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RNA Cap Methyltransferase Activity Assay
RNA帽结构的甲基转移酶活性测定   

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Nucleic Acids Research
Sep 2017

Abstract

Methyltransferases that methylate the guanine-N7 position of the mRNA 5’ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [32P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments.

Keywords: RNA (RNA), 5’ Cap ( 5’端帽结构), Cap methyltransferase (帽结构的甲基转移酶), RNMT (RNMT), Enzyme activity assay (酶活性测定), Subcellular fractionation (亚细胞分离), P1 nuclease (P1核酸酶), Thin-layer chromatography (薄层色谱法)

Background

The N7-methylguanosine cap at the 5’ end of an mRNA is a modification essential for proper eukaryotic mRNA processing, localization, and translation. The N7 methyl group is particularly critical for the mRNA life cycle, as it drastically increases the binding affinity of cap-binding proteins (Niedzwiecka et al., 2002) and protects mRNAs from cap-quality control surveillance mechanisms (Jiao et al., 2013). We recently reported that the mammalian RNA guanine-7 methyltransferase (RNMT) functions beyond its canonical role in nuclear co-transcriptional cap synthesis to participate in cytoplasmic RNA recapping (Trotman et al., 2017). We used the protocol presented here to demonstrate that the cap methyltransferase activity of cytoplasmic RNMT is unexpectedly robust relative to nuclear RNMT. Additionally, siRNA-mediated knockdown of RNMT greatly reduced the cap methyltransferase activity of cytoplasmic extracts, suggesting that RNMT is the predominant, if not only cap methyltransferase in the cytoplasm of mammalian cells. Nuclear RNMT exists as a heterodimer with RNMT-activating miniprotein (RAM, Gonatopoulos-Pournatzis et al., 2011), and we demonstrated that cytoplasmic RNMT also binds to RAM. Reduced cytoplasmic cap methyltransferase activity upon RAM knockdown indicated that RAM is a required cofactor for cytoplasmic RNMT.

This protocol is adapted from two earlier publications characterizing human RNMT (Cowling, 2010; Pillutla et al., 1998), with modifications that standardize generation of the substrate RNA, avoid cumbersome phenol-chloroform extractions with radioactive samples, and enable quantification of cap methyltransferase activity. We note that an alternative, nonradioactive assay has been reported for the analysis of cap methyltransferase reactions (Peyrane et al., 2007), but this method requires HPLC instrumentation that may not be available to all labs and may differ from the one presently reported in terms of sensitivity and sample compatibility. Additionally, a fluorescent assay for measuring cap methyltransferase activity was recently described (Aouadi et al., 2017), but this assay indirectly measures activity by monitoring the accumulation of S-adenosylhomocysteine (SAH) and may be incompatible with biological samples containing SAH. We hope that the level of detail in the protocol presented here enables future investigators to easily repeat and build upon our work.

Materials and Reagents

  1. 0.2 ml PCR tubes (e.g., BioExpress, GeneMate, catalog number: T-3225-1 )
  2. NucAway Spin Columns (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM10070 )
  3. 1.7 ml plastic, sterile, RNase-free microcentrifuge tubes (e.g., BioExpress, GeneMate, catalog number: C-3262-1 )
  4. 10 cm or 15 cm culture dishes (e.g., Alkali Scientific, catalog numbers: TD0100 , TD0150 )
  5. Sterile nitrile gloves
  6. Cell lifters (e.g., BioExpress, GeneMate, catalog number: T-2443-4 )
  7. Sterile, RNase-free tips for P10, P20, P100, and P1000 pipets
  8. Plastic, disposable cuvettes for Bradford assay (e.g., Fisher Scientific, catalog number: 14-955-127 )
  9. Plastic wrap (e.g., Saran or Stretch-Tite brand)
  10. Paper labeling tape (e.g., Fisher Scientific, catalog number: 15-901-20H)
    Manufacturer: Nevs, catalog number: 1590120H .
  11. Polyethylenimine (PEI) cellulose TLC plates (Macherey-Nagel, catalog number: 801053 ; see Note 2)
  12. Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Merck, catalog number: IPFL00010 )
  13. Scintillation vials compatible with liquid scintillation counter (e.g., DWK Life Sciences, WHEATON, catalog number: 225414 )
  14. U2OS cells (ATCC, catalog number: HTB-96 ) or HEK293 cells (ATCC, catalog number: CRL-1573 )
  15. P1 nuclease (United States Biological, catalog number: N7000 ), resuspended in RNase-free water to 0.625 U/μl
  16. Cap analog GpppG (New England Biolabs, catalog number: S1407 ), resuspended in RNase-free water to 10 mM
  17. Cap analog m7GpppG (New England Biolabs, catalog number: S1404 ), resuspended in RNase-free water to 10 mM
  18. RNase-free water (e.g., from a Millipore Synergy water purification system, 18.2 MΩ cm)
  19. Single-stranded DNA sense oligo
  20. Single-stranded DNA antisense oligo
  21. 5’ CATGCAAATTAACCCTCACTAAAGGGAGACCGGAATTCGAGCTCGCCCGGGGATC 3’ for T3 transcription template, resuspended in RNase-free water to 100 μM (e.g., synthesized by Integrated DNA Technologies (IDT); underlined is a T3 promoter sequence, bold sequence matches the transcribed 32-nucleotide (nt) pppRNA)
  22. 5’ GATCCCCGGGCGAGCTCGAATTCCGGTCTCCCTTTAGTGAGGGTTAATTTGCATG 3’ for T3 transcription template, resuspended in RNase-free water to 100 μM (e.g., synthesized by IDT)
  23. MEGAscript T3 Transcription Kit (Thermo Fisher Scientific, Ambion, catalog number: AM1138 )
  24. Pre-cast polyacrylamide mini-gels for urea polyacrylamide gel electrophoresis (urea-PAGE; e.g., Bio-Rad Laboratories, catalog number: 4566053 )
  25. Pre-cast polyacrylamide mini-gels for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; e.g., Bio-Rad Laboratories, catalog number: 4568094 )
  26. 2x Laemmli Sample Buffer (Bio-Rad Laboratories, catalog number: 1610737 )
  27. SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific, InvitrogenTM, catalog number: S11494 )
  28. RNA size marker (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: AM7778 )
  29. 2x RNA loading dye (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0641 )
  30. 40 U/μl RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777019 )
  31. Recombinant triphosphatase-guanylyltransferase capping enzyme (see Note 1)
  32. [α-32P]GTP (3,000 Ci/mmol, PerkinElmer, catalog number: BLU506H250UC )
  33. RNA Clean & Concentrator-5 kit (Zymo Research, catalog number: R1016 )
  34. ScintiSafe Econo 1 scintillation fluid (Fisher Scientific, catalog number: SX20-5 )
    Note: This product has been discontinued.
  35. McCoy’s 5A medium (for U2OS cells; Thermo Fisher Scientific, GibcoTM, catalog number: 16600082 )
  36. Dulbecco’s modified Eagle medium (DMEM; for HEK293 cells; Thermo Fisher Scientific, GibcoTM, catalog number: 21013024 )
  37. Fetal bovine serum (FBS; Atlanta Biologicals, catalog number: S10350 )
  38. Phosphate-buffered saline (PBS; e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 10010049 )
  39. Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
  40. Pre-stained protein marker (e.g., Bio-Rad Laboratories, catalog number: 1610393 )
  41. Bovine serum albumin (BSA; e.g., Fisher Scientific, catalog number: BP1600-100 ) dissolved in water to 1 μg/μl
  42. Antibody toward nuclear protein for Western blotting (e.g., rabbit polyclonal anti-nucleolin antibody, 1:5,000 working dilution, Sigma-Aldrich, catalog number: N2662 )
  43. Antibody toward cytoplasmic protein for Western blotting (e.g., mouse monoclonal anti-α-tubulin, 1:10,000 working dilution, Sigma-Aldrich, catalog number: T6199 )
  44. Appropriate secondary antibodies (e.g., for infrared imaging, Thermo Fisher Scientific, Invitrogen, catalog numbers: A-21109 and A-21058 , at 1:10,000 working dilutions)
  45. (Optional) Vaccinia Capping Enzyme (New England Biolabs, catalog number: M2080S )
  46. Dithiothreitol (DTT, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
  47. RNase-free 10x Tris/borate/EDTA (TBE) buffer (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: 15581 )
  48. Phenylmethylsulfonyl fluoride (PMSF; Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 36978 )
  49. Isopropanol
  50. Tris (e.g., VWR, AMRESCO, catalog number: 0497 )
  51. Glycine (e.g., VWR, AMRESCO, catalog number: 0167 )
  52. SDS
  53. Methanol (e.g., Fisher Scientific, catalog number: A452SK-4 )
  54. Sodium chloride (NaCl; e.g., Fisher Scientific, catalog number: BP358 )
  55. 5 M NaCl (e.g., Thermo Fisher Scientific, catalog number: AM9759 )
  56. Concentrated HCl
  57. Tween 20 (e.g., Fisher Scientific, catalog number: BP337 )
  58. 32 mM S-adenosylmethionine (SAM; New England Biolabs, catalog number: B9003 )
  59. 3 M sodium acetate, pH 5.2 (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R1181 )
  60. Ammonium sulfate (e.g., Sigma-Aldrich, catalog number: A4418 )
  61. IGEPAL CA-630 (Sigma-Aldrich, catalog number: I8896 )
  62. 1 M Tris-HCl, pH 7.5 (e.g., AMRESCO, catalog number: E691 )
  63. 1 M magnesium chloride (MgCl2, e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9530G )
  64. 500 mM ethylenediaminetetraacetic acid, pH 8.0 (EDTA; e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575020 )
  65. Glycerol (e.g., Fisher Scientific, catalog number: BP229-1 )
  66. 1 M HEPES pH 7.3 (e.g., AMRESCO, catalog number: J848 )
  67. 2 M potassium chloride (KCl; e.g., Alfa Aesar, catalog number: J75896 )
  68. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
  69. Phosphatase inhibitor cocktail 2 (Sigma-Aldrich, catalog number: P5726 )
  70. Phosphatase inhibitor cocktail 3 (Sigma-Aldrich, catalog number: P0044 )
  71. 1 M DTT (see Recipes)
  72. 1x Tris-buffered saline (TBS; see Recipes)
  73. 1x TBE (running buffer for urea-PAGE) (see Recipes)
  74. 20 mM DTT (see Recipes)
  75. 100 mM PMSF in isopropanol (see Recipes)
  76. 10x Tris/glycine (see Recipes)
  77. 10% (w/v) SDS (see Recipes)
  78. Tris/glycine/SDS running buffer for SDS-PAGE (see Recipes)
  79. Tris/glycine/methanol/SDS transfer buffer (see Recipes)
  80. 20x Tris-buffered saline (TBS) (see Recipes)
  81. 3% BSA in TBS (see Recipes)
  82. 40% (v/v) Tween 20 (see Recipes)
  83. TBS-T (see Recipes)
  84. 1 μM SAM (see Recipes)
  85. 500 mM sodium acetate, pH 5.2 (see Recipes)
  86. 0.4 M ammonium sulfate
  87. 10% IGEPAL CA-630 (v/v) in water (see Recipes)
  88. 10x annealing buffer (see Recipes)
  89. 4x capping buffer (see Recipes)
  90. YO Lysis Buffer (see Recipes)
  91. YO Buffer A (see Recipes)
  92. 10x cap methylation buffer (see Recipes)

Equipment

  1. Eye protection
  2. NanoDrop spectrophotometer (Thermo Fisher, model: NanoDropTM 1000 , catalog number: ND-1000)
  3. Lucite/Plexiglass acrylic benchtop shielding for handling 32P (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 6700-2418 )
  4. Geiger counter
  5. Metric (centimeter) ruler
  6. Scissors (e.g., Westcott, catalog number: ACM44217 )
  7. Graphite pencil
  8. Long (at least 18 cm) forceps or tongs (e.g., Fisher Scientific, catalog number: 15-186 )
  9. P10, P20, P100, and P1000 pipets
  10. Thermal cycler (e.g., MJ Research, model: PTC-200 )
  11. Heating block (e.g., Bioer, model: MB 101 )
  12. -80 °C freezer
  13. Handheld 254 nm UV light (e.g., UVP, model: 95-0016-14 )
  14. Liquid scintillation counter (e.g., Beckman Coulter, model: LS 6000IC )
  15. Water-jacketed incubator (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: Forma® Series II) set to 37 °C with 5% CO2
  16. Refrigerated centrifuge (Eppendorf, model: 5415 R )
  17. Programmable rotator-mixer (Grant Instruments, model: PTR-30 ) at 4 °C and set to 10 rpm orbital rotation
  18. UV-vis spectrophotometer (e.g., Beckman Coulter, model: DU 640 ) set to 595 nm
  19. Electrophoresis system for PAGE and membrane transfer (e.g., Bio-Rad Laboratories, catalog number: 1660828EDU )
  20. Opaque western blot incubation boxes (e.g., LI-COR, catalog number: 929-97205 )
  21. Western blot imaging system (e.g., LI-COR, model: Odyssey Imaging Systems )
  22. Rectangular glass TLC chamber (e.g., Miles Scientific, model: A70-22 , formerly Analtech)
  23. Electric hair dryer (optional)
  24. Storage phosphor screen (e.g., GE/Amersham Biosciences)
  25. Light eraser for storage phosphor screen (e.g., Molecular Dynamics Image Eraser)
  26. Typhoon imaging system (e.g., GE Healthcare, Amersham Biosciences, model: Typhoon 9200 )

Software

  1. Microsoft Excel software
  2. ImageQuant TL software

Procedure

A schematic overview of this protocol is presented in Figure 1. In short, an in-vitro-transcribed short RNA is guanylylated using [α-32P]GTP to generate a Gp*ppRNA substrate, with the radiolabeled phosphate indicated by an asterisk. This GpppRNA substrate is briefly incubated with nuclear or cytoplasmic extracts (or other biological samples) and SAM. Different levels of cap methyltransferase activity in each extract will result in differential N7-methylation of the GpppRNA substrate to produce m7GpppRNA. The mixtures of GpppRNA and m7GpppRNA are then digested to individual nucleotides with P1 nuclease, leaving intact the radiolabeled cap dinucleotide structures that are finally analyzed by TLC and phosphorimaging.


Figure 1. Schematic overview of cap methyltransferase activity assay

  1. Preparation of radiolabeled GpppRNA substrate for cap methyltransferase activity assay (see Note 3)
    1. Prepare a 30 μl mixture in a 0.2 ml PCR tube by combining the following: 15 μl of RNase-free water, 3 μl of 10x annealing buffer (see Recipes), 6 μl of 100 μM ssDNA sense oligo, 6 μl of 100 μM ssDNA antisense oligo.
    2. To denature and then slowly anneal the two oligos to create the dsDNA template for T3 transcription, place the mixture on a thermal cycler at 98 °C for 2 min, and ramp the temperature from 98 °C to 20 °C in 60 min (1.3 °C per min, down to 20 °C). Transfer this tube of dsDNA onto ice, and in a separate tube, prepare a 1 μM (1 pmol/μl) working stock by diluting it 20-fold with RNase-free water.
    3. Using the components provided in the MEGAscript T3 Transcription Kit, prepare a 20 μl reaction in a 0.2 ml PCR tube by combining in the following order: 6 μl of RNase-free water, 2 μl of 75 mM ATP, 2 μl of 75 mM CTP, 2 μl of 75 mM GTP, 2 μl of 75 mM UTP, 2 μl of 10x T3 reaction buffer, 2 μl of 1 μM dsDNA template, 2 μl of T3 RNA polymerase enzyme mix. Gently mix, and incubate at 37 °C for 16-24 h. This prolonged reaction time enables transcription of high yields of 32 nt 5’ triphosphate RNA (pppRNA).
    4. Bring the T3 transcription reaction product mixture to 50 μl by adding 30 μl of RNase-free water, and purify using a NucAway Spin Column according to manufacturer’s instructions.
    5. Measure the concentration of the purified pppRNA using a NanoDrop. In our experience, at least 7-8 μg of pppRNA should be recovered after a 24 h transcription reaction. Calculate the molar concentration of the pppRNA assuming a molecular mass of 10,830.3 g/mol.
    6. Further assess the purity of the pppRNA by 15% polyacrylamide urea-PAGE. As little as 150 fmol (1.6 ng) of pppRNA per lane should be sufficient to visualize under UV light after post-staining the gel with SYBR Gold at a dilution of 1:20,000 in 1x TBE. The pppRNA should run as a single band at the expected size.
    7. The pppRNA should be stored in small aliquots at -20 °C or -80 °C to avoid multiple freeze-thaw cycles.
    8. To prepare a 40 μl capping (guanylylation) reaction, combine in order the following components in a 1.7 ml tube on ice: RNase-free water (for 40 μl total volume), 10 μl of 4x capping buffer (see Recipes), 1 μl of RNaseOUT, 4.8 pmol of 32 nt pppRNA, 2 pmol of recombinant capping enzyme (see Note 1), 24 pmol (total GTP) of [α-32P]GTP (see Note 4). Gently mix and incubate at 30 °C for 3 h, then heat-inactivate by incubating at 65 °C for 10 min.
    9. Transfer 1.33 μl (1/30) of the capping reaction product to a new tube on ice to save as ‘input’ for scintillation counting. Bring the capping reaction to 50 μl by adding 11.33 μl of RNase-free water. Purify the RNA using the Zymo RNA Clean & Concentrator-5 kit according to manufacturer’s instructions, using 30 μl of RNase-free water for the final elution step. Return the purified RNA to ice, and transfer 1 μl (1/30) to a new tube on ice for scintillation counting.
    10. Mix the set-aside 1/30 capping reaction ‘input’ and purified RNA samples each with 1.5 ml of ScintiSafe Econo 1 scintillation fluid and transfer to scintillation vials. Measure the radioactivity in each mixture by scintillation counting. Determine the concentration of purified [32P]G-capped RNA according to the following equation: [GpppRNA] (in μM) = (24 pmol) x (purified counts/input counts)/(30 μl).
    11. The purified [32P]G-capped RNA may be stored at -20 °C but should be used within a few days to limit radioactive decay. Be sure to account for radioactive decay when determining the appropriate amount to use in the cap methyltransferase activity assay.

  2. Preparation of nuclear and cytoplasmic extracts of mammalian cells
    1. Two to three days in advance, seed 10-cm or 15-cm dishes with U2OS, HEK293, or other type of adherent mammalian cells such that the dishes will be ~80-90% confluent at the time of harvesting. U2OS cells should be grown in McCoy’s 5A medium containing 10% (v/v) FBS; HEK293 cells should be grown in DMEM containing 10% (v/v) FBS. Cells should be grown in an incubator set to 37 °C with 5% CO2.
    2. On the day of harvesting the cells, prepare YO Lysis Buffer and YO Buffer A (see Recipes) on ice, leaving out the DTT, PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktails until immediately before using the buffers.
    3. Remove culture medium from adherent cells, rinse with PBS and remove, and then harvest by scraping into 1 ml of PBS and transferring to 1.7 ml centrifuge tubes. Gently pellet the cells (70 x g for 10 min at 4 °C), and then carefully remove the supernatant PBS by aspiration. Make note of the approximate volume of the cell pellets, and place the tubes on ice.
    4. For the preparation of cytoplasmic extracts, immediately add YO Lysis Buffer equivalent to 4-5 times the volume of each cell pellet. Gently resuspend the cells with 10 up-down strokes of a P1000 pipet. Incubate the cells on ice for 10 min, and then mix with 5 additional up-down strokes of the P1000 pipet. Pellet nuclei (16,100 x g for 10 min at 4 °C), and then transfer the cytoplasmic extract supernatants to new, pre-chilled 1.7 ml tubes on ice.
    5. Carefully remove any residual cytoplasmic extract from the nuclear pellets using a P10 pipet. To prepare nuclear extracts, add 4-5 original-cell-pellet volumes of YO Buffer A, and resuspend each nuclear pellet with 15 up-down strokes of a P200 pipet. Incubate the tubes end-over-end at 4 °C for 20 min, and then pellet nuclear debris (16,100 x g for 5 min at 4 °C). Transfer the nuclear extract supernatants to new, pre-chilled 1.7 ml tubes on ice.
    6. Measure the concentration of protein in each extract by Bradford assay, using the Bio-Rad Protein Assay Dye Reagent Concentrate according to manufacturer’s instructions and using a standard curve of bovine serum albumin (BSA) at 0, 1, 2, 4, 6, and 8 μg/ml. Diluting the extracts 250- to 500-fold in the 1x dye reagent typically gives measurements within the linear range of the assay. Using a program such as Microsoft Excel, calculate the protein concentrations using the best-fit line of the BSA standard curve.
    7. For the cap methyltransferase activity assay, prepare 15-20 μl samples of each extract in new, pre-chilled tubes on ice. If comparing nuclear and cytoplasmic extracts, it is critical to ensure that these are in the same buffer composition. This can be accomplished by first bringing the extracts all to the same protein concentration (typically 0.5-1.0 μg/μl; such calculations can be greatly aided by software such as Microsoft Excel) with the same buffer used prior (i.e., YO Lysis Buffer for cytoplasmic extracts and YO Buffer A for nuclear extracts) and then mixing each sample with an equal volume of the other buffer (i.e., YO Buffer A for cytoplasmic extracts and YO Lysis Buffer for nuclear extracts). Store these extract samples in small aliquots at -80 °C.
    8. Assess the quality of subcellular fractionation by running the nuclear and cytoplasmic extracts on SDS-PAGE followed by Western blot analysis. The use of nuclear and cytoplasmic control antibodies (e.g., against nucleolin and tubulin) can determine whether cross-contamination occurred during the fractionation procedure. For an example of expected Western blot results, see Figure 1C of Trotman et al., 2017.

  3. Preparation of TLC plate for cap methyltransferase activity assay
    1. Wearing clean gloves, use a clean ruler and scissors to cut a 20 x 20 cm PEI cellulose TLC plate into four 10 x 10 cm squares. We recommend using clean forceps to handle the plate to limit contact with any contaminants that may be present on gloves. Store any unused plates at 4 °C. See Note 2 regarding TLC performance.
    2. Use the ruler and a pencil to lightly draw a faint, straight line (origin) 1.5 cm from the bottom edge of a 10 x 10 cm plate. Draw faint tick marks on the origin 1 cm apart to indicate where samples will be spotted. An example TLC plate is shown in Figure 2.


      Figure 2. Example of TLC plate preparation

    3. Pre-run the TLC plate with RNase-free water. To do so, fill a rectangular glass TLC chamber with just enough water to cover the bottom (approximately 60 ml in the model listed above) such that the water depth does not exceed 0.75 cm. Using long forceps, gently place the TLC plate upright in the water. Take care to ensure that the left and right edges enter the water simultaneously, as this will promote even migration of water up the TLC plate. This pre-running step forces contaminants to the top of the TLC plate and greatly improves performance for the subsequent chromatographic analysis of the cap methyltransferase activity assay samples.
    4. Once water has reached the top of the TLC plate, remove the TLC plate from the chamber and allow it to air-dry. Shaking the plate by hand or using a commercial hair dryer (without heating) can help speed up this process.
    5. Pre-run TLC plates can be stored at 4 °C for at least 2 weeks.

  4. Cap methyltransferase activity assay
    1. After thawing components on ice, prepare 10 μl cap methyltransferase reactions in 1.7 ml tubes on ice by combining the following in order: RNase-free water (for 10 μl total volume), 1 μl of 10x cap methylation buffer (see Recipes), 1 μl of 1 μM SAM, 1 μl of 20 mM DTT, 1 μl of RNaseOUT, nuclear or cytoplasmic extract for 1 μg of total protein, 5 fmol of [32P]G-capped RNA. Using a master mix of the reaction components (omitting the extracts) is recommended if working with a large number of extract samples. Gently mix, incubate at 37 °C for 30 min, and then immediately return to ice.
    2. Add 40 μl of RNase-free water to each tube and purify the RNA samples using the Zymo Clean & Concentrator-5 kit according to manufacturer’s instructions, using 7 μl RNase-free water for the final elution step. Place the purified RNA on ice.
    3. Prepare 7 μl P1 nuclease digestion reactions in 1.7 ml tubes on ice by combining the following in order: 5.0 μl of purified RNA from cap methyltransferase reactions, 0.6 μl of RNase-free water, 0.7 μl of 500 mM sodium acetate pH 5.2, 0.7 μl of 0.625 U/μl P1 nuclease. Gently mix and incubate at 37 °C for 30 min. The samples may now be stored at -20 °C or analyzed immediately.
    4. On the 1 cm tick marks on the origin of a pre-run PEI cellulose TLC plate, carefully spot 1 μl of the P1 nuclease digestion products using a P10 pipet. Allow the spots to completely air-dry, and then spot an additional 1 μl of the P1 digestion products on the same positions as before. As controls, also spot 2 μl (1 μl at a time) of 10 mM GpppG and 10 mM m7GpppG standards on a separate tick mark or marks (see Note 5).
    5. After allowing the spots to completely air-dry, use long forceps to evenly place the TLC plate upright in a rectangular TLC chamber containing approximately 60 ml 0.4 M ammonium sulfate mobile phase. Allow the mobile phase to develop upward, approximately 6-7 cm beyond the origin, and then remove the TLC plate from the chamber. Allow the TLC plate to completely air-dry, which can be sped up by hand-shaking or using a hair dryer without heat.
    6. Wearing proper eye and skin protection, observe the TLC plate under a handheld 254 nm UV light to determine the positions of the GpppG and m7GpppG standards. These can be noted by marking their positions with a pencil or by taking a photo with a handheld camera.
    7. Wrap the TLC plate in plastic wrap so that the front side is covered by one layer. Place the TLC plate in a storage phosphor screen cassette, taping the top edge to the cassette to keep it in place. Place a recently light-erased storage phosphor screen over the TLC plate, close the cassette, and expose at room temp for at least 16 h for maximum signal.
    8. Remove the TLC plate from the cassette and image the storage phosphor screen with a Typhoon imager set to phosphor mode. Typical results are shown in Figure 3.


      Figure 3. Typical phosphor image results. To demonstrate reproducibility, four technical replicates of the described activity assay were performed with U2OS cell nuclear and cytoplasmic extracts.

Data analysis

The extent of conversion of GpppRNA to m7GpppRNA during a cap methyltransferase reaction (and by extension, the cap methyltransferase activity present in the corresponding biological sample) is represented by the ratio of the m7GpppG spot intensity to the sum of the GpppG and m7GpppG spot intensities on the TLC plate phosphor image. The spot intensities can be determined using any image software that includes densitometry analysis, such as ImageQuant TL. To ensure consistent calculation of spot intensities for multiple samples, use lanes or boxes of the same size that are large enough to just fit the entire spot for densitometry analysis. Also ensure that any background signal is subtracted; in ImageQuant TL, we use the ‘rubberband’ setting for establishing the baseline for peak signal integration. To account for biological variability among samples when comparing multiple conditions, it is good practice to perform this assay with at least three biological replicates to enable appropriate statistical testing.

Notes

  1. The recombinant human capping enzyme we used to generate the radiolabeled GpppRNA was produced in-house as described in Trotman et al. (2017). Despite this enzyme stock having robust capping activity with non-radiolabeled GTP, the production of radiolabeled GpppRNA was rather inefficient. If a recombinant capping enzyme is not available, we recommend using a commercially available enzyme, such as the Vaccinia Capping Enzyme (NEB, catalog #M2080S), to produce the GpppRNA as was used in previous reports (Pillutla et al., 1998; Cowling, 2010). If Vaccinia Capping Enzyme is used to generate GpppRNA, be sure to omit SAM from this reaction, as this enzyme also contains cap methyltransferase activity.
  2. Unlike most types of chromatographic systems, the flow rate of TLC is variable and cannot be easily adjusted to optimize performance according to the van Deemter Equation (van Deemter et al., 1956; Guiochon et al., 1979). In TLC, the capillary flow rate is dependent on properties such as the particle size of the TLC plate, which can vary substantially from manufacturer to manufacturer. We have thus found that some manufacturers’ PEI cellulose plates give streakier, more poorly resolved results than others, with the Macherey-Nagel plates listed here giving the best performance of those we have tested under the given conditions.
  3. Any short, in-vitro-transcribed RNA (with a 5’ triphosphate) should be suitable for generating the radiolabeled GpppRNA substrate, and this protocol can be modified to produce other GpppRNA substrates if desired. The protocol presented here simply describes an easy way to generate the same 32 nt substrate RNA used in the initial characterization of human RNMT (Pillutla et al., 1998) and in our recent study (Trotman et al., 2017).
  4. Use fresh [α-32P]GTP to maximize the ratio of radioactive-to-nonradioactive GTP in the stock, which will allow for greater signal intensity during phosphorimaging. To account for radioactive decay of the [α-32P]GTP stock, we recommend using the PerkinElmer radioactivity calculator (https://www.perkinelmer.com/tools/calculatorrad) to determine the concentration of total GTP on the day of its use.
  5. As an additional control for visualizing the position of m7GpppG and ensuring success of the assay, we suggest preparing a parallel cap methyltransferase activity reaction using a recombinant cap methyltransferase (such as Vaccinia Capping Enzyme from NEB).
  6. The radioisotope 32P emits beta particles that can be potentially harmful if not handled properly. To ensure safety while handling 32P, always wear eye and skin protection, work behind an acrylic bench shield, and use a Geiger counter to frequently check surfaces for contamination. We recommend using a dedicated set of pipets for use with radioactive materials in case they become contaminated. Store all radioactive samples at 4 °C or -20 °C in secondary acrylic storage boxes. Clean up any spills, no matter how small, and notify lab personnel and appropriate safety officials in the case of a sufficiently large spill. All radioactive waste should be placed in acrylic waste bins for at least 10 half-lives (about 143 days) to decay in storage. Contact your institution’s radiation safety office for additional information.

Recipes

  1. 1 M DTT
    Dissolve 1.54 g of DTT in RNase-free water to a total volume of 10 ml. Aliquot and store at -20 °C
  2. 1x TBE (running buffer for urea-PAGE)
    Bring 100 ml of RNase-free 10x TBE (Reagent #47) to 1 L with RNase-free water. Store at room temp.
  3. 20 mM DTT
    Bring 20 μl of 1 M DTT to 1 ml with RNase-free water. Aliquot and store at -20 °C
  4. 100 mM PMSF in isopropanol
    Dissolve 1.74 g of PMSF in isopropanol to a total volume of 10 ml. Aliquot and store at -20 °C
  5. 10x Tris/glycine
    Dissolve 30 g of Tris and 144 g of glycine in RNase-free water to a total volume of 1 l. Store at room temp.
  6. 10% (w/v) SDS
    Dissolve 10 g of SDS in RNase-free water to a total volume of 100 ml. Store at room temp.
  7. Tris/glycine/SDS running buffer for SDS-PAGE
    890 ml RNase-free water
    100 ml 10x Tris/glycine
    10 ml 10% (w/v) SDS
  8. Tris/glycine/methanol/SDS transfer buffer
    To 200 ml of methanol, add 500 ml of RNase-free water and then 100 ml of 10x Tris/glycine and 1 ml of 10% (w/v) SDS. Add RNase-free water to a total volume of 1 L and store at 4 °C
  9. 20x Tris-buffered saline (TBS)
    Dissolve 48.4 g of Tris and 160 g of NaCl in 800 ml of RNase-free water and adjust the pH to 7.6 with concentrated HCl. Bring the total volume to 1 L with RNase-free water and store at room temp.
  10. 3% BSA in TBS
    To 1.5 g of BSA, add 2.5 ml of 20x TBS, and dissolve in RNase-free water to a total volume of 50 ml. Store at 4 °C
  11. 40% (v/v) Tween 20
    Mix 20 ml Tween 20 with RNase-free water to a total volume of 50 ml. Store at room temp.
  12. TBS-T
    474 ml RNase-free water
    25 ml 20x TBS
    1.25 ml 40% (v/v) Tween 20
  13. 1 μM SAM
    Dilute 32 mM SAM 320-fold with RNase-free water to produce a 100 μM stock, and then dilute this 100 μM stock 100-fold with RNase-free water. Aliquot and store at -20 °C
  14. 500 mM sodium acetate, pH 5.2
    Dilute 3 M sodium acetate, pH 5.2 6-fold with RNase-free water. Store at room temp.
  15. 0.4 M ammonium sulfate
    Dissolve 52.86 g of ammonium sulfate in RNase-free water to 1 L. Store at room temp.
  16. 10% IGEPAL CA-630 (v/v) in water
    Add RNase-free water to 1 ml IGEPAL CA-630 to a total volume of 10 ml. Store at room temp.
  17. 10x annealing buffer
    100 mM Tris-HCl pH 7.5
    500 mM NaCl
  18. 4x capping buffer
    40 mM Tris pH 7.5
    12 mM MgCl2
    4 mM DTT
    0.4 mM EDTA
    80% (v/v) glycerol
  19. YO Lysis Buffer
    10 mM HEPES pH 7.3
    10 mM KCl
    10 mM MgCl2
    0.2% (v/v) IGEPAL CA-630
    2 mM DTT*
    0.5 mM PMSF*
    7.5 μl/ml protease inhibitor cocktail (Sigma-Aldrich)*
    7.5 μl/ml phosphatase inhibitor cocktail 2 (Sigma-Aldrich)*
    7.5 μl/ml phosphatase inhibitor cocktail 3 (Sigma-Aldrich)*
  20. YO Buffer A
    10 mM HEPES pH 7.3
    25% (v/v) glycerol
    420 mM NaCl
    1.5 mM MgCl2
    0.2 mM EDTA
    1 mM DTT*
    0.5 mM PMSF*
    7.5 μl/ml protease inhibitor cocktail (Sigma-Aldrich)*
    7.5 μl/ml phosphatase inhibitor cocktail 2 (Sigma-Aldrich)*
    7.5 μl/ml phosphatase inhibitor cocktail 3 (Sigma-Aldrich)*
*Note: Add immediately before using the buffer.
  1. 10x cap methylation buffer
    500 mM Tris pH 8
    60 mM KCl
    12.5 mM MgCl2

Acknowledgments

This work was supported by R01 grant GM084177 from the National Institutes of Health (to D.R.S). J.B.T. was funded by NIH T32 training grant GM08512 (to The Ohio State University) and by a pre-doctoral fellowship from The Ohio State University Center for RNA Biology. The protocol presented here was adapted from those in Pillutla et al., 1998, Otsuka et al., 2009, and Cowling, 2010 and was used for experiments in Trotman et al., 2017. The authors declare no conflicts of interest or competing interests.

References

  1. Aouadi, W., Eydoux, C., Coutard, B., Martin, B., Debart, F., Vasseur, J. J., Contreras, J. M., Morice, C., Querat, G., Jung, M. L., Canard, B., Guillemot, J. C. and Decroly, E. (2017). Toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay. Antiviral Res 144: 330-339.
  2. Cowling, V. H. (2010). Enhanced mRNA cap methylation increases cyclin D1 expression and promotes cell transformation. Oncogene 29(6): 930-936.
  3. Gonatopoulos-Pournatzis, T., Dunn, S., Bounds, R. and Cowling, V. H. (2011). RAM/Fam103a1 is required for mRNA cap methylation. Mol Cell 44(4): 585-596.
  4. Guiochon, G., Bressolle, F. and Siouffi, A. (1979). Study of the performances of thin layer chromatography IV – Optimization of experimental conditions. J Chromatogr Sci 17: 368-386.
  5. Jiao, X., Chang, J. H., Kilic, T., Tong, L. and Kiledjian, M. (2013). A mammalian pre-mRNA 5' end capping quality control mechanism and an unexpected link of capping to pre-mRNA processing. Mol Cell 50(1): 104-115.
  6. Niedzwiecka, A., Marcotrigiano, J., Stepinski, J., Jankowska-Anyszka, M., Wyslouch-Cieszynska, A., Dadlez, M., Gingras, A. C., Mak, P., Darzynkiewicz, E., Sonenberg, N., Burley, S. K. and Stolarski, R. (2002). Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5’ cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins. J Mol Biol 319(3): 615-635.
  7. Otsuka, Y., Kedersha, N. L. and Schoenberg, D. R. (2009). Identification of a cytoplasmic complex that adds a cap onto 5'-monophosphate RNA. Mol Cell Biol 29(8): 2155-2167.
  8. Peyrane, F., Selisko, B., Decroly, E., Vasseur, J. J., Benarroch, D., Canard, B. and Alvarez, K. (2007). High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2'O positions. Nucleic Acids Res 35(4): e26.
  9. Pillutla, R. C., Yue, Z., Maldonado, E. and Shatkin, A. J. (1998). Recombinant human mRNA cap methyltransferase binds capping enzyme/RNA polymerase IIo complexes. J Biol Chem 273(34): 21443-21446.
  10. Trotman, J. B., Giltmier, A. J., Mukherjee, C. and Schoenberg, D. R. (2017). RNA guanine-7 methyltransferase catalyzes the methylation of cytoplasmically recapped RNAs. Nucleic Acids Res 45(18): 10726-10739.
  11. van Deemter, J. J., Zuiderweg, F. J. and Klinkenberg, A. (1956). Longitudinal diffusion and resistance to mass transfer as causes of nonideality in chromatography. Chem Eng Sci 5: 271-289.

简介

甲基化mRNA 5'帽结构的鸟嘌呤-N7位置的甲基转移酶在真核生物中普遍存在并且通常由病毒编码。这里我们提供生物样品的RNA帽甲基转移酶活性的生化分析的详细方案。该测定包括将含有帽 - 甲基转移酶的样品与[32 P] G-加帽的RNA底物和S-腺苷甲硫氨酸(SAM)温育以产生具有N7-甲基化帽的RNA。然后通过P1核酸酶消化,薄层色谱(TLC)和磷成像确定帽甲基化的程度。此处描述的方案包括用于产生[32 P] G-加帽的RNA底物和用于从哺乳动物细胞制备核和细胞质提取物的附加步骤。该分析也适用于分析其他生物样品(包括重组蛋白制剂和来自分析分离和免疫沉淀/下拉实验的级分)的帽甲基转移酶活性。

【背景】mRNA的5'端的N7-甲基鸟苷帽是适当的真核mRNA加工,定位和翻译所必需的修饰。 N7甲基对于mRNA的生命周期特别重要,因为它极大地增加了帽结合蛋白的结合亲和力(Niedzwiecka等,2002),并且保护mRNA免于帽质量控制监测机制(焦等人,2013年)。我们最近报道,哺乳动物RNA鸟嘌呤-7甲基转移酶(RNMT)的功能超出了其在核共转录帽合成中的规范作用以参与细胞质RNA重新表达(Trotman等人,2017)。我们使用此处提供的方案来证明细胞质RNMT的帽甲基转移酶活性相对于核RNMT出乎意料地稳健。此外,RNA介导的RNMT敲低大大降低了细胞质提取物的帽甲基转移酶活性,表明RNMT是哺乳动物细胞的细胞质中主要的,如果不是只有帽甲基转移酶。核RNMT作为与RNMT活化小蛋白(RAM,Gonatopoulos-Pournatzis等人,2011)的异二聚体存在,并且我们证实细胞质RNMT也与RAM结合。 RAM敲低后细胞质帽甲基转移酶活性降低表明RAM是细胞质RNMT所需的辅因子。

该协议改编自两个较早表征人类RNMT的出版物(Cowling,2010; Pillutla等人,1998),其修改标准化了底物RNA的产生,避免了用放射性样品繁琐的苯酚 - 氯仿抽提,并能够量化帽甲基转移酶活性。我们注意到另一种非放射性测定已被报道用于帽甲基转移酶反应的分析(Peyrane et al。,2007),但是这种方法需要所有实验室都不可用的HPLC仪器,并且可能与目前报道的灵敏度和样品兼容性不同。另外,最近描述了用于测量帽甲基转移酶活性的荧光测定法(Aouadi等,2017),但该测定法通过监测S-腺苷高半胱氨酸(SAH)的积累来间接测量活性并且可能不相容含有SAH的生物样品。我们希望在此提出的协议的详细程度能够使未来的调查人员轻松地重复并加强我们的工作。

关键字:RNA, 5’端帽结构, 帽结构的甲基转移酶, RNMT, 酶活性测定, 亚细胞分离, P1核酸酶, 薄层色谱法

材料和试剂

  1. 0.2ml PCR管(例如,BioExpress,GeneMate,目录号:T-3225-1)。
  2. NucAway Spin Columns(Thermo Fisher Scientific,Invitrogen TM,产品目录号:AM10070)
  3. 1.7ml塑料无菌无RNase微量离心管(例如BioExpress,GeneMate,目录号:C-3262-1)
  4. 10cm或15cm的培养皿(例如,Alkali Scientific,产品目录号:TD0100,TD0150)。
  5. 无菌丁腈手套
  6. 细胞提升器(例如,,BioExpress,GeneMate,目录号:T-2443-4)
  7. 无菌,无RNA酶的P10,P20,P100和P1000移液管吸头
  8. 用于Bradford测定的塑料一次性比色皿(例如,Fisher Scientific,目录号:14-955-127)
  9. 塑料包装(例如,Saran或Stretch-Tite品牌)
  10. 纸标签带(,例如,Fisher Scientific,目录号:15-901-20H)
    制造商:Nevs,目录号:1590120H。
  11. 聚乙烯亚胺(PEI)纤维素TLC板(Macherey-Nagel,目录号:801053;参见注释2)
  12. Immobilon-FL聚偏二氟乙烯(PVDF)膜(Merck,目录号:IPFL00010)
  13. 与液体闪烁计数器兼容的闪烁瓶(例如,DWK Life Sciences,WHEATON,目录号:225414)

  14. U2OS细胞(ATCC,目录号:HTB-96)或HEK293细胞(ATCC,目录号:CRL-1573)
  15. P1核酸酶(美国生物公司,目录号:N7000),重悬于无RNase的水中至0.625U /μl
  16. Cap类似物GpppG(New England Biolabs,目录号:S1407),重悬于无RNA酶的水中至10mM。
  17. Cap类似物7GpppG(New England Biolabs,目录号:S1404),重悬浮于无RNA酶的水中至10mM。
  18. 无RNase的水(例如,来自Millipore Synergy水纯化系统,18.2MΩcm)。
  19. 单链DNA正义寡核苷酸
  20. 单链DNA反义寡核苷酸
  21. 5'CATGCAAATTAACCCTCACTAAA GGGAGA CCGGAATTCGAGCTCGCCCGGGGATC 3'用于T3转录模板,重悬于无RNA酶的水中至100μM(例如,由Integrated DNA Technologies(IDT)合成;加下划线的是T3启动子序列,粗体序列与转录的32个核苷酸(nt)pppRNA相匹配)
  22. 5'GATCCCCGGGCGAGCTCGAATTCCGGTCTCCCTTTAGTGAGGGTTAATTTGCATG 3'用于T3转录模板,重悬浮于无RNA酶的水中至100μM(例如,由IDT合成)。
  23. MEGAscript T3转录试剂盒(Thermo Fisher Scientific,Ambion,目录号:AM1138)
  24. 用于尿素聚丙烯酰胺凝胶电泳的预制聚丙烯酰胺微型凝胶(尿素-PAGE;例如,Bio-Rad Laboratories,目录号:4566053)
  25. 用于十二烷基硫酸钠聚丙烯酰胺凝胶电泳的预制聚丙烯酰胺微型凝胶(SDS-PAGE;例如,Bio-Rad Laboratories,目录号:4568094)
  26. 2个Laemmli样品缓冲液(Bio-Rad Laboratories,目录号:1610737)
  27. SYBR Gold核酸凝胶染色(Thermo Fisher Scientific,Invitrogen TM,目录号:S11494)
  28. RNA大小标记(例如,Thermo Fisher Scientific,Invitrogen TM,目录号:AM7778)。
  29. 2x RNA加载染料(例如,Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0641)
  30. 40U /μlRNaseOUT重组核糖核酸酶抑制剂(Thermo Fisher Scientific,Invitrogen TM,目录号:10777019)
  31. 重组三磷酸酶 - 鸟苷酸转移酶加帽酶(见注1)
  32. [α-32P] GTP(3000Ci / mmol,PerkinElmer,目录号:BLU506H250UC)
  33. RNA Clean& Concentrator-5试剂盒(Zymo Research,目录号:R1016)
  34. ScintiSafe Econo 1闪烁液(Fisher Scientific,目录号:SX20-5)
    注意:此产品已停产。
  35. McCoy的5A培养基(用于U2OS细胞; Thermo Fisher Scientific,Gibco TM,目录号:16600082)
  36. Dulbecco改良的Eagle培养基(DMEM;用于HEK293细胞; Thermo Fisher Scientific,Gibco TM,目录号:21013024)
  37. 胎牛血清(FBS; Atlanta Biologicals,目录号:S10350)
  38. 磷酸盐缓冲盐水(PBS;例如,Thermo Fisher Scientific,Gibco TM,目录号:10010049)
  39. Bio-Rad蛋白分析染料试剂浓缩物(Bio-Rad Laboratories,目录号:5000006)
  40. 预染的蛋白质标记物(例如,Bio-Rad Laboratories,目录号:1610393)
  41. 将牛血清白蛋白(BSA;例如,Fisher Scientific,目录号:BP1600-100)溶于水中至1μg/μl
  42. 用于蛋白质印迹的核蛋白抗体(例如,兔多克隆抗核仁抗体,1:5,000工作稀释度,Sigma-Aldrich,目录号:N2662)。
  43. 用于蛋白质印迹的抗细胞质蛋白抗体(例如,小鼠单克隆抗α-微管蛋白,1:10,000工作稀释度,Sigma-Aldrich,目录号:T6199)
  44. 用于红外成像的适当二级抗体(例如,用于红外成像的Thermo Fisher Scientific,Invitrogen,目录号:A-21109和A-21058,以1:10,000的工作稀释度)。
  45. (可选)痘苗加帽酶(新英格兰生物实验室,目录号:M2080S)
  46. 二硫苏糖醇(DTT,Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0861)
  47. 无RNase的10x Tris /硼酸盐/ EDTA(TBE)缓冲液(例如,Thermo Fisher Scientific,Invitrogen TM,目录号:15581)。
  48. 苯基甲基磺酰氟(PMSF; Thermo Fisher Scientific,Thermo Scientific TM,目录号:36978)
  49. 异丙醇
  50. Tris(例如,VWR,AMRESCO,目录号:0497)
  51. 甘氨酸(例如,VWR,AMRESCO,目录号:0167)
  52. SDS
  53. 甲醇(例如,Fisher Scientific,目录号:A452SK-4)
  54. 氯化钠(NaCl;例如,Fisher Scientific,目录号:BP358)
  55. 5M NaCl(例如,Thermo Fisher Scientific,目录号:AM9759)。
  56. 浓缩HCl
  57. 吐温20(例如,Fisher Scientific,目录号:BP337)
  58. 32mM S-腺苷甲硫氨酸(SAM; New England Biolabs,目录号:B9003)
  59. pH5.2的3M乙酸钠(例如,Thermo Fisher Scientific,Thermo Scientific TM,目录号:R1181)。
  60. 硫酸铵(例如,Sigma-Aldrich,目录号:A4418)
  61. IGEPAL CA-630(Sigma-Aldrich,目录号:I8896)
  62. 1M Tris-HCl,pH 7.5(例如,AMRESCO,目录号:E691)。
  63. 1M氯化镁(MgCl 2,例如,Thermo Fisher Scientific,Invitrogen TM,目录号:AM9530G)。
  64. 500mM乙二胺四乙酸,pH8.0(EDTA;例如,Thermo Fisher Scientific,Invitrogen TM,目录号:15575020)。
  65. 甘油(例如,Fisher Scientific,目录号:BP229-1)
  66. 1M HEPES pH 7.3(例如,AMRESCO,目录号:J848)
  67. 2M氯化钾(KCl;例如,Alfa Aesar,目录号:J75896)
  68. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)
  69. 磷酸酶抑制剂混合物2(Sigma-Aldrich,目录号:P5726)
  70. 磷酸酶抑制剂混合物3(Sigma-Aldrich,目录号:P0044)
  71. 1 M DTT(见食谱)
  72. 1x Tris缓冲盐水(TBS;见食谱)
  73. 1×TBE(尿素-PAGE的运行缓冲液)(见食谱)
  74. 20 mM DTT(见食谱)
  75. 在异丙醇中的100mM PMSF(见食谱)
  76. 10倍Tris /甘氨酸(见食谱)
  77. 10%(w / v)SDS(见食谱)
  78. 用于SDS-PAGE的Tris /甘氨酸/ SDS运行缓冲液(见配方)
  79. Tris /甘氨酸/甲醇/ SDS转移缓冲液(见食谱)
  80. 20倍Tris缓冲盐水(TBS)(见食谱)
  81. 3%BSA在TBS(见食谱)
  82. 40%(v / v)吐温20(见食谱)
  83. TBS-T(见食谱)
  84. 1μMSAM(见食谱)
  85. 500 mM乙酸钠,pH 5.2(见食谱)
  86. 0.4 M硫酸铵
  87. 10%IGEPAL CA-630(v / v)在水中(见食谱)
  88. 10倍退火缓冲液(见食谱)
  89. 4倍加盖缓冲液(见食谱)
  90. YO裂解缓冲液(见食谱)
  91. YO缓冲区A(请参阅食谱)
  92. 10倍帽甲基化缓冲液(见食谱)

设备

  1. 眼睛保护
  2. NanoDrop分光光度计(Thermo Fisher,型号:NanoDrop TM 1000,目录号:ND-1000)
  3. 用于处理32P(例如,Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:6700-2418)的璐彩特/有机玻璃丙烯酸台面屏蔽 br />
  4. 盖革计数器
  5. 公制(厘米)标尺
  6. 剪刀( ,Westcott,目录号:ACM44217)
  7. 石墨铅笔
  8. 长的(至少18厘米)镊子或夹钳(例如,Fisher Scientific,产品目录号:15-186)

  9. P10,P20,P100和P1000移液管
  10. 热循环仪(例如,MJ Research,型号:PTC-200)
  11. 加热块(例如,Bioer,型号:MB 101)
  12. -80°C冷冻机
  13. 手持式254nm紫外光(例如,UVP,型号:95-0016-14)
  14. 液体闪烁计数器(例如,Beckman Coulter,型号:LS 6000IC)
  15. 水套培养箱(Thermo Fisher Scientific Thermo Scientific TM ,型号:Forma II系列)设置为37°C,5 %CO 2
  16. 冷冻离心机(Eppendorf,型号:5415 R)
  17. 可编程旋转混合器(Grant Instruments,型号:PTR-30)在4°C下设置为10 rpm轨道旋转
  18. 紫外可见分光光度计(例如,Beckman Coulter,型号:DU 640)设置为595nm。
  19. 用于PAGE和膜转移的电泳系统(例如,Bio-Rad Laboratories,目录号:1660828EDU)
  20. 不透明的蛋白质印迹孵育盒(例如,LI-COR,目录号:929-97205)
  21. 蛋白质印迹成像系统(例如,LI-COR,模型:奥德赛成像系统)
  22. 矩形玻璃TLC腔室(例如,Miles Scientific,型号:A70-22,以前为Analtech)
  23. 电吹风机(可选)
  24. 存储荧光屏(例如,GE / Amersham Biosciences)
  25. 用于存储荧光屏(例如,分子动力学图像擦除器)的轻擦。
  26. 台风成像系统(例如,GE Healthcare,Amersham Biosciences,型号:Typhoon 9200)

软件

  1. Microsoft Excel软件
  2. ImageQuant TL软件

程序

图1给出了该方案的示意图。简而言之,使用[α-32 P] GTP将体外转录的短RNA进行鸟苷酸化以产生Gp * ppRNA底物,放射性标记的磷酸盐用星号表示。将该GpppRNA底物与核或细胞质提取物(或其他生物样品)和SAM短暂温育。每种提取物中不同水平的帽甲基转移酶活性将导致GpppRNA底物产生差异N7-甲基化以产生mβ7GpppRNA。然后用P1核酸酶将GpppRNA和mt7 GpppRNA的混合物消化为单个核苷酸,保留放射性标记的帽二核苷酸结构完整,最终通过TLC和磷成像分析。


图1.帽甲基转移酶活性测定的示意图

  1. 用于帽甲基转移酶活性测定的放射性标记的GpppRNA底物的制备(参见注释3)
    1. 准备一个30毫升混合物在一个0.2毫升的PCR管结合以下内容:15微升无RNase的水,3微升10x退火缓冲液(见食谱),6微升100μMssDNA有义寡核苷酸,6微升100微米ssDNA反义寡核苷酸。
    2. 为了使两种寡聚物变性然后缓慢退火以产生用于T3转录的双链DNA模板,将混合物置于98℃的热循环仪中2分钟,并在60分钟内将温度从98℃升至20℃(1.3 °C每分钟,降至20°C)。将此dsDNA管转移到冰上,并在另一个试管中,用无RNase的水稀释20倍,制备1μM(1pmol/μl)工作物。
    3. 使用MEGAscript T3转录试剂盒中提供的组分,通过按以下顺序组合,在0.2 ml PCR管中制备20μl反应:6μl无RNA酶的水,2μl75 mM ATP,2μl75 mM CTP ,2μl75mM GTP,2μl75mM UTP,2μl10×T3反应缓冲液,2μl1μMdsDNA模板,2μlT3 RNA聚合酶混合物。轻轻混合,并在37°C孵育16-24小时。这种延长的反应时间可以转录高产量的32 nt 5'三磷酸RNA(pppRNA)。
    4. 通过加入30μl无RNase的水将T3转录反应产物混合物加至50μl,并根据制造商的说明使用NucAway Spin Column进行纯化。
    5. 使用NanoDrop测量纯化的pppRNA的浓度。根据我们的经验,在24小时的转录反应后应该回收至少7-8微克的pppRNA。计算假设分子量为10,830.3 g / mol的pppRNA摩尔浓度。
    6. 通过15%聚丙烯酰胺尿素-PAGE进一步评估pppRNA的纯度。在1x TBE中以1:20,000的稀释度用SYBR Gold进行后染色后,在紫外光下低至150fmol(1.6ng)的pppRNA /泳道应足以可见。
      pppRNA应该以预期的大小运行
    7. pppRNA应储存在-20℃或-80℃的小等分试样中以避免多次冻融循环。
    8. 为了制备40μl封端(鸟苷酸化)反应,将以下组分在冰上的1.7ml试管中:RNase-free水(总共40μl),10μl4x加帽缓冲液(参见食谱),1μl (参见注释1),24 pmol(总GTP)[α-32 P] GTP(参见注释4)。轻轻混合并在30°C孵育3 h,然后通过在65°C孵育10 min进行加热灭活。
    9. 将1.33μl(1/30)封端反应产物转移至冰上的新管中以作为闪烁计数的“输入”。通过加入11.33μl无RNase的水使加盖反应物达到50μl。使用Zymo RNA Clean& Purification TM纯化RNA。 Concentrator-5试剂盒按照制造商的说明使用30μl无RNase的水进行最终洗脱步骤。将纯化的RNA返回到冰上,并将1μl(1/30)转移至冰上的新试管用于闪烁计数。
    10. 将预留的1/30封盖反应“输入”和纯化的RNA样品各自与1.5ml ScintiSafe Econo 1闪烁液混合并转移至闪烁瓶中。通过闪烁计数测量每种混合物的放射性。根据下式确定纯化的[32 P] G加帽的RNA的浓度:[GpppRNA](单位μM)=(24pmol)×(纯化计数/输入计数)/(30μl )。
    11. 纯化的[32 P] G加帽RNA可以保存在-20°C,但应在几天内使用以限制放射性衰变。确定在甲基转移酶活性测定中使用的适当量时,请务必考虑放射性衰变。

  2. 哺乳动物细胞的核和细胞质提取物的制备
    1. 提前2至3天,将含有U2OS,HEK293或其他类型的贴壁哺乳动物细胞的10-cm或15-cm种子播种,使得收集时皿将达到〜80-90%汇合。 U2OS细胞应该在含有10%(v / v)FBS的McCoy's 5A培养基中生长; HEK293细胞应该在含有10%(v / v)FBS的DMEM中生长。细胞应在培养箱中培养,温度设定为37℃,5%CO 2。
    2. 在收获细胞的当天,在冰上制备YO裂解缓冲液和YO缓冲液A(参见食谱),留下DTT,PMSF,蛋白酶抑制剂混合物和磷酸酶抑制剂混合物,直至使用缓冲液之前。
    3. 从贴壁细胞中取出培养基,用PBS冲洗并除去,然后通过刮入1ml PBS收集并转移到1.7ml离心管中。轻轻沉淀细胞(70℃×g,10分钟,4℃),然后通过抽吸小心取出上清液PBS。记下细胞团的大致体积,并将管置于冰上。
    4. 为了制备细胞质提取物,立即加入相当于每个细胞沉淀体积4-5倍的YO裂解缓冲液。使用P1000移液器的10个上下笔划轻轻地重悬细胞。将细胞在冰上孵育10分钟,然后与P1000移液器的另外5个上下冲程混合。沉淀细胞核(16,100×g,4℃10分钟),然后将细胞质提取物上清液转移到新的预先冷冻的1.7ml冰上。
    5. 使用P10移液器小心地去除核颗粒中残留的细胞质提取物。为了制备核提取物,加入4-5个原始细胞沉淀体积的YO缓冲液A,并用P200移液管的15个上下冲程重新悬浮每个核沉淀。将试管在4℃下颠倒孵育20分钟,然后沉淀核碎片(4,100℃下16,100μg×g 5分钟)。
      将核提取物上清液转移至新的预先冷冻的1.7ml冰管中。
    6. 使用Bio-Rad蛋白质测定染料试剂浓缩物,根据制造商的说明书并使用牛血清白蛋白(BSA)在0,1,2,4,6和6的标准曲线,通过Bradford测定法测量每种提取物中的蛋白质浓度8微克/毫升。在1x染料试剂中稀释250倍至500倍的提取物通常在测定的线性范围内进行测量。使用Microsoft Excel等程序,使用BSA标准曲线的最佳拟合线计算蛋白质浓度。
    7. 对于帽甲基转移酶活性测定,准备15-20μL每个提取物样品在新的,预先冷却的管冰上。如果比较核提取物和细胞质提取物,确保这些提取物处于相同的缓冲液组成中是至关重要的。这可以通过首先将所有提取物与相同的蛋白质浓度(通常为0.5-1.0μg/μl;这样的计算可以通过诸如Microsoft Excel的软件极大地辅助)与使用相同的缓冲液之前(即,用于细胞质提取物的YO裂解缓冲液和用于核提取物的YO缓冲液A),然后将每个样品与等体积的其他缓冲液(即,YO缓冲液A用于细胞质提取物和YO裂解缓冲液核提取物)。
      在-80°C下将这些提取物样品以小份储存。
    8. 通过在SDS-PAGE上运行核和细胞质提取物然后进行蛋白质印迹分析来评估亚细胞分级的质量。使用核和细胞质对照抗体(例如针对核仁素和微管蛋白)可以确定在分离过程期间是否发生交叉污染。有关预期的Western印迹结果的例子,请参见2017年Trotman等人图1C。

  3. 制备用于帽甲基转移酶活性测定的TLC板
    1. 戴上干净的手套,用干净的尺子和剪刀将20×20厘米的PEI纤维素薄层板切成四块10×10厘米的方块。我们建议使用清洁镊子来处理印版,以限制与手套上可能存在的任何污染物接触。将所有未使用的培养板储存在4°C。请参阅关于TLC性能的注释2。
    2. 用尺子和铅笔从10×10厘米板的底部边缘轻轻画出距离底部边缘1.5厘米的微弱直线(原点)。在相距1厘米的原点上绘制微弱的刻度标记,以指示样品将被点样的位置。图2显示了一个薄层色谱板的例子。


      图2.薄层色谱板制备示例

    3. 用无RNase水预先运行TLC板。为此,用足够的水填充矩形玻璃TLC室以覆盖底部(上述模型中大约60毫升),使得水深不超过0.75厘米。使用长镊子,轻轻地将TLC板直立放入水中。注意确保左右边缘同时进入水中,因为这将促进水向TLC板上的均匀迁移。该预运行步骤将污染物强制到TLC板的顶部,并极大地提高了对甲基转移酶活性测定样品进行后续色谱分析的性能。
    4. 一旦水已经到达TLC板的顶部,从室中取出TLC板并使其风干。
      。用手摇动板子或使用商用吹风机(不加热)可以加速这一过程。
    5. 预润滑的TLC板可以在4°C下储存至少2周。

  4. Cap甲基转移酶活性测定
    1. 在冰上解冻组分后,按照下列顺序合并下列物质,在冰上1.7ml试管中制备10μl帽甲基转移酶反应:无RNA酶的水(总体积10μl),1μl10x帽甲基化缓冲液(参见食谱),1 μl1μMSAM,1μl20mM DTT,1μlRNaseOUT,1μg总蛋白的核或胞质提取物,5fmol [32 P] G-加帽的RNA。如果使用大量提取物样品,建议使用反应组分的主混合物(省略提取物)。轻轻混合,在37°C孵育30分钟,然后立即回到冰上。
    2. 向每个管中加入40μl无RNA酶的水,并使用Zymo Clean& Concentrator-5试剂盒按照制造商的说明使用7μl无RNase的水进行最终洗脱步骤。将纯化的RNA置于冰上。
    3. 通过依次组合以下步骤,在1.7ml管中的冰上制备7μlP1核酸酶消化反应:将5.0μl来自帽甲基转移酶反应的纯化RNA,0.6μl不含RNase的水,0.7μl500mM乙酸钠pH5.2,0.7μl的0.625U /μlP1核酸酶。轻轻混合并在37°C孵育30分钟。样品现在可以储存在-20°C或立即分析。
    4. 在预先运行的PEI纤维素薄层色谱板的原点上的1厘米刻度线上,使用P10移液管仔细检测1μlP1核酸酶消化产物。让斑点完全空气干燥,然后在与之前相同的位置上再添加1μlP1消化产物。作为对照,还在单独的刻度标记或标记上点出2μl(每次1μl)10mMGpppG和10mMm7GpppG标准(参见注释5)。
    5. 使斑点完全空气干燥后,使用长镊子将TLC平板直立放置在包含约60ml 0.4M硫酸铵流动相的矩形TLC室中。让流动相向上发展,离原点约6-7厘米,然后从腔室中取出TLC板。让TLC板完全空气干燥,可以通过手摇或使用吹风机加热而不加热。
    6. 佩戴适当的眼睛和皮肤保护,在手持254nm紫外光下观察TLC板以确定GpppG和mpp7GpppG标准的位置。这些可以通过用铅笔标记位置或用手持相机拍摄照片来指出。
    7. 用薄膜包裹TLC板,使正面覆盖一层。将TLC板放入存储荧光屏幕盒中,将磁带的顶部边缘贴在磁带上以保持其位置。将最近清除光的存储荧光屏置于TLC板上,关闭盒子,并在室温下暴露至少16小时以获得最大信号。
    8. 从盒中取出薄层色谱板,并将Typhoon成像仪设置为荧光模式,对存储荧光屏进行成像。典型的结果如图3所示。


      图3.典型的荧光图像结果为了证明重现性,用U2OS细胞核和细胞质提取物进行所述活性测定的四个技术重复。

数据分析

在帽甲基转移酶反应过程中(并且通过延伸,存在于相应生物样品中的帽甲基转移酶活性),GpppRNA向mβ7GpppRNA的转化程度由mβ7 GpppG斑点强度与TLC板荧光图像上的GpppG和m7GpppG斑点强度的总和。斑点强度可以使用任何包括密度测定分析的图像软件来确定,例如ImageQuant TL。为了确保对多个样品的斑点强度进行一致的计算,请使用尺寸相同的通道或箱子,以便适合整个光斑进行密度测定分析。同时确保减去任何背景信号;在ImageQuant TL中,我们使用“橡皮筋”设置来确定峰值信号积分的基线。为了在比较多种条件时解释样品之间的生物变异性,最好使用至少三个生物学重复实施该测定以便进行适当的统计测试。

笔记

  1. 我们用于产生放射性标记的GpppRNA的重组人类加帽酶如Trotman等人所述在室内生产。 (2017)。尽管这种酶库与非放射性标记的GTP具有强大的封闭活性,放射性标记的GpppRNA的产生效率相当低。如果重组加帽酶不可用,我们推荐使用商业上可获得的酶,如牛痘加帽酶(NEB,目录号M2080S),以产生如先前报道中所用的GpppRNA(Pillutla等人, 1998年; Cowling,2010年)。如果使用痘苗加帽酶来产生GpppRNA,请确保从此反应中省略SAM,因为此酶也含有帽甲基转移酶活性。
  2. 与大多数类型的色谱系统不同,TLC的流速是可变的并且不能根据van Deemter方程式(van Deemter等人,1956; Guiochon等人。,1979)。在TLC中,毛细管流速取决于诸如TLC板的粒度之类的性质,其可以从制造商到制造商显着变化。因此,我们发现一些生产商的PEI纤维素板比其他生产商的纤维板分辨率更糟糕,分辨率更差,这里列出的Macherey-Nagel板可以给出我们在给定条件下测试的最佳性能。
  3. 任何短的体外转录RNA(带有5'三磷酸)应该适合于产生放射性标记的GpppRNA底物,并且如果需要,可以修改该方案以产生其他GpppRNA底物。这里提出的方案简单地描述了一种简单的方法来生成用于人RNMT(Pillutla等,,1998)和我们最近的研究(Trotman等,1998)的初始表征中的相同的32nt底物RNA。 et al。,2017)。
  4. 使用新鲜的[α-32 P] GTP以最大化原料中放射性与非放射性GTP的比例,这将允许在成像过程中获得更大的信号强度。为了考虑[α-32 P] GTP库存的放射性衰减,我们推荐使用PerkinElmer放射性计算器( https://www.perkinelmer.com/tools/calculatorrad )来确定使用当天的总GTP浓度。
  5. 作为用于显现mGpppG位置并确保检测成功的额外对照,我们建议使用重组帽甲基转移酶(例如来自NEB的痘苗加帽酶)制备平行帽甲基转移酶活性反应。
  6. 放射性同位素32 P发射β粒子,如果处理不当,可能有害。为确保在处理 32 P时的安全性,请始终佩戴护目镜和皮肤保护装置,在丙烯酸屏蔽罩后工作,并使用盖革计数器经常检查表面是否有污染。我们建议使用一套专用的移液管以防放射性物质被污染。将所有放射性样品在4°C或-20°C储存在二级压克力储存盒中。清理溢出物,无论多小,并在泄漏量足够大的情况下通知实验室人员和相应的安全官员。所有放射性废物应放置在丙烯酸废物箱中至少10个半衰期(大约143天)以使其储存衰变。
    请联系您所在机构的辐射安全办公室

食谱

  1. 1 M DTT
    将1.54克DTT溶于无RNA酶的水中,使其总体积为10毫升。分装并储存在-20°C
  2. 1x TBE(尿素-PAGE的运行缓冲液)
    将100毫升不含RNase的10x TBE(试剂#47)用无RNase的水加至1 L。在室温下储存。
  3. 20 mM DTT
    将20μl1 M DTT加入1 ml无RNase的水中。分装并储存在-20°C
  4. 在异丙醇中的100mM PMSF
    将1.74g PMSF溶于异丙醇中至总体积10ml。分装并储存在-20°C
  5. 10x Tris /甘氨酸
    将30g Tris和144g甘氨酸溶于无RNA酶的水中至总体积1μl。在室温下储存。
  6. 10%(w / v)SDS
    将10g SDS溶于无RNA酶的水中至总体积100ml。在室温下储存。
  7. 用于SDS-PAGE的Tris /甘氨酸/ SDS运行缓冲液 890毫升无RNase水
    100毫升10x Tris /甘氨酸
    10 ml 10%(w / v)SDS
  8. Tris /甘氨酸/甲醇/ SDS转移缓冲液
    向200ml甲醇中加入500ml无RNase的水,然后加入100ml 10x Tris /甘氨酸和1ml 10%(w / v)SDS。
    加入无RNase的水至总体积1 L,并储存于4°C
  9. 20倍Tris缓冲盐水(TBS)
    将48.4克Tris和160克NaCl溶解在800毫升无RNase的水中并用浓HCl将pH调节至7.6。用不含RNase的水将总体积加至1L并储存在室温下。
  10. 3%BSA在TBS
    向1.5克BSA中加入2.5毫升20×TBS,并溶于无RNA酶的水中至总体积为50毫升。在4°C储存
  11. 40%(v / v)吐温20
    将20ml吐温20与无RNase水混合至总体积50ml。在室温下储存。
  12. TBS-T

    474毫升无RNase水 25毫升20x TBS

    1.25毫升40%(V / V)吐温20
  13. 1μMSAM
    用无RNase的水稀释32 mM SAM 320倍,产生100μM的原液,然后用无RNase的水稀释100μM的原液100倍。分装并储存在-20°C
  14. 500mM乙酸钠,pH 5.2
    用无RNase的水稀释3M柠檬酸钠,pH5.2 6倍。在室温下储存。
  15. 0.4 M硫酸铵

    将52.86克硫酸铵溶于无RNA酶的水中溶解至1升。室温下储存

  16. 10%IGEPAL CA-630(v / v) 加入不含RNase的水至1 ml IGEPAL CA-630至总体积10 ml。在室温下储存。
  17. 10倍退火缓冲液
    100mM Tris-HCl pH 7.5
    500 mM NaCl
  18. 4倍加盖缓冲液
    40 mM Tris pH 7.5
    12mM MgCl 2·/ 2 4 mM DTT
    0.4 mM EDTA
    80%(v / v)甘油
  19. YO裂解缓冲液
    10 mM HEPES pH 7.3
    10 mM KCl
    10mM MgCl 2 2/2 0.2%(v / v)IGEPAL CA-630
    2 mM DTT *
    0.5毫米PMSF *
    7.5μl/ ml蛋白酶抑制剂混合物(Sigma-Aldrich)*
    7.5μl/ ml磷酸酶抑制剂混合物2(Sigma-Aldrich)*
    7.5μl/ ml磷酸酶抑制剂混合物3(Sigma-Aldrich)*
  20. YO缓冲区A
    10 mM HEPES pH 7.3
    25%(v / v)甘油
    420 mM NaCl
    1.5mM MgCl 2 2/2 0.2mM EDTA
    1 mM DTT *
    0.5毫米PMSF *
    7.5μl/ ml蛋白酶抑制剂混合物(Sigma-Aldrich)*
    7.5μl/ ml磷酸酶抑制剂混合物2(Sigma-Aldrich)*
    7.5μl/ ml磷酸酶抑制剂混合物3(Sigma-Aldrich)*
*注意:在使用缓冲区之前立即添加。
  1. 10倍帽甲基化缓冲液
    500 mM Tris pH 8
    60 mM KCl
    12.5mM MgCl2·2/2

致谢

这项工作得到了美国国立卫生研究院(至D.R.S)R01拨款GM084177的支持。 J.B.T.由美国国立卫生研究院T32培训资助GM08512(俄亥俄州立大学)和俄亥俄州立大学RNA生物学中心的博士后研究员资助。这里介绍的协议是从Pillutla et al。,1998年,Otsuka et al。,2009年和Cowling 2010年的那些改编的,并用于Trotman et al。,2017。作者声明不存在利益冲突或利益冲突。

参考

  1. Aouadi W. Eydoux C. Coutard B. Martin Martin B. Debart F. Vasseur JJ Contreras JM Morice C. C. Querat G. Jung Jung ML ML Canard B 。,Guillemot,JC和Decroly,E.(2017)。 通过荧光筛选实验鉴定病毒帽甲基转移酶抑制剂。 抗病毒药物 144:330-339。
  2. Cowling,V.H。(2010)。 增强的mRNA帽甲基化可增加细胞周期蛋白D1的表达并促进细胞转化。致癌基因 29(6):930-936。
  3. Gonatopoulos-Pournatzis,T.,Dunn,S.,Bounds,R.和Cowling,V.H。(2011)。 RAM / Fam103a1是mRNA帽甲基化所必需的。分子细胞
    44(4):585-596。
  4. Guiochon,G.,Bressolle,F.和Siouffi,A。(1979)。 薄层色谱的性能研究IV - 优化的实验条件。
  5. Jiao,X.,Chang,J.H。,Kilic,T.,Tong,L。和Kiledjian,M.(2013)。 哺乳动物pre-mRNA 5'末端封盖质量控制机制以及意想不到的封盖前环节, mRNA处理。
  6. Niedzwiecka,A.,Marcotrigiano,J.,Stepinski,J.,Jankowska-Anyszka,M.,Wyslouch-Cieszynska,A.,Dadlez,M.,Gingras,AC,Mak,P.,Darzynkiewicz,E.,Sonenberg, N.,Burley,SK和Stolarski,R。(2002)。 eIF4E帽结合蛋白的生物物理学研究:识别mRNA 5'帽结构和eIF4G的合成片段和4E-BP1蛋白。 J Mol Biol 319(3):615-635。
  7. Otsuka,Y.,Kedersha,N.L。和Schoenberg,D.R。(2009)。 鉴定在5'-单磷酸RNA上加帽的细胞质复合体 < Mol Cell Biol 29(8):2155-2167。
  8. Peyrane,F.,Selisko,B.,Decroly,E.,Vasseur,J.J。,Benarroch,D.,Canard,B。和Alvarez,K。(2007)。 高产量生产短GpppA-和7MeGpppA-封端的RNA和高效液相色谱监测甲基转移反应鸟嘌呤-N7和腺苷-2'O位置。核酸研究35(4):e26。
  9. Pillutla,R.C.,Yue,Z.,Maldonado,E。和Shatkin,A.J。(1998)。 重组人类mRNA甲基转移酶结合加帽酶/ RNA聚合酶IIo复合物。 J Biol Chem 273(34):21443-21446。
  10. Trotman,J.B.,Giltmier,A.J.,Mukherjee,C。和Schoenberg,D.R。(2017)。 RNA鸟嘌呤-7甲基转移酶催化细胞质重新封装的RNA的甲基化。 Nucleic Acids Res 45(18):10726-10739。
  11. van Deemter,J.J.,Zuiderweg,F.J。和Klinkenberg,A。(1956)。 纵向扩散和传质阻力是导致色谱中非理想性的原因 Chem Eng Sci 5:271-289。
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引用:Trotman, J. and Schoenberg, D. R. (2018). RNA Cap Methyltransferase Activity Assay. Bio-protocol 8(6): e2767. DOI: 10.21769/BioProtoc.2767.
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