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Protocol
Mapping RNA–Protein Interactions Using Hydroxyl-Radical Footprinting Timothy W. Nilsen
The binding of a protein to an RNA sequence protects the region of the RNA from cleavage by chemicals or RNases; this protected region is known as the protein’s “footprint.” In the footprinting protocol presented here, end-labeled RNAs with and without bound protein are cleaved using chemical methods. Fe(II)–EDTA is used to generate hydroxyl radicals in the presence of a reducing agent. These hydroxyl radicals indiscriminately cleave ribose groups in regions of the ribose–phosphate backbone that are exposed to solvent. After termination of cleavage, the resulting RNA fragments are analyzed by gel electrophoresis on denaturing polyacrylamide gels. Because hydroxyl radicals are smaller and cleave less specifically than RNases, this approach, if feasible, is often the method of choice for monitoring sites of RNA–protein interactions.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPE: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Ammonium iron(II) sulfate (50 mM) Freeze in 1-mL aliquots and store at −20˚C.
Ascorbic acid (0.25 M) Freeze in 1-mL aliquots and store at −20˚C.
Binding buffer specific for the protein and RNA of interest Before carrying out hydroxyl-radical footprinting, determine the optimum binding parameters for the protein and RNA using gel shifts (see Electrophoretic Mobility Shift Assays for RNA–Protein Complexes [Rio 2014c]) or filter binding (see Filter-Binding Assay for Analysis of RNA–Protein Interactions [Rio 2012]). It may be necessary to modify the binding buffer used in these assays to find conditions that are compatible with hydroxyl-radical cleavage. Use buffers such as HEPES instead of Tris and omit glycerol and bovine serum albumin (BSA). Glycerol and Tris must be absent or their concentrations greatly reduced for hydroxyl-radical footprinting. The glycerol concentration in the final binding reaction should be no more than 0.5%.
Denaturing polyacrylamide gels and electrophoresis reagents EDTA (0.2 M, pH 8.0)
Adapted from RNA: A Laboratory Manual by Donald C. Rio, Manuel Ares Jr, Gregory J. Hannon, and Timothy W. Nilsen. CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080952
1333
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
T.W. Nilsen
Ethanol (100%) Extract or protein for binding reactions GlycoBlue (10 µg/µL) Hydrogen peroxide (H2O2; 30%) (Fisher H325–100) Store 30% stock tightly capped at 4˚C.
Phenol RNA of interest, 32P-labeled and gel-purified Either 5′ - or 3′ -end-labeled RNA can be used, but it is often advisable to analyze 5′ - and 3′ -end-labeled RNA in parallel. To prepare labeled RNA, see 5′ -End Labeling of RNA with [γ- 32P]ATP and T4 Polynucleotide Kinase (Rio 2014a), 3′ -End Labeling of RNA with [5′ - 32P]Cytidine 3′ ,5′ -Bis(phosphate) and T4 RNA Ligase 1 (Nilsen 2014a), or 3′ -End Labeling of RNA with Yeast Poly(A) Polymerase and 3′ -Deoxyadenosine 5′ -[γ- 32P]Triphosphate (Rio 2014b).
Sodium acetate (3 M, pH 5.2) Thiourea (0.1 M; Sigma-Aldrich T-7875) Urea loading dye Equipment
Chemical fume hood Dry ice Gel dryer Microcentrifuge Microcentrifuge tubes (1.5 mL) Phosphorimager or autoradiography materials Polyacrylamide gel electrophoresis system and power supply Gel plates must be thoroughly cleaned with a solution of 5% SDS before use.
Water baths or heat blocks at binding temperature (see Step 1) and at 95˚C (see Step 7) Whatman 3MM paper
METHOD Carry out the procedure in a chemical fume hood.
1. Allow the protein to bind to labeled RNA in 25 µL of binding buffer in a 1.5-mL microcentrifuge tube for the appropriate amount of time. Include a control reaction with no protein. Binding must be close to 100% or the footprint will not be clear (purify the complex if necessary).
2. Set up the cleavage reaction. i. Prepare a fresh solution of 2.5% H2O2 by diluting a 30% stock of H2O2 in H2O. ii. Initiate the cleavage reaction by adding 1 µL of each of the following reagents to the side of the 1.5-mL microcentrifuge tube containing the binding reaction: Ammonium iron(II) sulfate EDTA Ascorbic acid H2O2
50 mM 100 mM 250 mM 2.5%
Increase cleavage by adding more Fe(II)–EDTA. However, do not increase the amount of H2O2 as this may affect binding of the protein to the RNA.
iii. Centrifuge the tube in a microcentrifuge for 2–5 sec to collect the reagents in the bottom of the tube with the reaction mix. 1334
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080952
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
Hydroxyl-Radical Footprinting
3. Allow the cleavage reaction to proceed for 5 min at room temperature. It will be necessary to optimize the cleavage times and temperature. Cleavage can be conducted on ice instead of at room temperature, but it will take longer.
4. Stop the reaction by adding 10 µL of 0.1 M thiourea. 5. Recover the RNAs by phenol extraction and ethanol precipitation (adding 10 µg of GlycoBlue as carrier). See Purification of RNA by SDS Solubilization and Phenol Extraction (Rio et al. 2010a) and Ethanol Precipitation of RNA and the Use of Carriers (Rio et al. 2010b).
6. Resuspend the RNA pellet in 200 µL of 2 mM EDTA. Add 600 µL of 100% cold (–20˚C) ethanol and 3 M sodium acetate (pH 5.2) to a final concentration of 0.3 M. Freeze the mixture on dry ice, thaw, and recover the RNAs by centrifuging in a microcentrifuge for 15 min at room temperature. Carefully remove the ethanol supernatant and discard. 7. Resuspend the RNA in 5–10 µL of H2O. Denature in urea loading dye and analyze the cleavage products on the appropriate denaturing polyacrylamide gel. See Polyacrylamide Gel Electrophoresis of RNA (Rio et al. 2010c). Depending on the size of the RNA, it may be necessary to use two different gel percentages for best resolution.
8. Subject the gel to phosphorimaging or autoradiography. Compare the lanes having no protein to the lanes with RNA samples bound to protein (see Fig. 1).
RELATED INFORMATION
For alternative footprinting techniques, see Mapping RNA–Protein Interactions Using Iodine Footprinting (Nilsen 2014b) and RNase Footprinting to Map Sites of RNA–Protein Interactions (Nilsen 2014c).
FIGURE 1. Example of hydroxyl-radical footprinting. The gel shows results obtained when an end-labeled nematode SL RNA without associated proteins (lanes 0), bound by Sm proteins (lane −), or bound by Sm proteins plus SL RNP-specific proteins (lane +) was exposed to hydroxyl radicals. A clear footprint is present in the − and + lanes. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080952
1335
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
T.W. Nilsen
RECIPE Urea Loading Dye
Reagent Urea Tris–HCl (1 M, pH 8.0) EDTA (0.25 M) Bromphenol blue/xylene cyanol (1%, w/v) ddH2O
Quantity (for 10 mL)
Final concentration
4.8 g 200 µL 40 µL 0.5 mL to 10 mL
8M 20 mM 1 mM 0.05% (w/v)
REFERENCES Nilsen TW. 2014a. 3′ -End labeling of RNA with [5′ -32P]cytidine 3′ ,5′ -bis (phosphate) and T4 RNA ligase 1. Cold Spring Harb Protoc doi: 10.1101/ pdb.prot080713. Nilsen TW. 2014b. Mapping RNA–protein interactions using iodine footprinting. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080960. Nilsen TW. 2014c. RNase footprinting to map sites of RNA–protein interactions. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080788. Rio DC. 2012. Filter-binding assay for analysis of RNA–protein interactions. Cold Spring Harb Protoc doi: 10.1101/pdb.prot071449. Rio DC. 2014a. 5′ -End labeling of RNA with [γ-32P]ATP and T4 polynucleotide kinase. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080739. Rio DC. 2014b. 3′ -End labeling of RNA with yeast poly(A) polymerase and 3′ -deoxyadenosine 5′ -[α-32P]triphosphate. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080770.
1336
Rio DC. 2014c. Electrophoretic mobility shift assays for RNA-protein complexes. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080721. Rio DC, Ares M, Hannon GJ, Nilsen TW. 2010a. Purification of RNA by SDS solubilization and phenol extraction. Cold Spring Harb Protoc doi: 10.1101/pdb.prot5438. Rio DC, Ares M, Hannon GJ, Nilsen TW. 2010b. Ethanol precipitation of RNA and the use of carriers. Cold Spring Harb Protoc doi: 10.1101/pdb .prot5440. Rio DC, Ares M, Hannon GJ, Nilsen TW. 2010c. Polyacrylamide gel electrophoresis of RNA. Cold Spring Harb Protoc doi: 10.1101/pdb .prot5444.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080952
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
Mapping RNA−Protein Interactions Using Hydroxyl-Radical Footprinting Timothy W. Nilsen Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080952 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Characterization of Protein Complexes (66 articles) Electrophoresis (22 articles) Electrophoresis of Nucleic Acids (38 articles) Electrophoresis of RNA (14 articles) Molecular Biology, general (1020 articles) RNA (226 articles) RNA, general (187 articles)
To subscribe to Cold Spring Harbor Protocols go to:
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© 2014 Cold Spring Harbor Laboratory Press
Protocol
Mapping RNA–Protein Interactions Using Hydroxyl-Radical Footprinting Timothy W. Nilsen
The binding of a protein to an RNA sequence protects the region of the RNA from cleavage by chemicals or RNases; this protected region is known as the protein’s “footprint.” In the footprinting protocol presented here, end-labeled RNAs with and without bound protein are cleaved using chemical methods. Fe(II)–EDTA is used to generate hydroxyl radicals in the presence of a reducing agent. These hydroxyl radicals indiscriminately cleave ribose groups in regions of the ribose–phosphate backbone that are exposed to solvent. After termination of cleavage, the resulting RNA fragments are analyzed by gel electrophoresis on denaturing polyacrylamide gels. Because hydroxyl radicals are smaller and cleave less specifically than RNases, this approach, if feasible, is often the method of choice for monitoring sites of RNA–protein interactions.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPE: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Ammonium iron(II) sulfate (50 mM) Freeze in 1-mL aliquots and store at −20˚C.
Ascorbic acid (0.25 M) Freeze in 1-mL aliquots and store at −20˚C.
Binding buffer specific for the protein and RNA of interest Before carrying out hydroxyl-radical footprinting, determine the optimum binding parameters for the protein and RNA using gel shifts (see Electrophoretic Mobility Shift Assays for RNA–Protein Complexes [Rio 2014c]) or filter binding (see Filter-Binding Assay for Analysis of RNA–Protein Interactions [Rio 2012]). It may be necessary to modify the binding buffer used in these assays to find conditions that are compatible with hydroxyl-radical cleavage. Use buffers such as HEPES instead of Tris and omit glycerol and bovine serum albumin (BSA). Glycerol and Tris must be absent or their concentrations greatly reduced for hydroxyl-radical footprinting. The glycerol concentration in the final binding reaction should be no more than 0.5%.
Denaturing polyacrylamide gels and electrophoresis reagents EDTA (0.2 M, pH 8.0)
Adapted from RNA: A Laboratory Manual by Donald C. Rio, Manuel Ares Jr, Gregory J. Hannon, and Timothy W. Nilsen. CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080952
1333
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
T.W. Nilsen
Ethanol (100%) Extract or protein for binding reactions GlycoBlue (10 µg/µL) Hydrogen peroxide (H2O2; 30%) (Fisher H325–100) Store 30% stock tightly capped at 4˚C.
Phenol RNA of interest, 32P-labeled and gel-purified Either 5′ - or 3′ -end-labeled RNA can be used, but it is often advisable to analyze 5′ - and 3′ -end-labeled RNA in parallel. To prepare labeled RNA, see 5′ -End Labeling of RNA with [γ- 32P]ATP and T4 Polynucleotide Kinase (Rio 2014a), 3′ -End Labeling of RNA with [5′ - 32P]Cytidine 3′ ,5′ -Bis(phosphate) and T4 RNA Ligase 1 (Nilsen 2014a), or 3′ -End Labeling of RNA with Yeast Poly(A) Polymerase and 3′ -Deoxyadenosine 5′ -[γ- 32P]Triphosphate (Rio 2014b).
Sodium acetate (3 M, pH 5.2) Thiourea (0.1 M; Sigma-Aldrich T-7875) Urea loading dye Equipment
Chemical fume hood Dry ice Gel dryer Microcentrifuge Microcentrifuge tubes (1.5 mL) Phosphorimager or autoradiography materials Polyacrylamide gel electrophoresis system and power supply Gel plates must be thoroughly cleaned with a solution of 5% SDS before use.
Water baths or heat blocks at binding temperature (see Step 1) and at 95˚C (see Step 7) Whatman 3MM paper
METHOD Carry out the procedure in a chemical fume hood.
1. Allow the protein to bind to labeled RNA in 25 µL of binding buffer in a 1.5-mL microcentrifuge tube for the appropriate amount of time. Include a control reaction with no protein. Binding must be close to 100% or the footprint will not be clear (purify the complex if necessary).
2. Set up the cleavage reaction. i. Prepare a fresh solution of 2.5% H2O2 by diluting a 30% stock of H2O2 in H2O. ii. Initiate the cleavage reaction by adding 1 µL of each of the following reagents to the side of the 1.5-mL microcentrifuge tube containing the binding reaction: Ammonium iron(II) sulfate EDTA Ascorbic acid H2O2
50 mM 100 mM 250 mM 2.5%
Increase cleavage by adding more Fe(II)–EDTA. However, do not increase the amount of H2O2 as this may affect binding of the protein to the RNA.
iii. Centrifuge the tube in a microcentrifuge for 2–5 sec to collect the reagents in the bottom of the tube with the reaction mix. 1334
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080952
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
Hydroxyl-Radical Footprinting
3. Allow the cleavage reaction to proceed for 5 min at room temperature. It will be necessary to optimize the cleavage times and temperature. Cleavage can be conducted on ice instead of at room temperature, but it will take longer.
4. Stop the reaction by adding 10 µL of 0.1 M thiourea. 5. Recover the RNAs by phenol extraction and ethanol precipitation (adding 10 µg of GlycoBlue as carrier). See Purification of RNA by SDS Solubilization and Phenol Extraction (Rio et al. 2010a) and Ethanol Precipitation of RNA and the Use of Carriers (Rio et al. 2010b).
6. Resuspend the RNA pellet in 200 µL of 2 mM EDTA. Add 600 µL of 100% cold (–20˚C) ethanol and 3 M sodium acetate (pH 5.2) to a final concentration of 0.3 M. Freeze the mixture on dry ice, thaw, and recover the RNAs by centrifuging in a microcentrifuge for 15 min at room temperature. Carefully remove the ethanol supernatant and discard. 7. Resuspend the RNA in 5–10 µL of H2O. Denature in urea loading dye and analyze the cleavage products on the appropriate denaturing polyacrylamide gel. See Polyacrylamide Gel Electrophoresis of RNA (Rio et al. 2010c). Depending on the size of the RNA, it may be necessary to use two different gel percentages for best resolution.
8. Subject the gel to phosphorimaging or autoradiography. Compare the lanes having no protein to the lanes with RNA samples bound to protein (see Fig. 1).
RELATED INFORMATION
For alternative footprinting techniques, see Mapping RNA–Protein Interactions Using Iodine Footprinting (Nilsen 2014b) and RNase Footprinting to Map Sites of RNA–Protein Interactions (Nilsen 2014c).
FIGURE 1. Example of hydroxyl-radical footprinting. The gel shows results obtained when an end-labeled nematode SL RNA without associated proteins (lanes 0), bound by Sm proteins (lane −), or bound by Sm proteins plus SL RNP-specific proteins (lane +) was exposed to hydroxyl radicals. A clear footprint is present in the − and + lanes. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080952
1335
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
T.W. Nilsen
RECIPE Urea Loading Dye
Reagent Urea Tris–HCl (1 M, pH 8.0) EDTA (0.25 M) Bromphenol blue/xylene cyanol (1%, w/v) ddH2O
Quantity (for 10 mL)
Final concentration
4.8 g 200 µL 40 µL 0.5 mL to 10 mL
8M 20 mM 1 mM 0.05% (w/v)
REFERENCES Nilsen TW. 2014a. 3′ -End labeling of RNA with [5′ -32P]cytidine 3′ ,5′ -bis (phosphate) and T4 RNA ligase 1. Cold Spring Harb Protoc doi: 10.1101/ pdb.prot080713. Nilsen TW. 2014b. Mapping RNA–protein interactions using iodine footprinting. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080960. Nilsen TW. 2014c. RNase footprinting to map sites of RNA–protein interactions. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080788. Rio DC. 2012. Filter-binding assay for analysis of RNA–protein interactions. Cold Spring Harb Protoc doi: 10.1101/pdb.prot071449. Rio DC. 2014a. 5′ -End labeling of RNA with [γ-32P]ATP and T4 polynucleotide kinase. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080739. Rio DC. 2014b. 3′ -End labeling of RNA with yeast poly(A) polymerase and 3′ -deoxyadenosine 5′ -[α-32P]triphosphate. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080770.
1336
Rio DC. 2014c. Electrophoretic mobility shift assays for RNA-protein complexes. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080721. Rio DC, Ares M, Hannon GJ, Nilsen TW. 2010a. Purification of RNA by SDS solubilization and phenol extraction. Cold Spring Harb Protoc doi: 10.1101/pdb.prot5438. Rio DC, Ares M, Hannon GJ, Nilsen TW. 2010b. Ethanol precipitation of RNA and the use of carriers. Cold Spring Harb Protoc doi: 10.1101/pdb .prot5440. Rio DC, Ares M, Hannon GJ, Nilsen TW. 2010c. Polyacrylamide gel electrophoresis of RNA. Cold Spring Harb Protoc doi: 10.1101/pdb .prot5444.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080952
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
Mapping RNA−Protein Interactions Using Hydroxyl-Radical Footprinting Timothy W. Nilsen Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080952 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Characterization of Protein Complexes (66 articles) Electrophoresis (22 articles) Electrophoresis of Nucleic Acids (38 articles) Electrophoresis of RNA (14 articles) Molecular Biology, general (1020 articles) RNA (226 articles) RNA, general (187 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
