9. Dialysis tube (Sigma-Aldrich, catalog number: D6191 )
  10. Dialysis tube clip (Sigma-Aldrich, catalog number: Z371092 )
  11. Dialysis tubing (Sigma-Aldrich, catalog number: D9652 )
  12. pGEM®-T Easy vector systems (Promega, catalog number: A1360 )
  13. One Shot TOP10 competent cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: C404003 )
  14. pET-28a(+) plasmid (EMD Millipore, catalog number: 69864 )
  15. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  16. FBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
  17. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  18. TRIzol reagent (Thermo Fisher Scientific, AmbionTM, catalog number: 15596026 )
  19. Chloroform, biotechnology grade (VWR, catalog number: 97064-678 )
  20. 2-propanol, biotechnology grade (VWR, catalog number: 97065-048 )
  21. Ethanol, absolute (Sigma-Aldrich, catalog number: E7023 )
  22. DEPC-treated water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 750023 )
  23. 5x iScript cDNA Synthesis Kit (Bio-Rad Laboratories, catalog number: 1708890 )
  24. Nuclease-free water (New England Biolabs, catalog number: B1500 )
  25. PCR primers (IDT)
  26. Phusion high-fidelity DNA polymerase (New England Biolabs, catalog number: M0530 )
  27. dNTP (New England Biolabs, catalog number: N0447 )
  28. Taq DNA polymerase (New England Biolabs, catalog number: M0273 )
  29. Magnesium chloride (MgCl2) solution (New England Biolabs, catalog number: B9021 )
  30. dATP (100 mM) (New England Biolabs, catalog number: N0440S )
  31. T4 DNA ligase (New England Biolabs, catalog number: M0202 )
  32. X-Gal (β-galactoside) (Promega, catalog number: V3941 )
  33. SOC medium (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15544034 )
  34. LB plate (Thermo Fisher Scientific, GibcoTM, catalog number: 10855021 )
  35. Ampicillin (Sigma-Aldrich, catalog number: A9393 )
  36. NEBuffer 3.1 (New England Biolabs, catalog number: B7203S )
  37. SalI restriction enzyme (New England Biolabs, catalog number: R0138 )
  38. NotI restriction enzyme (New England Biolabs, catalog number: R0189 )
  39. Qiagen QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28706 )
  40. T4 DNA ligase buffer (New England Biolabs, catalog number: B0202S )
  41. IPTG (Sigma-Aldrich, catalog number: I6758 )
  42. Liquid nitrogen (Midwest Liquid Nitrogen Service)
  43. Lysozyme (Sigma-Aldrich, catalog number: L6876 )
  44. RNase A (Sigma-Aldrich, catalog number: R4642 )
  45. DNase I (New England Biolabs, catalog number: M0303 )
  46. LDS loading buffer (4x) (Thermo Fisher Scientific, NovesTM, catalog number: NP0007 )
  47. Ni-NTA agarose (QIAGEN, catalog number: 30230 )
  48. Qubit Protein Assay (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: Q33211 )
  49. λ-PPase (New England Biolabs, catalog number: P0753 )
  50. Phosphatase reaction buffer (New England Biolabs, catalog number: B6022S )
  51. Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
  52. Poly I:C (Sigma-Aldrich, catalog number: P1530 )
  53. NuPAGE 4-12% Bis-Tris protein gel (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0322BOX )
  54. Silver staining kit (Thermo Fisher Scientific, NovesTM, catalog number: LC6100 )
  55. 1% bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
  56. PKR primary antibody (Santa Cruz Biotechnology, catalog number: sc-6282 )
  57. Phosphorylated-PKR primary antibody (Abcam, catalog number: ab32036 )
  58. Peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, InvitrogenTM, catalog number: 31460 )
  59. ECL reagents (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 32132 )
  60. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S8282 )
  61. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  62. Imidazole (Sigma-Aldrich, catalog number: I5513 )
  63. PMSF (Sigma-Aldrich, catalog number: P7626 )
  64. Protease inhibitor (Roche Diagnostics, catalog number: 11697498001 )
  65. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
  66. Tris (pH 7.6) (VWR, catalog number: 97061-260 )
  67. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  68. Magnesium chloride (MgCl2) powder (Sigma-Aldrich, catalog number: M8266 )
  69. Glycerol (VWR, BDH®, catalog number: BDH1172-1LP )
  70. EDTA (Sigma-Aldrich, catalog number: E6758 )
  71. DTT (Sigma-Aldrich, catalog number: D0632 )
  72. HEPES (pH 7.5) (Sigma-Aldrich, catalog number: H3375 )
  73. ATP (New England Biolabs, catalog number: P0756 )
  74. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  75. Tween-20 (Sigma-Aldrich, catalog number: P9416 )
  76. E. coli lysis buffer (see Recipes)
  77. Protein purification wash buffer (see Recipes)
  78. Protein purification elution buffer (see Recipes)
  79. Protein storage buffer (see Recipes)
  80. Phosphatase reaction buffer (see Recipes)
  81. PKR activation buffer (see Recipes)
  82. TE buffer (10x) (see Recipes)
  83. Tween-20 phosphate buffered saline (pH 7.2) (see Recipes)

Equipment

  1. Cell culture incubator (Panasonic Biomedical, model: MCO-230AIC )
  2. Pipette (VWR, catalog number: 89079-970 )
  3. Centrifuge (Eppendorf, model: 5424 )
  4. Qubit 3.0 Fluorometer (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q33216 )
  5. PCR machine (Bio-Rad Laboratories, model: S1000TM Thermal Cycler , catalog number: 1852148)
  6. Vortex (VWR, catalog number: 97043-562 )
  7. Sonicator (Branson, model: Digital Sonifier 450 or from Fisher Scientific, catalog number: 15-338-553)
  8. 600 ml beaker (VWR, catalog number: 10754-956 )
  9. Typhoon scanner (GE Healthcare, model: Amersham Molecular Dynamics Typhoon 9410 or from GMI, catalog number: 8149-30-9410)

Procedure

  1. Human PKR gene cloning
    1. RNA isolation and cDNA library generation
      1. Grow the cells in a 35 mm dish with 10 ml DMEM with 10% FBS and 1% penicillin-streptomycin in a 37 °C incubator with 5% CO2 until 80% confluence.
      2. Remove the media from the culture dish.
      3. Add 1 ml TRIzol reagent directly to the cells in the culture dish.
      4. Pipette the cells up and down for 5 times in the culture dish to lyse the cells.
      5. Incubate the sample at room temperature for 5 min to allow for complete nuclear lysis.
      6. Add 0.2 ml of chloroform and share the tube vigorously by hand for 15 sec.
      7. Incubate for 2 min at room temperature.
      8. Centrifuge the sample at 12,000 x g for 15 min at 4 °C.
      9. Remove the aqueous supernatant by angling the tube at 45° to avoid contacting the interphase or the organic layer.
      10. Transfer the aqueous phase into a new tube and add 0.5 ml of 100% isopropanol.
      11. Incubate at room temperature for 10 min.
      12. Centrifuge at 12,000 x g for 10 min at 4 °C.
        Note: RNA should be visible as a white pellet.
      13. Remove the supernatant carefully from the tube without touching the RNA pellet.
      14. Wash the pellet with 1 ml of 75% ethanol pre-chilled at 4 °C.
      15. Vortex the sample briefly, followed by centrifugation at 7,600 x g for 5 min at 4 °C.
      16. Remove the ethanol carefully and air dry the RNA pellet for 15 min on ice.
      17. Re-suspend the RNA pellet in 100 μl DEPC-treated water and incubate at 37 °C for 10 min.
      18. Measure the RNA concentration using a Qubit 3.0 Fluorometer, NanoDrop spectrophotometer or Bioanalyzer.
      19. Prepare the 5x iScript cDNA Synthesis reaction mix on ice for reverse transcription.


      20. Incubate the reaction mix on a PCR machine with the following program:
        5 min at 25 °C
        30 min at 42 °C
        5 min at 85 °C
        Hold at 4 °C
      21. Store the cDNA in aliquots at -20 °C. Alternatively, store the cDNA at 4 °C if used repeatedly in days. Avoid repeated freezing and thawing cycles.
    2. PCR amplification and subcloning into pET-28a+ plasmid
      Other bacterial expression plasmids may be used. Different restriction enzymes may be used in other plasmids depending on the cloning sites.
      1. Amplify the human PKR gene with the following primers:
        Forward: 5’ TAGTCGACATGGCTGGTGATCTTTCAGC 3’
        Reverse: 5’ GTCAGCCTAACATGTGTGTCGTTCATTTTTCTC 3’
        Notes: 
        1. The forward primer contains a SalI restriction site, as underlined, before the start codon.
        2. Only a single band will be obtained.
      2. Prepare a PCR reaction mix on ice as follow


      3. Incubate the reaction mix on a PCR machine with the following program:


      4. Incubate the following reaction mix to produce the product with an overhang


      5. Prepare the following reaction mix for subcloning the PKR gene into the pGEM-T Easy vector  


      6. Incubate the ligation reaction mix at 4 °C overnight. .
    3. Transformation into E. coli
      1. Thaw One Shot TOP 10 chemically competent cells on ice for 3 min.
      2. Add 5 μl ligation product to the cells.
      3. Incubate the tube on ice for 30 min.
      4. Place the tube at room temperature for 10 min.
      5. Mix 50 μl β-galactoside (X-Gal) (50 mg/ml) to 150 μl SOC medium and add the mixture to the LB plate with ampicillin.
      6. Spread the transformed cells on the LB plate with ampicillin.
      7. Incubate the plate at 37 °C overnight.
      8. Pick the white colonies to check for successful ligation of PKR gene.
    4. Digestion and ligation of PKR gene into pET-28a+
      1. Set up SalI and NotI digestion reactions for pET-28a+ and PKR




      2. Run the double digestion products pET-28a+ and PKR separately on a 1% agarose gel.
        Note: Look for the bands for pET-28a and PKR at 5,369 bp and 1,656 bp, respectively.
      3. Carefully excise the bands from the gel using clean razor blades.
      4. Perform gel extraction using the QIAGEN Gel Extraction Kit.
      5. Weigh the gel slice in a tube and add 3 volumes buffer QG to 1 volume gel.
      6. Incubate at 50 °C for 10 min.
      7. Add 1 gel volume isopropanol to the sample and vortex.
      8. Transfer the sample to the QlAquick column and centrifuge for 1 min at 18,000 x g.
      9. Add 750 ml buffer PE to the QlAquick column and centrifuge for 1 min at 18,000 x g.
      10. Add 50 μl buffer EB to the center of the QlAquick membrane to elute the DNA.
      11. Set up a ligation reaction to subclone PKR gene into the pET-28a plasmid


      12. Incubate at 16 °C overnight and transform 2 μl of the reaction into 25 μl competent cells.

  2. PKR protein purification from E. coli
    1. Grow the cells in LB broth at 37 °C until OD600 reaches 0.4-0.6.
    2. Induce with 40 µl of a 100 mM IPTG (final concentration 400 µM) for 3 to 5 h at 37 °C.
    3. Harvest the cells and proceed immediately to cell lysis or freeze the cells in liquid nitrogen followed by storing them at -80 °C.
    4. Resuspend the cells in lysis buffer at 2-5 ml per gram wet weight.
    5. Add lysozyme (1 mg/ml) to the lysis buffer with the cells and incubate on ice for 30 min.
    6. Sonicate on ice using a sonicator equipped with a microtip.
      Note: Use five 15 sec bursts at 30x with a 45 sec cooling period between each burst.
    7. (Optional) If the lysate is very viscous, add RNase A (10 μg/ml) and DNase I (5 μg/ml) and incubate on ice for 10-15 min.
    8. Centrifuge lysate at 10,000 x g for 20-30 min at 4 °C to pellet the cellular debris and keep the supernatant.
    9. Add 5 μl 2x LDS-PAGE sample buffer to 5 μl supernatant and store at -20 °C for LDS-PAGE analysis.
    10. Pipette 10 ml of Ni-NTA slurry to a 50 ml tube and briefly centrifuge. Remove supernatant and add 20 ml of lysis buffer. Mix gently by inverting. Repeat centrifugation step and remove the supernatant.
      Note: Adjust the amount of Ni-NTA used depending on the yield of the target protein.
    11. Add cleared lysate to this equilibrated matrix and mix gently by shaking at 4 °C for 1 h.
    12. Load 10 ml lysate-Ni-NTA mixture into a column.
    13. Remove bottom cap and collect the flow-through. Save and store for Western blotting.
    14. Wash 10 times with wash buffer. Collect wash factions for Western blotting.
    15. Elute protein 4 times with 1 ml elution buffer.
    16. Perform dialysis in storage buffer overnight at 4 °C.
    17. Wet the dialysis tube with distilled water and close the tube with a clip near one end.
    18. Load the sample into the dialysis tube and clip another end.
    19. Immerse the knotted tube in storage buffer in a 500 ml beaker overnight.
    20. Measure protein concentration by Qubit Protein Assay and check the purity of protein (Figure 1).


      Figure 1. Silver staining of purified PKR protein. The purified PKR protein (65 kDa) was visualized by silver staining. IPTG was added to induce the protein expression in E. coli. Lysate from cells without IPTG induction was used as a negative control.

  3. PKR dephosphorylation and activation
    Human PKR protein is activated by the RNAs from E. coli upon cell lysis; therefore, protein dephosphorylation is necessary to obtain the non-phosphorylated form of PKR before activation assays.
    1. Dephosphorylation of PKR
      1. Incubate 2 μg of PKR with 400 U λ-PPase (1 μl) in 10 μl phosphatase reaction buffer for 1 h at 30 °C.
      2. Aliquot the dephosphorylated PKR into 10 tubes each containing 0.2 μg PKR protein
      3. Add freshly prepared 2 mM sodium orthovanadate to inhibit the λ-PPase.
    2. In vitro PKR activation assay
      1. Prepare 10 μg/ml Poly I:C from stock (as a positive control) (10 mg/ml).
      2. Make the reaction mixture on ice.


      3. Incubate dephosphorylated PKR with 10 ng Poly I:C or dsRNA in PKR activation buffer at 30 °C for 0.5-2 h.
      4. Add 1x LDS loading buffer with 5% β-mercaptoethanol to stop the reaction.
      5. Perform Western blotting to determine the PKR activation by probing for level p-PKR against total PKR.
      6. Electrophorese the samples through a NuPAGE 4-12% Bis-Tris protein gel.
      7. Transfer the resolved proteins onto an immobilon-P polyvinylidene difluoride (PVDF) membrane at 100 V for 90 min at 4 °C.
      8. Block the membrane with 1% bovine serum albumin in 0.5% Tween-20 phosphate buffered saline (PBST) for 60 min.
      9. Incubate the membrane with primary antibody overnight at 4 °C.
      10. Wash the membrane three times with PBST for 5 min each.
      11. Add horseradish peroxidase-conjugated secondary antibody at a dilution of 1:3,000 for 1 h at room temperature.
      12. Wash the membrane three times with PBST for 5 min each.
      13. Add enhanced chemiluminescence detection system (ECL reagents).
      14. Scan the membrane with a Typhoon scanner (Figure 2).


        Figure 2. Representative results of PKR activation in vitro. PKR activation assay was used to study different types of RNAs (mRNAs, small RNAs and rRNAs) isolated from WT and NIPBL-MS (missense) human lymphoblastoid cell lines (LCLs). Both ncRNAs and rRNAs isolated from NIPBL-MS LCLs are capable of activating recombinant PKR in vitro; 10 ng Poly I:C was used as a positive control for PKR activation. *P < 0.001 compared to untreated control. (Please refer to Yuen et al., 2016 for details)

Data analysis

All experiments were repeated independently at least in triplicate, and the data are presented as mean ± SD. Statistical significance was determined using the Student’s t-test. A P value of < 0.05 was considered to be statistically significant. The experiments described in this protocol has been performed and their data have been published in Yuen et al., 2016, NIPBL controls RNA biogenesis to prevent activation of the stress kinase PKR. Cell Rep 14(1): 93-102, which can be accessed by: http://www.cell.com/cell-reports/fulltext/S2211-1247(15)01425-4.

Recipes

  1. E. coli lysis buffer (1 L)
    50 mM NaH2PO4
    300 mM NaCl
    10 mM imidazole
    1 mM PMSF
    10 mM protease inhibitor
    Adjust pH to 8.0 using NaOH
  2. Protein purification wash buffer (1 L)
    50 mM NaH2PO4
    300 mM NaCl
    20 mM imidazole
    1 mM PMSF
    10 mM protease inhibitor
    Adjust pH to 8.0 using NaOH
  3. Protein purification elution buffer (1 L)
    50 mM NaH2PO4
    300 mM NaCl
    250 mM imidazole
    1 mM PMSF
    10 mM protease inhibitor
    Adjust pH to 8.0 using NaOH
  4. Protein storage buffer
    10 mM Tris (pH 7.6)
    50 mM KCl
    2 mM MgCl2
    10% glycerol
    7 mM β-mercaptoethanol
  5. Phosphatase reaction buffer
    50 mM Tris-HCl, pH 7.5
    2 mM MgCl2
    0.1 mM EDTA
    5 mM DTT
  6. PKR activation buffer
    20 mM HEPES (pH 7.5)
    4 mM MgCl2
    100 mM KCl
    1 mM ATP
  7. TE buffer (10x)
    100 mM Tris-Cl
    10 mM EDTA (pH 8.0)
  8. Tween-20 phosphate buffered saline (pH 7.2) (1 L)
    8 g of NaCl
    0.2 g of KCl
    1.44 g of Na2HPO4
    0.24 g of KH2PO4
    2 ml of Tween-20

Acknowledgments

I would like to thank Dr. Jennifer Gerton for reading this protocol. This study was supported by Stowers Institute for Medical Research, the Cornelia de Lange Syndrome (CdLS) Foundation, and the March of Dimes (MOD) Foundation (6-FY14-434).

References

  1. Bevilacqua, P. C., George, C. X., Samuel, C. E. and Cech, T. R. (1998). Binding of the protein kinase PKR to RNAs with secondary structure defects: role of the tandem A-G mismatch and noncontiguous helixes. Biochemistry 37(18): 6303-6316.
  2. Hinnebusch, A. G. (2005). eIF2α kinases provide a new solution to the puzzle of substrate specificity. Nat Struct Mol Biol 12(10): 835-838.
  3. Holcik, M. and Sonenberg, N. (2005). Translational control in stress and apoptosis. Nat Rev Mol Cell Biol 6(4): 318-327.
  4. Manche, L., Green, S. R., Schmedt, C. and Mathews, M. B. (1992). Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol Cell Boil 12(11): 5238-5248.
  5. Nallagatla, S. R. and Bevilacqua, P. C. (2008). Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure-specific manner. RNA 14(6): 1201-1213.
  6. Yuen, K. C., Xu, B., Krantz, I. D. and Gerton, J. L. (2016). NIPBL controls RNA biogenesis to prevent activation of the stress kinase PKR. Cell Rep 14(1): 93-102.
  7. Zheng, X. and Bevilacqua, P. C. (2004). Activation of the protein kinase PKR by short double-stranded RNAs with single-stranded tails. RNA 10(12): 1934-1945.

简介

蛋白激酶R(PKR)是先天免疫的核心RNA激活传感器之一。 PKR由致病性或异常RNA如短双链RNA或具有不完全二级结构的RNA激活,以及RNA修饰的量和数量的减少。 PKR的激活可能是人类疾病发病机制的潜在机制。在本协议中,我描述了一种在体外研究RNA诱导的PKR激活水平的方法。

背景 PKR是四种哺乳动物激酶之一,其响应于应激信号磷酸化真核起始因子2-α亚基(eIF2α)。 PKR主要是响应于病毒感染而激活(Holcik和Sonenberg,2005)。 PKR是识别和结合病原RNA的先天免疫的关键组成部分。 RNA与PKR的相互作用促进并稳定其二聚化。然后PKR经历自身磷酸化,随后磷酸化eIF2α以切断一般翻译,同时激活下游信号级联,包括增加的ATF4应激反应转录因子的翻译(Hinnebusch,2005)。
&nbsp;已知PKR被短双链RNA激活(Manche等人,1992; Zheng和Bevilacqua,2004)以及具有一些不完全二级结构的RNA,例如发夹环(Bevilacqua 等人,1998)。此外,RNA生物发生缺陷,包括较低水平的m A修饰,通过激活PKR导致应激反应(Nallagatla和Bevilacqua,2008)。降低水平的重组随后激活PKR介导的应激反应的改变可以作为人类疾病如Cornelia de Lange综合征的基础分子病因学(Yuen等, em>。,2016)。本协议中描述的方法允许我们通过检查其对体外PKR活化的影响来研究外源或异常RNA引发的应激反应。

关键字:PKR, 应激反应, 先天免疫, 异常RNA

材料和试剂

  1. 35毫升TC处理培养皿(康宁,目录号:430165)
  2. 剃刀刀片
  3. 蛋白质纯化柱(Kimble Chase Life Science and Research Products,目录号:420400-1010)
  4. 移液器提示(VWR,目录号:53508-794)
  5. 50ml管(Corning,目录号:430290)
  6. VWR 10 ml血清移液管(VWR,目录号:89130-888)
  7. Parafilm M(Bemis,目录号:PM996)
  8. Immobilon-P聚偏二氟乙烯膜(EMD Millipore,目录号:IPVH00010)
  9. 透析管(Sigma-Aldrich,目录号:D6191)
  10. 透析管夹(Sigma-Aldrich,目录号:Z371092)
  11. 透析管(Sigma-Aldrich,目录号:D9652)
  12. pGEM ® -T Easy矢量系统(Promega,目录号:A1360)
  13. One Shot TOP10感受态细胞(Thermo Fisher Scientific,Invitrogen TM,目录号:C404003)
  14. pET-28a(+)质粒(EMD Millipore,目录号:69864)
  15. DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:11965092)
  16. FBS(Thermo Fisher Scientific,Gibco TM ,目录号:10437028)
  17. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  18. TRIzol试剂(Thermo Fisher Scientific,Ambion TM ,目录号:15596026)
  19. 氯仿,生物技术级(VWR,目录号:97064-678)
  20. 2-丙醇,生物技术级(VWR,目录号:97065-048)
  21. 乙醇,绝对(Sigma-Aldrich,目录号:E7023)
  22. DEPC处理过的水(Thermo Fisher Scientific,Invitrogen TM,目录号:750023)
  23. 5x iScript cDNA合成试剂盒(Bio-Rad Laboratories,目录号:1708890)
  24. 无核酸酶水(New England Biolabs,目录号:B1500)
  25. PCR引物(IDT)
  26. Phusion高保真DNA聚合酶(New England Biolabs,目录号:M0530)
  27. dNTP(New England Biolabs,目录号:N0447)
  28. Taq DNA聚合酶(New England Biolabs,目录号:M0273)
  29. 氯化镁(MgCl 2)溶液(New England Biolabs,目录号:B9021)
  30. dATP(100mM)(New England Biolabs,目录号:N0440S)
  31. T4 DNA连接酶(New England Biolabs,目录号:M0202)
  32. X-Gal(β-半乳糖苷)(Promega,目录号:V3941)
  33. SOC培养基(Thermo Fisher Scientific,Invitrogen TM,目录号:15544034)
  34. LB板(Thermo Fisher Scientific,Gibco TM ,目录号:10855021)
  35. 氨苄青霉素(Sigma-Aldrich,目录号:A9393)
  36. NEBuffer 3.1(New England Biolabs,目录号:B7203S)
  37. 限制酶(New England Biolabs,目录号:R0138)
  38. I限制酶(New England Biolabs,目录号:R0189)
  39. Qiagen QIAquick凝胶提取试剂盒(QIAGEN,目录号:28706)
  40. T4 DNA连接酶缓冲液(New England Biolabs,目录号:B0202S)
  41. IPTG(Sigma-Aldrich,目录号:I6758)
  42. 液氮(中西部液氮服务)
  43. 溶菌酶(Sigma-Aldrich,目录号:L6876)
  44. RNase A(Sigma-Aldrich,目录号:R4642)
  45. DNase I(New England Biolabs,目录号:M0303)
  46. LDS加载缓冲液(4x)(Thermo Fisher Scientific,Noves TM,目录号:NP0007)
  47. Ni-NTA琼脂糖(QIAGEN,目录号:30230)
  48. Qubit蛋白测定(Thermo Fisher Scientific,Molecular Probes TM ,目录号:Q33211)
  49. λ-PPase(New England Biolabs,目录号:P0753)
  50. 磷酸酶反应缓冲液(New England Biolabs,目录号:B6022S)
  51. 原钒酸钠(Sigma-Aldrich,目录号:S6508)
  52. 聚I:C(Sigma-Aldrich,目录号:P1530)
  53. NuPAGE 4-12%Bis-Tris蛋白凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:NP0322BOX)
  54. 银染试剂盒(Thermo Fisher Scientific,Noves TM ,目录号:LC6100)
  55. 1%牛血清白蛋白(BSA)(Sigma-Aldrich,目录编号:A9418)
  56. PKR一抗(Santa Cruz Biotechnology,目录号:sc-6282)
  57. 磷酸化PKR一抗(Abcam,目录号:ab32036)
  58. 过氧化物酶缀合的第二抗体(Thermo Fisher Scientific,Invitrogen TM,目录号:31460)
  59. ECL试剂(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:32132)
  60. 磷酸二氢钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S8282)
  61. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  62. 咪唑(Sigma-Aldrich,目录号:I5513)
  63. PMSF(Sigma-Aldrich,目录号:P7626)
  64. 蛋白酶抑制剂(Roche Diagnostics,目录号:11697498001)
  65. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045)
  66. Tris(pH 7.6)(VWR,目录号:97061-260)
  67. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  68. 氯化镁(MgCl 2)粉末(Sigma-Aldrich,目录号:M8266)
  69. 甘油(VWR,BDH ,目录号:BDH1172-1LP)
  70. EDTA(Sigma-Aldrich,目录号:E6758)
  71. DTT(Sigma-Aldrich,目录号:D0632)
  72. HEPES(pH 7.5)(Sigma-Aldrich,目录号:H3375)
  73. ATP(New England Biolabs,catalog number:P0756)
  74. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  75. 吐温-20(Sigma-Aldrich,目录号:P9416)
  76. E。大肠杆菌裂解缓冲液(参见食谱)
  77. 蛋白质纯化洗涤缓冲液(参见食谱)
  78. 蛋白质纯化洗脱缓冲液(参见食谱)
  79. 蛋白质储存缓冲液(参见食谱)
  80. 磷酸酶反应缓冲液(参见食谱)
  81. PKR激活缓冲液(见配方)
  82. TE缓冲区(10x)(见配方)
  83. 吐温-20磷酸盐缓冲盐水(pH 7.2)(见配方)

设备

  1. 细胞培养箱(Panasonic Biomedical,型号:MCO-230AIC)
  2. 移液器(VWR,目录号:89079-970)
  3. 离心机(Eppendorf,型号:5424)
  4. Qubit 3.0荧光计(Thermo Fisher Scientific,Invitrogen TM,目录号:Q33216)
  5. PCR机(Bio-Rad Laboratories,型号:S1000 TM热循环仪,目录号:1852148)
  6. 涡旋(VWR,目录号:97043-562)
  7. 超声波仪(Branson,型号:Digital Sonifier 450或Fisher Scientific,目录号:15-338-553)
  8. 600毫升烧杯(VWR,目录号:10754-956)
  9. 台风扫描仪(GE Healthcare,型号:Amersham Molecular Dynamics Typhoon 9410或GMI,目录号:8149-30-9410)

程序

  1. 人PKR基因克隆
    1. RNA分离和cDNA文库生成
      1. 在具有10%FBS和1%青霉素 - 链霉素的10ml DMEM的35mm培养皿中,在具有5%CO 2的37℃培养箱中将细胞生长至80%汇合。
      2. 从培养皿中取出培养基。
      3. 将1ml TRIzol试剂直接加入培养皿中的细胞
      4. 在培养皿中上下移动细胞5次以裂解细胞。
      5. 在室温下孵育样品5分钟以允许完全的核裂解
      6. 加入0.2毫升氯仿,用手强力共用管15秒
      7. 在室温下孵育2分钟。
      8. 在4℃下将样品以12,000×g离心15分钟。
      9. 通过以45°角将管倾斜取出上清液,以避免接触相间或有机层
      10. 将水相转移到新管中,加入0.5ml 100%异丙醇
      11. 在室温下孵育10分钟
      12. 在4℃下以12,000 x g离心10分钟。
        注意:RNA应作为白色颗粒可见。
      13. 从管中小心取出上清液,不要接触RNA颗粒
      14. 用4ml预冷的1ml 75%乙醇洗涤沉淀物
      15. 短暂旋转样品,然后在4℃下以7,600×g离心5分钟。
      16. 小心取出乙醇,并在冰上将RNA颗粒空气干燥15分钟。
      17. 将RNA沉淀重新悬浮于100μlDEPC处理过的水中,37℃孵育10分钟
      18. 使用Qubit 3.0荧光计,NanoDrop分光光度计或Bioanalyzer测量RNA浓度。
      19. 在冰上准备5x iScript cDNA合成反应混合物进行逆转录

      20. 使用以下程序在PCR机上孵育反应混合物:
        25°C 5分钟
        42°C 30分钟
        85°C 5分钟
        保持在4°C
      21. 将cDNA以等分试样储存在-20℃。或者,如果在几天内反复使用,将cDNA存储在4℃。避免反复冻融循环。
    2. PCR扩增和亚克隆到pET-28a +质粒中 可以使用其他细菌表达质粒。根据克隆位点,不同的限制酶可以用于其他质粒。
      1. 用以下引物扩增人PKR基因:
        转发:5'TA GGGGAC ATGGCTGGTGATCTTTCAGC 3'
        反向:5'GTCAGCCTAACATGTGTGTCGTTCATTTTTCTC 3'
        注意:
        1. 正向引物在起始密码子之前含有SalI限制性位点,如下划线所示。

        2. 只能单个乐队
      2. 在冰上准备PCR反应混合物,如下所示

      3. 使用以下程序在PCR机上孵育反应混合物:


      4. 孵育以下反应混合物以产生具有悬伸的产品


      5. 准备以下反应混合物,将PKR基因亚克隆到pGEM-T Easy载体中, 


      6. 将连接反应混合物在4℃下孵育过夜。
    3. 转化为E。大肠杆菌
      1. 解冻一击TOP 10化学感受态细胞在冰上3分钟。
      2. 向细胞中加入5μl连接产物
      3. 在冰上孵育管30分钟。
      4. 将管置于室温下10分钟
      5. 将50μlβ-半乳糖苷(X-Gal)(50mg/ml)与150μlSOC培养基混合,并将混合物加入含有氨苄青霉素的LB平板上。
      6. 用氨苄青霉素在LB平板上转染转化细胞。
      7. 将板在37℃孵育过夜。
      8. 选择白色菌落检查PKR基因的连接是否成功。
    4. 将PKR基因消化和连接到pET-28a +中
      1. 设置 Sal I和我对pET-28a +和PKR的消化反应




      2. 在1%琼脂糖凝胶上分别运行双重消化产物pET-28a +和PKR。
        注意:分别在5,369 bp和1,656 bp处寻找pET-28a和PKR的条带。
      3. 使用干净的剃须刀刀片小心地从凝胶上切除带子。
      4. 使用QIAGEN凝胶提取试剂盒进行凝胶提取。
      5. 称取管中的凝胶切片,并加入3体积缓冲液QG至1体积凝胶
      6. 在50°C孵育10分钟。
      7. 向样品中加入1体积异丙醇并涡旋
      8. 将样品转移到QlAquick色谱柱,并以18,000 x g离心1分钟。
      9. 向QlAquick柱中加入750ml缓冲液PE,并以18,000 x g离心1分钟。
      10. 加入50μl缓冲液EB到QlAquick膜的中心洗脱DNA
      11. 建立连接反应,将PKR基因克隆到pET-28a质粒中

      12. 在16℃孵育过夜,并将2μl反应转化成25μl感受态细胞
  2. 来自E的PKR蛋白质纯化。大肠杆菌
    1. 在LB培养液中在37℃下培养细胞,直到OD 600达到0.4-0.6
    2. 用40μl100 mM IPTG(终浓度400μM)在37°C诱导3〜5 h。
    3. 收获细胞并立即进行细胞裂解或将液体冷冻细胞,然后将其储存在-80°C。
    4. 将细胞重悬于裂解缓冲液中,每克湿重为2-5毫升
    5. 用细胞加入溶菌酶(1 mg/ml)到裂解缓冲液,并在冰上孵育30分钟。
    6. 使用配有微尖头的超声波仪器在冰上超声波测量。
      注意:在每次突发之间,以30秒的时间间隔使用5秒15秒的冷却时间。
    7. (可选)如果裂解物非常粘稠,加入RNase A(10μg/ml)和DNase I(5μg/ml),并在冰上孵育10-15分钟。
    8. 在4℃下以10,000×g离心裂解物20-30分钟以沉淀细胞碎片并保持上清液。
    9. 加入5μl2x LDS-PAGE样品缓冲液至5μl上清液,储存于-20°C进行LDS-PAGE分析。
    10. 移取10ml的Ni-NTA浆液至50ml管中,并短暂离心。除去上清液并加入20 ml裂解缓冲液。反转轻轻混合。重复离心步骤并除去上清液 注意:根据目标蛋白质的产量调整使用的Ni-NTA量。
    11. 将澄清的裂解物加入到平衡的基质中,并在4℃下摇动1小时
    12. 将10ml裂解物-NN-NTA混合物装入柱中
    13. 取下底盖并收集流通。保存并储存用于Western印迹。
    14. 用洗涤缓冲液洗10次。收集洗涤系统进行Western印迹。
    15. 用1ml洗脱缓冲液洗脱蛋白质4次
    16. 在4℃下,在储存缓冲液中进行一次透析。
    17. 用蒸馏水润湿透析管,用一个夹子靠近一端关闭管子。
    18. 将样品装入透析管并夹住另一端。
    19. 将打结的管子放入储存缓冲液中,放入500ml烧杯中过夜
    20. 通过Qubit蛋白测定法测定蛋白质浓度,并检查蛋白质的纯度(图1)

      图1.纯化的PKR蛋白的银染色。 纯化的PKR蛋白(65kDa)通过银染色显现。加入IPTG以诱导E中的蛋白质表达。大肠杆菌。使用不含IPTG诱导细胞的裂解物作为阴性对照
  3. PKR去磷酸化和活化
    人PKR蛋白由来自E的RNA激活。大肠杆菌细胞裂解后;因此,蛋白质去磷酸化是激活测定前获得非磷酸化形式的PKR所必需的
    1. PKR的脱磷酸化
      1. 在10μl磷酸酶反应缓冲液中,在30℃下孵育2μg具有400Uλ-PPase(1μl)的PKR 1小时。
      2. 将去磷酸化的PKR等分成10个管,每个管含有0.2μgPKR蛋白质
      3. 加入新制备的2mM原钒酸钠以抑制λ-PPase
    2. PKR活化分析
      1. 从储备(作为阳性对照)(10 mg/ml)准备10μg/ml Poly I:C
      2. 将反应混合物置于冰上。


      3. 用10ng Poly I:C或dsRNA在PKR活化缓冲液中于30℃孵育脱磷酸化的PKR 0.5-2h。
      4. 加入1x LDS加载缓冲液与5%β-巯基乙醇停止反应
      5. 进行蛋白质印迹以通过探测p-PKR水平达到总PKR来确定PKR激活
      6. 通过NuPAGE 4-12%Bis-Tris蛋白凝胶电泳样品。
      7. 将解析的蛋白质在4℃在100V下将固定-P聚偏二氟乙烯(PVDF)膜转移90分钟。
      8. 用0.5%Tween-20磷酸缓冲盐水(PBST)中的1%牛血清白蛋白封闭膜60分钟。
      9. 在4℃下将膜与一抗孵育过夜。
      10. 用PBST洗三次,每次5分钟。
      11. 将辣根过氧化物酶缀合的二抗体以1:3,000稀释度在室温下加热1小时
      12. 用PBST洗三次,每次5分钟。
      13. 添加增强化学发光检测系统(ECL试剂)
      14. 用台风扫描仪扫描膜片(图2)

        图2.体外PKR激活的代表性结果。使用PKR激活测定法研究从WT分离的不同类型的RNA(mRNA,小RNA和rRNA) > NIPBL -MS(错义)人类淋巴母细胞样细胞系(LCL)。从NIPBL-MS LCLs分离的ncRNA和rRNA都能够在体外激活重组PKR;使用10ng聚I:C作为PKR活化的阳性对照。 * 0.001与未处理对照相比。 (详情请参阅Yuen等等,,2016)

数据分析

所有实验至少一式三份重复,数据以平均值±SD表示。统计学意义使用学生的测试来确定。 A

值< 0.05被认为具有统计学意义。本协议中描述的实验已经完成,其数据已经在Yuen等人发布,2016年,NIPBL控制RNA生物发生以防止应激激酶PKR的激活。 Cell Rep 14(1):93-102,可以通过以下方式访问: http://www.cell.com/cell-reports/fulltext/S2211-1247(15)01425-4


食谱

  1. E。大肠杆菌裂解缓冲液(1L)
    50mM NaH 2 PO 4
    300 mM NaCl
    10 mM咪唑
    1 mM PMSF
    10 mM蛋白酶抑制剂
    使用NaOH调节pH至8.0
  2. 蛋白纯化洗涤缓冲液(1升)
    50mM NaH 2 PO 4
    300 mM NaCl
    20mM咪唑
    1 mM PMSF
    10 mM蛋白酶抑制剂
    使用NaOH调节pH至8.0
  3. 蛋白纯化洗脱缓冲液(1升)
    50mM NaH 2 PO 4
    300 mM NaCl
    250毫克咪唑
    1 mM PMSF
    10 mM蛋白酶抑制剂
    使用NaOH调节pH至8.0
  4. 蛋白质储存缓冲液
    10mM Tris(pH 7.6)
    50 mM KCl
    2mM MgCl 2
    10%甘油
    7mMβ-巯基乙醇
  5. 磷酸酶反应缓冲液
    50mM Tris-HCl,pH7.5
    2mM MgCl 2
    0.1 mM EDTA
    5 mM DTT
  6. PKR激活缓冲区
    20 mM HEPES(pH 7.5)
    4mM MgCl 2
    100 mM KCl
    1 mM ATP
  7. TE缓冲区(10x)
    100mM Tris-Cl
    10 mM EDTA(pH 8.0)
  8. 吐温-20磷酸盐缓冲盐水(pH7.2)(1L)
    8克NaCl
    0.2克KCl
    1.44g的Na 2 HPO 4
    0.24g KH 2 PO 4
    2毫升Tween-20

致谢

感谢Jennifer Gerton博士阅读这个协议。本研究得到了Stowers医学研究所,Cornelia de Lange综合征(CdLS)基金会和3月份的Dimes(MOD)基金会(6-FY14-434)的支持。

参考文献

  1. Bevilacqua,PC,George,CX,Samuel,CE和Cech,TR(1998)。蛋白激酶PKR与具有二级结构缺陷的RNA的结合:串联AG失配和非连续螺旋的作用。生物化学 37(18):6303-6316 。
  2. Hinnebusch,AG(2005)。eIF2α激酶提供新的解决了底物特异性的难题。 Nat Struct Mol Biol 12(10):835-838。
  3. Holcik,M。和Sonenberg,N。(2005)。应激和细胞凋亡中的翻译控制。 Nat Rev Mol Cell Biol 6(4):318-327。
  4. Manche,L.,Green,SR,Schmedt,C.and Mathews,MB(1992)。< a class ="ke-insertfile"href ="http://mcb.asm.org/content/12/11/5238.short"target ="_ blank">双链RNA调节剂和蛋白激酶DAI之间的相互作用。分子细胞沸腾 12(11):5238-5248。 >
  5. Nallagatla,SR和Bevilacqua,PC(2008)。  NIPBL控制RNA生物发生以防止应激激酶PKR的激活。细胞Rep 14(1):93-102。
  6. Zheng,X.和Bevilacqua,PC(2004)。  通过具有单链尾的短双链RNA激活蛋白激酶PKR。 10(12):1934-1945。
  • English
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yuen, K. C. (2017). Measurement of RNA-induced PKR Activation in vitro. Bio-protocol 7(6): e2178. DOI: 10.21769/BioProtoc.2178.
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