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Measurement of RNA-induced PKR Activation in vitro

RNA诱导的PKR活化的体外测定

Kobe C. Yuen Kobe C. Yuen

发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2178 浏览次数: 7865

评审: Antoine de MorreeQiangjun ZhouAnonymous reviewer(s)

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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) search Stress response (应激反应) search Innate immunity (先天免疫) search Aberrant RNA (异常RNA) search

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

English
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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

生物化学 > 蛋白质 > 活性

生物化学 > RNA > RNA-蛋白质相互作用

分子生物学 > 蛋白质 > 去磷酸化

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