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Because covalent bond can form between RNA and its binding proteins after UV irradiation, UV cross-linking is widely used to identify the specific RNA binding proteins. This protocol is described in details as follows.

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In vitro RNA-protein Binding Assay by UV Crosslinking

Biochemistry > RNA > RNA-protein interaction
Authors: Hailong Zhang
Hailong ZhangAffiliation: Department of Pediatrics, Emory University, Atlanta, USA
For correspondence: hzhan52@emory.edu
Bio-protocol author page: a151
 and Muxiang Zhou
Muxiang ZhouAffiliation: Department of Pediatrics, Emory University, Atlanta, USA
Bio-protocol author page: a152
Vol 2, Iss 21, 11/5/2012, 8410 views, 1 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.287

[Abstract] Because covalent bond can form between RNA and its binding proteins after UV irradiation, UV cross-linking is widely used to identify the specific RNA binding proteins. This protocol is described in details as follows.

Materials and Reagents

  1. Linearized DNA template for transcription of the RNA of interest
  2. MAXIscript in vitro Transcription Kit (Applied Biosciences, catalog number: AM1308M) (This kit contain ATP, CTP, GTP, α-P32UTP and DNase I)
  3. RNasin (Promega Corporation, catalog number: N2611)
  4. RNase T1 (Applied Biosciences, catalog number: 10109193001)
  5. 0.5 M EDTA (Life Technologies, Gibco®, catalog number: 15575-038)
  6. ddH2O (GeneMate Loyalty, catalog number: UPW-1000)
  7. HEPES (Sigma-Aldrich, catalog number: H3375)
  8. MgCl2 (Sigma-Aldrich, catalog number: M-2393)
  9. Glycerol (Sigma-Aldrich, catalog number: G7893-1L)
  10. DTT (Sigma-Aldrich, catalog number: D9779-5G)
  11. Tris-Base (Thermo Fisher Scientific, catalog number: BP152-1)
  12. SDS (Sigma-Aldrich, catalog number: L-4390)
  13. Bromphenol blue (Sigma-Aldrich, catalog number: B5525)
  14. TEMED
  15. Ammoniampersulfate
  16. 5x binding buffer (see Recipes)
  17. 2x SDS loading buffer (see Recipes)
  18. 10x TGE buffer (see Recipes)
  19. 6% TGE gel (see Recipes)

Equipment

  1. SPIN-PureTM column (G-50) (PireBiotech SCW50-50 DEPC-water)
  2. SDS-PAGE system (Bio-Rad, catalog number: 165-1802)
  3. UV Stratalinker 1800 (Stratagene, catalog number: 474645)
  4. Film processor (Konica Minolta, model: SRX-101A)
  5. Centrifuges (Eppendorf, catalog number: 5810R)
  6. Scintillation Counter (Beckman, catalog number: LS6500)
  7. Bench top Radiation Shield with 6-1/4" base (Cole-Parmer, catalog number: WU-36218-00)
  8. 37 °C water bath

Procedure

  1. In vitro transcription of 32P-labled RNA probes
    1. DNA templates for synthesis of the RNA probe are generated by PCR using specific primers.
    2. Thaw frozen reagents, place RNA polymerase on ice and vortex the 10x Transcription buffer and keep the buffer at room temperature. All reagents should be centrifuged briefly before opening.
    3. Assemble transcription reaction at room temperature as the following sequence:
      1 μg DNA template
      ddH2O to 20 μl
      2 μl 10x transcription buffer (after addition of water and DNA)
      1 μl 10 mM ATP
      1 μl 10 mM CTP
      1 μl 10 mM GTP
      5 μl [α-32P]UTP
      2 μl T7 enzyme mix
      2 μl RNasin (40 U/μl)
      Mix thoroughly.
    4. Incubate 1 h at 37 °C.
    5. Add 1 μl of 5 mg/ml DNase I to digest the rest template DNA and incubate 10 min in 37 °C water bath.
    6. Add 1 μl of 0.5 M EDTA, mix and add 78 μl ddH2O to adjust final volume to 100 μl and mix.

  2. Purification of 32P-labled RNA probes with SPIN-Pure column (G-50)
    1. Allow a minimum of 30 min of room temperature to warm the columns before the following steps.
    2. Gently invert the column several times to suspend the column buffer (DEPC-Water).
    3. Remove the top cap from the column, and then remove the bottom tip.
    4. Allow the column buffer to drain by gravity before proceeding.
    5. Place this column/tube apparatus into an adaptor tube. Centrifuge at 1,100 x g for 2 min at room temperature.
    6. Repeat centrifugation again to let the column dry completely and discard the collection tube and the eluted buffer.
    7. Put the column in a second collection tube in upright position and apply the RNA sample (20 to 50 μl) to the center of the column gel very slowly and carefully (the rest RNA sample can be stored at -80 °C for one week).
    8. Centrifuge at 1,100 x g for 4 min. The purified 32P-labled RNA probe is collected in the bottom of the collection tube. Discard the spin column and continue with the following procedure.
    9. Pick 1 μl of the RNA sample and count the cpm value.

  3. RNA and protein binding reaction
    1. Prepare the following mixture on ice:
      32P-labled RNA probe (2 x 105 cpm)
      1 μl 12.5 mM ATP
      3μl 5x binding buffer
      3 μl 0.5 M KCl
      Protein (100 ng) (this protein can be a GST fusion protein purified according to the protocol prepared by GE Healthcare Life Sciences)
      H2O to final volume of 15 μl.
    2. Mix gently by pipetting up and down for 10 times, avoiding vortex.
    3. Place the uncovered tube containing the reaction mixture on ice, directly underneath the bulb (about 10 cm from the surface) of a 254-nm UV light source, irradiation with 4 x 105 μJ/cm2 energy.
    4. Incubate at room temperature for 30 min then keep on ice for another 3 h.

  4. Remove the non-incorporated 32P
    1. Add 1 μl of RNase T1 (1 U/μl) to each tube and incubate for additional 10 min at 37 °C to degrade the free RNA.
    2. Add an equal volume (16 μl) of 2x SDS loading dye andseparate the sample in TGE-gel or SDS-PAGE (no boiling need).
    3. Autoradiography for 12 h to 7 days for visualization.


Recipes

  1. 5x binding buffer
    50 mM HEPES (pH 7.2)
    15 mM MgCl2
    25% glycerol
    5 mM DTT
    Stored at -20 °C
  2. 2x SDS loading buffer
    125 mM Tris-Cl (pH 7.6)
    20% (v/v) glycerol
    4% (w/v) SDS
    0.008% (w/v) bromphenol blue
    20 mM DTT
  3. 10x TGE buffer
    Tris 100 mM
    Glycine 1 M
    EDTA 10 mM
    pH 8.3
  4. 6% TGE gel
    6 ml 30% acrylamide
    5 ml 10x TGE buffer
    35 ml H2O
    300 μl ammoniampersulfate
    22 μl TEMED
    Notes:
    1. Avoiding RNase contamination is the key for successful.
    2. As 32P is radioactive, this experiment should be performed under the protection of Benchtop Radiation Shield.

Acknowledgments

This work was supported by the National Institutes of Health (R01 CA123490 and R01CA143107 to MZ) and CURE (MZ and LG).

References

  1. Gu, L., Zhang, H., He, J., Li, J., Huang, M. and Zhou, M. (2012). MDM2 regulates MYCN mRNA stabilization and translation in human neuroblastoma cells. Oncogene 31(11): 1342-1353.
  2. Walker, J., de Melo Neto, O. and Standart, N. (1998). Gel retardation and UV-crosslinking assays to detect specific RNA-protein interactions in the 5' or 3' UTRs of translationally regulated mRNAs. Methods Mol Biol 77: 365-378.


How to cite this protocol: Zhang, H. and Zhou, M. (2012). In vitro RNA-protein Binding Assay by UV Crosslinking. Bio-protocol 2(21): e287. DOI: 10.21769/BioProtoc.287; Full Text



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1/21/2016 7:08:48 AM  

Guosheng Qu
UAlbany

1. I notice that ATP is added in the RNA-protein binding mixture before UV-crosslinking. Is it necessary for UV-crosslinking? If yes, why?

2. I did iCLIP by mixing RNA with protein in a different buffer (50 mM Tris-HCl, pH 6.8; 140 mM NaCl; 0.05% Triton-X100) and UV-cross-linking with 400 mJ/cm2. Under this condition, IP works and UV caused RNA damage is seen but no UV-crosslinks between RNA and protein have been detected (I use reverse transcription to detect peptidyl ligands left on the RNA after proteinase K digestion). Do you think what my troubles could be?

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