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Detecting RNA−RNA Interactions Using Psoralen Derivatives Timothy W. Nilsen Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080861 Email Alerting Service Subject Categories
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Protocol
Detecting RNA–RNA Interactions Using Psoralen Derivatives Timothy W. Nilsen
Psoralens are tricyclic compounds that intercalate into double-stranded DNA or RNA and, on irradiation with long-wavelength (365-nm) UV light, covalently link pyrimidines on adjacent strands. More rarely, psoralen cross-links can be observed at the ends of helices (i.e., double-stranded–single-stranded boundaries). Although psoralens can, in some instances, cross-link protein to RNA, their primary application is to detect RNA–RNA base-pairing interactions. The most useful psoralen derivative is 4′ -aminomethyl trioxsalen (AMT), which is soluble in H2O. This protocol describes the use of AMT to detect RNA–RNA interactions in tissue culture cells or in extracts. Cross-linked RNAs are detectable by their reduced mobility in polyacrylamide gels. Cross-links can be reversed by exposure to short-wavelength (254 nm) UV light.
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
4′ -Aminomethyl trioxsalen (AMT; 1 mg/mL in H2O) Cell culture and appropriate growth medium (see Step 7) Cell extract (see Preparation of Nuclear Extracts from HeLa Cells [Nilsen 2013]) Ethanol (100%) In vitro transcription reagents for preparation of RNA sample (see Step 1) The plasmid to be transcribed should encode the RNA of interest.
Oligodeoxyribonucleotides complementary to the candidate target RNAs (see Step 13) Phenol:chloroform:isoamyl alcohol (25:24:1) Phosphate-buffered saline (PBS) Polyacrylamide gels and electrophoresis reagents Proteinase K (10 mg/mL) RNase H and reagents for cleavage of nuclease-resistant RNA (see Step 13) SDS extraction buffer Sodium acetate (3 M, pH 5.2) TRIzol 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.prot080861
996
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIFORNIA Santa Cruz Library on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Detecting RNA–RNA Interactions
Equipment
Conical centrifuge tube (see Step 10) Denaturing polyacrylamide gel electrophoresis system and power supply Glass plate (2-mm thick, such as a gel plate) Ice Incubator or water bath at 42˚C (see Step 6) Metal rack or square pipette can lid Microcentrifuge Microcentrifuge tubes (1.5 mL) Parafilm Petri dish or chilled metal block on ice Phosphorimager or X-ray film Styrofoam or 3-inch-high plastic box Tissue culture dish (multiwell with lid) UV lamp (365 nm) or Stratalinker with 365-nm bulbs METHOD Cross-Linking in Cell Extracts
1. Prepare 32P-labeled RNA by in vitro transcription. See In Vitro Transcription of Labeled RNA: Synthesis, Capping, and Substitution (Nilsen and Rio 2012). Unlabeled RNA can be prepared by omitting the label from the transcription reaction. If desired, this unlabeled RNA product may be labeled at either the 5′ or 3′ end. 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′ -[α-32P]Triphosphate (Rio 2014b).
2. Incubate the RNA in the cellular extract using the appropriate reaction conditions. Include controls of unincubated reactions or reactions with conditions designed to be inhibitory. 3. Chill the samples on ice, and then add psoralen (AMT) to 40 µg/mL. 4. Spot the reaction mixes (10–50 µL) onto Parafilm in an open Petri dish or on a chilled metal block kept on ice. 5. Place a 2-mm glass plate between the UV source and the samples to block shortwave irradiation. Irradiate the solution on ice with 365-nm UV light (handheld or in a Stratalinker with 365-nm bulbs) for times to be determined empirically (10–30 min). If longer times are required, make sure that the samples remain ice cold. Be careful not to irradiate too long or the RNA will be damaged. Cross-links are reversible by exposure to shortwave UV light (254 nm) for 10–15 min at 4˚C.
6. After cross-linking, transfer the extract to a 1.5-mL microcentrifuge tube, dilute with 200 µL of SDS extraction buffer, and digest with 1 mg/mL proteinase K for 15 min at 42˚C. Recover the RNA by extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitation with sodium acetate and ethanol. For details, 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). Proceed to Step 12 for analysis of cross-linked RNA.
Cross-Linking in Cells
7. Add AMT to cells as follows: i. For suspension cell cultures, centrifuge and resuspend the cells in growth medium at a concentration of 2 × 107 cells/mL. Aliquot the resuspended cells into a multiwell tissue culture plate and add AMT to each well. Titrate the amount of AMT by using 0, 5, 10, 20, and 50 µg/mL. Use a Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080861
997
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIFORNIA Santa Cruz Library on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
T.W. Nilsen
concentrated stock of AMT diluted in fresh media and mix by pipetting up and down. Set one sample aside as a nonirradiated control. ii. For monolayer cultures, remove the medium from the cell monolayers and replace it with medium containing the desired amount of AMT. Use just enough medium to cover the monolayer surface and prevent the cells from drying out. 8. Incubate the cells under growth conditions for 5 min and then chill on ice. 9. Remove the plate lids and place the plates on ice on a level metal block. Cover with a 2-mm-thick glass plate and irradiate the cells for 15 min from a distance of 2.5 cm with a handheld 365-nm light or in a Stratalinker with 365-nm bulbs. Be careful not to irradiate too long or the RNA will be damaged. Cross-links are reversible by exposure to shortwave UV light (254 nm) for 10–15 min at 4˚C.
10. After cross-linking, transfer the suspension cells to a conical centrifuge tube and pellet the cells at 1000g for 5 min. If using adherent cells, leave on the plate until the cells are lysed. 11. Wash the cells with PBS and then lyse the cells and extract the RNA using TRIzol (see Purification of RNA Using TRIzol [TRI Reagent] [Rio et al. 2010c]). Recover the RNA by precipitation with ethanol (see Ethanol Precipitation of RNA and the Use of Carriers [Rio et al. 2010b]). Analyzing Cross-Linked RNA
12. Resuspend the precipitated RNA in H2O, add denaturing loading dye, and analyze the samples (including the nonirradiated control from Step 7) by electrophoresis through a denaturing polyacrylamide gel as described in Polyacrylamide Gel Electrophoresis of RNA (Rio et al. 2010d). Compare the cross-linked and noncross-linked control RNAs.
A
B 1 2 3 4 5 6 7
1 2 3 4 5 6 7 a
M 622 527 404 309
b c
240 217 201 190 180 160 147 122
FIGURE 1. Example of psoralen cross-linking to identify interacting RNA. In this case, a radiolabeled nematode SL RNA was incubated in whole-cell extract and then cross-linked with psoralen. (A) Bands a, b, and c appeared to be crosslinked species because of their mobility. To determine which, if any, of the cross-linked species resulted from interaction with another snRNP, the RNAs were digested with RNase H in the presence of a panel of oligodeoxynucleotides complementary to various RNAs. Lane 1 was undigested, and lane 2 was digested with an oligodeoxynucleotide complementary to the SL RNA. Although oligodeoxynucleotides complementary to U2, U4, U5, and U1 snRNAs (lanes 3–6) did not elicit degradation, an oligodeoxynucleotide complementary to U6 snRNA (lane 7) did cause fragmentation in the presence of RNase H. (B) Digestion of a purified band (b) with RNase H as in A. These results, among others, showed that the SL RNA interacts with U6 snRNP via base-pairing. (Reprinted, with permission, from Hannon et al. 1992.)
998
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080861
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIFORNIA Santa Cruz Library on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Detecting RNA–RNA Interactions
TABLE 1. Advantages and disadvantages of using psoralens for cross-linking Advantages
Disadvantages
Can be used in vitro or in vivo
Less effective at detecting RNA–RNA interactions not mediated by base-pairing Poor for cross-linking protein to RNA
Effective for cross-linking double-stranded RNAs Cross-links readily reversible by irradiation with short (254nm)-wavelength UV light
The cross-linked RNAs will behave like structured RNA on higher-percentage gels (>6%) and will likely run slower than the sum of the sizes of the two RNAs. For example, two RNAs each 100 nucleotides long will migrate slower than a 200-nucleotide RNA in an 8% denaturing polyacrylamide gel. RNA from unlabeled reactions or from tissue culture cells can be detected by northern blot (see Hausner et al. 1990). The RNAs from the gels described in this step are transferred to a hybridization membrane and probed with probes complementary to candidate RNAs.
13. For further analysis of cross-linked RNAs, identify the nonlabeled RNA cross-linked to the substrate by performing an RNase H digestion in the presence of oligodeoxyribonucleotides complementary to candidate target RNAs. After digestion and resolution by electrophoresis, the cross-linked species will show increased mobility (see Fig. 1). Cross-linked RNAs can also be analyzed by primer extension to map the positions of cross-linking. Reverse transcription will “stop” at the cross-linked nucleotides.
DISCUSSION
Cross-linking with psoralen derivatives is the preferred method to identify RNA–RNA interactions, particularly those that are mediated by base-pairing (for the particular advantages and disadvantages of psoralens, see Table 1). AMT has been used successfully to elucidate RNA–RNA interactions in the spliceosome as well as to identify double-strand regions in RNA. It is the agent of choice whenever two RNAs (intermolecular interactions) or double-stranded regions of a single RNA (intramolecular interactions) are suspected to interact via base-pairing.
RELATED INFORMATION
The use of formaldehyde, a more versatile agent for promoting protein-RNA cross-links, is described in Preparation of Cross-linked Cellular Extracts with Formaldehyde (Nilsen 2014b).
RECIPE SDS Extraction Buffer
Reagent
Quantity (for 500 mL)
Final concentration
10 mL 1 mL 25 mL 464 mL
20 mM 1 mM 0.5% (w/v) –
Tris–HCl (1 M, pH 7.5) EDTA (0.5 M, pH 8.0) SDS (10% w/v) H2O Store indefinitely at room temperature. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080861
999
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIFORNIA Santa Cruz Library on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
T.W. Nilsen
REFERENCES Hannon GJ, Maroney PA, Yu YT, Hannon GE, Nilsen TW. 1992. Interaction of U6 snRNA with a sequence required for function of the nematode SL RNA in trans-splicing. Science 258: 1775–1780. Hausner T-P, Giglio LM, Weiner AM. 1990. Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles. Genes Dev 4: 2146–2156. Nilsen TW. 2013. Preparation of nuclear extracts from HeLa cells. Cold Spring Harb Protoc doi:10.1101/pdb.prot075176. 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. Preparation of cross-linked cellular extracts with formaldehyde. Cold Spring Harb Protoc doi:10.1101/pdb.prot080879. Nilsen TW, Rio DC. 2012. In vitro transcription of labeled RNA: Synthesis, capping, and substitution. Cold Spring Harb Protoc doi:10.1101/pdb .prot072066.
1000
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. 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. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc doi:10.1101/pdb .prot5439. Rio DC, Ares M, Hannon GJ, Nilsen TW. 2010d. 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.prot080861
Detecting RNA−RNA Interactions Using Psoralen Derivatives Timothy W. Nilsen Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080861 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. Molecular Biology, general (1013 articles) RNA (217 articles) RNA, general (179 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIFORNIA Santa Cruz Library on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Protocol
Detecting RNA–RNA Interactions Using Psoralen Derivatives Timothy W. Nilsen
Psoralens are tricyclic compounds that intercalate into double-stranded DNA or RNA and, on irradiation with long-wavelength (365-nm) UV light, covalently link pyrimidines on adjacent strands. More rarely, psoralen cross-links can be observed at the ends of helices (i.e., double-stranded–single-stranded boundaries). Although psoralens can, in some instances, cross-link protein to RNA, their primary application is to detect RNA–RNA base-pairing interactions. The most useful psoralen derivative is 4′ -aminomethyl trioxsalen (AMT), which is soluble in H2O. This protocol describes the use of AMT to detect RNA–RNA interactions in tissue culture cells or in extracts. Cross-linked RNAs are detectable by their reduced mobility in polyacrylamide gels. Cross-links can be reversed by exposure to short-wavelength (254 nm) UV light.
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
4′ -Aminomethyl trioxsalen (AMT; 1 mg/mL in H2O) Cell culture and appropriate growth medium (see Step 7) Cell extract (see Preparation of Nuclear Extracts from HeLa Cells [Nilsen 2013]) Ethanol (100%) In vitro transcription reagents for preparation of RNA sample (see Step 1) The plasmid to be transcribed should encode the RNA of interest.
Oligodeoxyribonucleotides complementary to the candidate target RNAs (see Step 13) Phenol:chloroform:isoamyl alcohol (25:24:1) Phosphate-buffered saline (PBS) Polyacrylamide gels and electrophoresis reagents Proteinase K (10 mg/mL) RNase H and reagents for cleavage of nuclease-resistant RNA (see Step 13) SDS extraction buffer Sodium acetate (3 M, pH 5.2) TRIzol 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.prot080861
996
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIFORNIA Santa Cruz Library on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Detecting RNA–RNA Interactions
Equipment
Conical centrifuge tube (see Step 10) Denaturing polyacrylamide gel electrophoresis system and power supply Glass plate (2-mm thick, such as a gel plate) Ice Incubator or water bath at 42˚C (see Step 6) Metal rack or square pipette can lid Microcentrifuge Microcentrifuge tubes (1.5 mL) Parafilm Petri dish or chilled metal block on ice Phosphorimager or X-ray film Styrofoam or 3-inch-high plastic box Tissue culture dish (multiwell with lid) UV lamp (365 nm) or Stratalinker with 365-nm bulbs METHOD Cross-Linking in Cell Extracts
1. Prepare 32P-labeled RNA by in vitro transcription. See In Vitro Transcription of Labeled RNA: Synthesis, Capping, and Substitution (Nilsen and Rio 2012). Unlabeled RNA can be prepared by omitting the label from the transcription reaction. If desired, this unlabeled RNA product may be labeled at either the 5′ or 3′ end. 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′ -[α-32P]Triphosphate (Rio 2014b).
2. Incubate the RNA in the cellular extract using the appropriate reaction conditions. Include controls of unincubated reactions or reactions with conditions designed to be inhibitory. 3. Chill the samples on ice, and then add psoralen (AMT) to 40 µg/mL. 4. Spot the reaction mixes (10–50 µL) onto Parafilm in an open Petri dish or on a chilled metal block kept on ice. 5. Place a 2-mm glass plate between the UV source and the samples to block shortwave irradiation. Irradiate the solution on ice with 365-nm UV light (handheld or in a Stratalinker with 365-nm bulbs) for times to be determined empirically (10–30 min). If longer times are required, make sure that the samples remain ice cold. Be careful not to irradiate too long or the RNA will be damaged. Cross-links are reversible by exposure to shortwave UV light (254 nm) for 10–15 min at 4˚C.
6. After cross-linking, transfer the extract to a 1.5-mL microcentrifuge tube, dilute with 200 µL of SDS extraction buffer, and digest with 1 mg/mL proteinase K for 15 min at 42˚C. Recover the RNA by extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitation with sodium acetate and ethanol. For details, 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). Proceed to Step 12 for analysis of cross-linked RNA.
Cross-Linking in Cells
7. Add AMT to cells as follows: i. For suspension cell cultures, centrifuge and resuspend the cells in growth medium at a concentration of 2 × 107 cells/mL. Aliquot the resuspended cells into a multiwell tissue culture plate and add AMT to each well. Titrate the amount of AMT by using 0, 5, 10, 20, and 50 µg/mL. Use a Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080861
997
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIFORNIA Santa Cruz Library on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
T.W. Nilsen
concentrated stock of AMT diluted in fresh media and mix by pipetting up and down. Set one sample aside as a nonirradiated control. ii. For monolayer cultures, remove the medium from the cell monolayers and replace it with medium containing the desired amount of AMT. Use just enough medium to cover the monolayer surface and prevent the cells from drying out. 8. Incubate the cells under growth conditions for 5 min and then chill on ice. 9. Remove the plate lids and place the plates on ice on a level metal block. Cover with a 2-mm-thick glass plate and irradiate the cells for 15 min from a distance of 2.5 cm with a handheld 365-nm light or in a Stratalinker with 365-nm bulbs. Be careful not to irradiate too long or the RNA will be damaged. Cross-links are reversible by exposure to shortwave UV light (254 nm) for 10–15 min at 4˚C.
10. After cross-linking, transfer the suspension cells to a conical centrifuge tube and pellet the cells at 1000g for 5 min. If using adherent cells, leave on the plate until the cells are lysed. 11. Wash the cells with PBS and then lyse the cells and extract the RNA using TRIzol (see Purification of RNA Using TRIzol [TRI Reagent] [Rio et al. 2010c]). Recover the RNA by precipitation with ethanol (see Ethanol Precipitation of RNA and the Use of Carriers [Rio et al. 2010b]). Analyzing Cross-Linked RNA
12. Resuspend the precipitated RNA in H2O, add denaturing loading dye, and analyze the samples (including the nonirradiated control from Step 7) by electrophoresis through a denaturing polyacrylamide gel as described in Polyacrylamide Gel Electrophoresis of RNA (Rio et al. 2010d). Compare the cross-linked and noncross-linked control RNAs.
A
B 1 2 3 4 5 6 7
1 2 3 4 5 6 7 a
M 622 527 404 309
b c
240 217 201 190 180 160 147 122
FIGURE 1. Example of psoralen cross-linking to identify interacting RNA. In this case, a radiolabeled nematode SL RNA was incubated in whole-cell extract and then cross-linked with psoralen. (A) Bands a, b, and c appeared to be crosslinked species because of their mobility. To determine which, if any, of the cross-linked species resulted from interaction with another snRNP, the RNAs were digested with RNase H in the presence of a panel of oligodeoxynucleotides complementary to various RNAs. Lane 1 was undigested, and lane 2 was digested with an oligodeoxynucleotide complementary to the SL RNA. Although oligodeoxynucleotides complementary to U2, U4, U5, and U1 snRNAs (lanes 3–6) did not elicit degradation, an oligodeoxynucleotide complementary to U6 snRNA (lane 7) did cause fragmentation in the presence of RNase H. (B) Digestion of a purified band (b) with RNase H as in A. These results, among others, showed that the SL RNA interacts with U6 snRNP via base-pairing. (Reprinted, with permission, from Hannon et al. 1992.)
998
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080861
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIFORNIA Santa Cruz Library on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
Detecting RNA–RNA Interactions
TABLE 1. Advantages and disadvantages of using psoralens for cross-linking Advantages
Disadvantages
Can be used in vitro or in vivo
Less effective at detecting RNA–RNA interactions not mediated by base-pairing Poor for cross-linking protein to RNA
Effective for cross-linking double-stranded RNAs Cross-links readily reversible by irradiation with short (254nm)-wavelength UV light
The cross-linked RNAs will behave like structured RNA on higher-percentage gels (>6%) and will likely run slower than the sum of the sizes of the two RNAs. For example, two RNAs each 100 nucleotides long will migrate slower than a 200-nucleotide RNA in an 8% denaturing polyacrylamide gel. RNA from unlabeled reactions or from tissue culture cells can be detected by northern blot (see Hausner et al. 1990). The RNAs from the gels described in this step are transferred to a hybridization membrane and probed with probes complementary to candidate RNAs.
13. For further analysis of cross-linked RNAs, identify the nonlabeled RNA cross-linked to the substrate by performing an RNase H digestion in the presence of oligodeoxyribonucleotides complementary to candidate target RNAs. After digestion and resolution by electrophoresis, the cross-linked species will show increased mobility (see Fig. 1). Cross-linked RNAs can also be analyzed by primer extension to map the positions of cross-linking. Reverse transcription will “stop” at the cross-linked nucleotides.
DISCUSSION
Cross-linking with psoralen derivatives is the preferred method to identify RNA–RNA interactions, particularly those that are mediated by base-pairing (for the particular advantages and disadvantages of psoralens, see Table 1). AMT has been used successfully to elucidate RNA–RNA interactions in the spliceosome as well as to identify double-strand regions in RNA. It is the agent of choice whenever two RNAs (intermolecular interactions) or double-stranded regions of a single RNA (intramolecular interactions) are suspected to interact via base-pairing.
RELATED INFORMATION
The use of formaldehyde, a more versatile agent for promoting protein-RNA cross-links, is described in Preparation of Cross-linked Cellular Extracts with Formaldehyde (Nilsen 2014b).
RECIPE SDS Extraction Buffer
Reagent
Quantity (for 500 mL)
Final concentration
10 mL 1 mL 25 mL 464 mL
20 mM 1 mM 0.5% (w/v) –
Tris–HCl (1 M, pH 7.5) EDTA (0.5 M, pH 8.0) SDS (10% w/v) H2O Store indefinitely at room temperature. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080861
999
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIFORNIA Santa Cruz Library on September 8, 2014 - Published by Cold Spring Harbor Laboratory Press
T.W. Nilsen
REFERENCES Hannon GJ, Maroney PA, Yu YT, Hannon GE, Nilsen TW. 1992. Interaction of U6 snRNA with a sequence required for function of the nematode SL RNA in trans-splicing. Science 258: 1775–1780. Hausner T-P, Giglio LM, Weiner AM. 1990. Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles. Genes Dev 4: 2146–2156. Nilsen TW. 2013. Preparation of nuclear extracts from HeLa cells. Cold Spring Harb Protoc doi:10.1101/pdb.prot075176. 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. Preparation of cross-linked cellular extracts with formaldehyde. Cold Spring Harb Protoc doi:10.1101/pdb.prot080879. Nilsen TW, Rio DC. 2012. In vitro transcription of labeled RNA: Synthesis, capping, and substitution. Cold Spring Harb Protoc doi:10.1101/pdb .prot072066.
1000
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. 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. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc doi:10.1101/pdb .prot5439. Rio DC, Ares M, Hannon GJ, Nilsen TW. 2010d. 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.prot080861
