rna–protein interaction protocols

TM TM Methods in Molecular Biology RNA–Protein Interaction Protocols Edited by Susan R. Haynes VOLUME 118 HUMANA PRESS HUMANA PRESS Methods in Molecular Biology Edited by Susan R. Haynes RNA–Protein Interaction Protocols Labeling and Purification of RNA 1 1 From: Methods in Molecular Biology, Vol. 118: RNA-Protein Interaction Protocols Edited by: S. Haynes © Humana Press Inc., Totowa, NJ 1 Labeling and Purification of RNA Synthesized by In Vitro Transcription Paul A. Clarke 1. Introduction The problems of isolating sufficient quantities of rare RNAs for detailed biochemical analysis can be circumvented by synthesis of the desired RNA in vitro (1–3). Early methods of in vitro transcription included the use of eukary- otic cell extracts or Escherichia coli RNA polymerase to transcribe DNA tem- plates containing the appropriate promoter. Ideally, however, the optimal in vitro transcription system should require simple buffer components without need for preparation of extracts and should precisely initiate and terminate tran- scription at definable sites. In vitro bacteriophage transcription systems fulfill these criteria. Single-stranded RNA of the desired sequence can now be syn- thesized using commercially available SP6, T3, and T7 bacteriophage RNA polymerases that have a very high specificity for their respective promoters. Large quantities of RNA can be synthesized and used as substrates in assays involving translation, RNA processing, microinjection, or transfection. The RNA can be end-labeled for structural analysis or for examining RNA–protein interactions. Alternatively, the RNA can be internally labeled during transcrip- tion and used as a riboprobe for Southern/Northern blots, for RNase protection assays, or in the assays described previously. 1.1. Principle of the Procedure SP6, T3, and T7 bacteriophage RNA polymerases and plasmid vectors con- taining multiple cloning sites flanked by bacteriophage promoters are now available from a large number of commercial sources. The DNA to be tran- scribed is inserted into the polylinker site of the plasmid vector using a restric- tion endonuclease site downstream of the promoter element. The cloned 2 Clarke plasmid is then used as the DNA template for transcription. In preparation for transcription, the template is linearized by restriction endonuclease digestion (see Note 1). The template is then purified and transcribed. Uniform RNA of a single defined length is produced because the RNA polymerase initiates at the defined promoter site and “runs off” the end of the linear template. The 3' end of the RNA is defined by the choice of restriction endonuclease used to linear- ize the template. There are, however, several potential drawbacks to the plas- mid-based approach. In most instances it is difficult to insert the DNA fragment exactly at the site of transcription initiation, while at the 3' end of the template, convenient restriction endonuclease sites located exactly at the 3' end of the sequence to be transcribed are often lacking. This results in RNAs containing additional vector sequences at both the 5' and 3' ends of the RNA (Fig. 1). In some circumstances these extra sequences will not influence the outcome of the experiment. However, if the RNA is used for structural analysis, protein binding, or functional studies the extra sequences may influence both structure and function. This is especially critical in the case of small RNAs in which the extra sequence can exert a large influence. In these circumstances it is crucial that the transcribed sequence is free of vector sequences. This can be achieved easily by using the polymerase chain reaction (PCR) to place the template ex- actly at the transcription initiation site by using a PCR primer containing the core bacteriophage promoter sequence (Fig. 1). The 3' end of the RNA is de- fined by the position of the downstream PCR primer. The PCR fragment can be used directly in the transcription reaction or if desired can be cloned into a plasmid vector by the addition of restriction endonuclease recognition se- quences to the PCR primers (see Note 2). It is also possible to produce small RNAs using a single-stranded oligonucleotide as the template, provided the core promoter sequence is double-stranded (4). The protocols outlined in this chapter describe the in vitro synthesis and gel purification of unlabeled, internally labeled, or end-labeled RNA. 2. Materials 1. RNase inhibitor (RNasin or equivalent). 2. T3, T7, or SP6 bacteriophage RNA polymerases. 3. T4 polynucleotide kinase. 4. T4 RNA ligase. 5. Calf intestinal phosphatase. 6. RNase-free DNase I. 7. RNase-free bovine serum albumin. 8. Distilled phenol, phenol/chloroform (1:1 v/v), and chloroform. 9. Acrylamide, bis-acrylamide, 10% ammonium persulfate (w/v) and TEMED (N,N,N’,N’-tetramethylethylenediamine). Labeling and Purification of RNA 3 10. Nucleotide triphosphates (ATP, CTP, GTP, and UTP). Prepare a stock solution containing each NTP at 25 mM. 11. Dichlorodimethylsilane. 12. Dimethyl sulfoxide (DMSO). 13. 10 mM CaCl 2. 14. 5 M NaCl. 15. Rad tape (Sigma-Aldrich Ltd, Poole, Dorset, BH12 4QH, UK). 16. Siliconized 0.5- and 1.5-mL tubes. 17. Scotch 3M electrical tape (Life Technologies, Paisley, PA4 9RF, UK). 18. [_- 32 P]NTP (800 Ci/mmol; 10 mCi/mL). 19. [a- 32 P]ATP (3000 Ci/mmol; 10 mCi/mL). 20. [ 32 P]pCp (3000 Ci/mmol; 10 mCi/mL). 21. DEPC-H 2 O: add 1 mL of diethyl pyrocarbonate (DEPC) to 1 L of double-dis- tilled H 2 O, mix vigorously, and autoclave (see Note 3). Fig. 1. The DNA template (i) to be transcribed is either (A; ii) cloned into site A of the multiple cloning site (black box) of a vector containing a bacteriophage RNA poly- merase promoter (gray box; transcription initiates at the arrow) or (B; ii) is used as a PCR template with primers 1 and 2. Primer 1 also contains a T7 RNA polymerase promoter sequence (gray box). The templates for transcription (iii) are prepared either by (A) linearizing the vector by restriction endonuclease digestion at site B or (B) by PCR. The templates (iii) are transcribed to give RNA products (iv). The RNA product from (A) will contain additional sequences from the multiple cloning site sequences (black boxes), whereas the RNA product from the PCR-generated template (B) will contain no extra sequences. 4 Clarke 22. 0.5 M EDTA, pH 8.0: dissolve 186 g of ethylenediaminetetraacetic acid·2H 2 O in 800 mL of DEPC-H 2 O and adjust to pH 8.0 with NaOH (approx 20 g). Adjust to 1 L with DEPC-H 2 O, autoclave, aliquot, and store at room temperature. 23. 10× TBE: dissolve 54 g of Tris base and 27.5 g of boric acid in 20 mL of EDTA, pH 8.0, and add DEPC-H 2 O to 1 L. Autoclave and store at room temperature. 24. 30% Acrylamide/bis-acrylamide mix: dissolve 145 g of acrylamide and 5 g of bis- acryla-mide in DEPC-H 2 O to 500 mL, filter sterilize, and store at 4°C in a light- tight bottle. 25. Gel mix: for 500 mL of a denaturing 10% gel mix dissolve 240 g of urea in 25 mL of 10× TBE and 167 mL of 30% acrylamide/bis-acrylamide mix and add DEPC- H 2 O to 500 mL. Some gentle heating may be required to dissolve the urea. Filter sterilize and store at 4°C in a light-tight bottle. Occasionally the urea will crystal- lize out of solution during storage, but can be redissolved by gentle warming to room temperature. Care must be taken to pour the gel quickly, as warming the gel mix will significantly increase the rate of polymer-ization. This is more of a prob- lem with high percentage gels. The percentage of poly-acrylamide in the gel is adjusted according to the size of product being purified (see Subheading 3.5.). 26. 5× Transcription reaction buffer: 200 mM Tris-HCl, pH 7.8, 20 mM dithiothreitol (DTT), 100 mM spermidine, 7 mM MgCl 2 ; filter sterilize and store at –20°C. 27. TE pH 7.4: 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0. 28. 10× Phosphatase reaction buffer: 0.5 M Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0; autoclave and store at –20°C. 29. 10× T4 polynucleotide kinase buffer: 0.5 M Tris-HCl, pH 7.6, 100 mM MgCl 2 , 50 mM DTT, 1 mM spermidine, 1 mM EDTA, pH 8.0; filter sterilize and store at –20°C. 30. 1.5× gel loading buffer: 10 M urea, 1.5× TBE, 0.015% (w/v) bromophenol blue, 0.015% (w/v) xylene cyanol; filter sterilize and store at –20°C. 31. 10× T4 RNA ligase reaction buffer: 0.5 M HEPES-KOH, pH 7.5, 33 mM DTT, 150 mM MgCl 2 ; filter sterilize and store at –20°C. 32. TE/sodium dodecyl sulfate (SDS): add 2.5 mL of 20% (w/v) SDS to 97.5 mL of TE, pH 7.4. 3. Methods 3.1. In Vitro Transcription The following protocol has been optimized for a 160-nucleotide RNA using T7 bacteriophage RNA polymerase (5,6). If large amounts of RNA are required the reaction can be scaled up accordingly. 1. Either digest 10 µg of the transcription vector containing the sequence to be tran- scribed with the appropriate restriction endonuclease or amplify the appropriate sequence by PCR as outlined in Subheading 1.1. (see Notes 1 and 2). Purify the DNA template on an aga-rose gel following digestion or PCR (see Note 4). 2. At room temperature (see Note 5) combine 4 µL of 5× transcription reaction buffer, 3.2 µL of 25 mM NTPs, 0.8 µL (25 U) of RNasin, 1 µL of 1 mg/mL gel- Labeling and Purification of RNA 5 purified DNA, 2 µL (20 U) of T7 bacteriophage RNA polymerase, and 9 µL of DEPC-treated H 2 O. Incubate at 37°C for 2 h (see Note 6). To avoid problems with condensation use a cabinet incubator or overlay with RNase-free mineral oil and incubate in a water bath or heating block. 3. The DNA template is removed by the addition of 2 µL of 10 mM CaCl 2 and RNase- free DNase I to 20 µg/mL followed by incubation at 37°C for 15 min. Add 1 µL of 0.5 M EDTA, pH 8.0, 3 µL of 5 M NaCl, and TE, pH 7.4, to a final volume of 100 µL (see Note 7). 4. Add an equal volume of phenol/chloroform. Vortex the mixture vigorously for 1–2 min and microcentrifuge at room temperature for 10 min at full speed (ap- prox 16,000g). Recover the upper aqueous phase and extract with an equal vol- ume of chloroform as before. 5. Collect the upper aqueous phase and recover the RNA by the addition of 2.5 vol of ethanol. Incubate in a dry ice/ethanol bath for 10–20 min. Precipitate the RNA by microcentrifugation at 4°C for 15 min at 16,000g. Wash the RNA pellet once with ice-cold 70% ethanol and pellet as before. 6. Resuspend the pellet in DEPC-treated H 2 O and store at –80°C. A number of factors will influence the yield; 25–50 µg/reaction is usual, but can be as high as 100 µg/reaction (see Notes 6 and 8). 7. The quality of the RNA should be checked by electrophoresis on a 2% agarose minigel (see Note 9). If required, the RNA can be purified on a polyacrylamide gel (see Notes 4 and 10) (Subheading 3.5.). 3.2. Internal Labeling of Substrate RNA The RNA can be internally labeled during the transcription process by in- corporation of a radiolabeled precursor (see Note 11). 1. Perform the in vitro transcription exactly as described under Subheading 3.1. in the presence of 5 µL of [_< 32 P]UTP or CTP (800 Ci/mmol; 10 mCi/mL). Omit the unlabeled UTP or CTP or keep at a concentration of 10–50 µM (see Note 10). Incu- bate for a maximum of 1 h. 2. Following DNase digestion load the RNA on a denaturing polyacrylamide gel and purify the full-length labeled RNA as described in Subheading 3.5. (see Note 7). 3.3. 5' End-Labelling of Substrate RNA RNA is labeled at its 5' end by T4 polynucleotide kinase. This enzyme trans- fers 32 P from [a- 32 P]ATP onto the 5' end of the RNA molecule. However, to 5' end-label one has to first remove the 5' phosphate using calf intestinal phos- phatase. 1. Dilute 1–2 µg of RNA to a final volume of 16 µL in DEPC-treated H 2 O. Denature the RNA by incubation at 95°C for 2 min followed by quenching on ice for 5 min. 2. Add the remaining components: 2 µL of 10× phosphatase reaction buffer and 2 µL of calf intestinal phosphatase (1 U/µL); incubate at 37°C for 30 min. 6 Clarke 3. Dilute the RNA to 200 µL with TE, pH 7.4, add 6 µL of 5 M NaCl, and extract/ precipitate the RNA as described in Subheading 3.1. 4. Resuspend the RNA to 0.5 µg/µL in TE, pH 7.6. Denature 2 µL of the RNA at 95°C for 2 min and quench on ice for 5 min (see Note 12). 5. Add 1 µL of 10× T4 polynucleotide kinase buffer, 4 µL of [a- 32 P]ATP (3000 Ci/ mmol; 10 mCi/mL), and 1 µL of T4 polynucleotide kinase (3–6 U/µL). Incubate for 30 min at 37°C. 6. Add 20 µL of 1.5× gel loading buffer and purify the labeled RNA by denaturing polyacrylamide gel electrophoresis (Subheading 3.5.). 3.4. 3' End-Labeling of Substrate RNA The addition of label to the 3' ends of RNA uses T4 RNA ligase and an excess of [ 32 P]pCp that is ligated to the 3' end of the RNA. 1. Denature 1–2 µg of RNA by heating to 68°C for 2 min and then quenching on ice for 5 min (see Note 13). 2. Add 4 µL of 10× T4 RNA ligase reaction buffer, 4 µL of DMSO, 1 µL of 20 µM ATP, 1 µL of 10 µg/mL RNase-free bovine serum albumin, 1–2 µg of RNA, 5–10 µL of [ 32 P]pCp (3000 Ci/mmol; 10 mCi/mL), and DEPC-treated H 2 O to a final volume of 40 µL. Incubate overnight at 4°C (see Note 13). 3. Add 80 µL of 1.5× gel loading buffer and purify the labeled RNA by denaturing polyacrylamide gel electrophoresis (Subheading 3.5.). 3.5. Polyacrylamide Gel Purification of Labeled RNA For almost all purposes gel purification of the full-length RNA is essential. The majority of commercially available or homemade sequencing systems can be used. The protocol described here is for the Gibco-BRL S2 system using 31 cm × 38.5 cm × 0.4 mm gels. 1. Clean the gel plates scrupulously with soap and water and rinse in deionized water. Treat the gel plates by wiping with 5% dichlorodimethylsilane (in chloro- form), allow to dry in a fume hood, and finally wipe with ethanol and acetone. Assemble the plates with two spacers and secure using bulldog clips or Scotch 3M electrical tape. 2. Mix 75 mL of gel mix (Subheading 2.5.) with 75 µL of TEMED and 450 µL of fresh 10% ammonium persulfate. Immediately pour the mix between the plates. A number of alternate techniques for pouring gels can be employed according to personal preference. Insert the gel comb and allow 15–45 min for the gel to poly- merize. To accommodate the larger volumes of the labeling reactions use a comb that forms a 2–4 cm well (usually cut from an old sharkstooth comb or spacer). 3. Remove the comb and immediately remove unpolymerized acrylamide by flush- ing the wells with deionized water or running buffer (0.5× TBE). Assemble the apparatus using 0.5× TBE (Subheading 2.5.) as the running buffer and prerun the gel at a constant 55 W power setting until the external gel plate is hot to touch (30–60 min). Incubate the sample for 10 min at 68°C and load immediately. Elec- Labeling and Purification of RNA 7 trophorese the sample at 55 W constant power until the full-size product migrates to approximately a third to a halfway down the gel. The time of running depends on the size of the product and percentage of gel used. On 5%, 10%, and 20% gels RNAs of 135, 60, and 25 nucleotides, respectively, migrate with the xylene cyanol dye front, while RNAs 35, 10, and 5 nucleotides in length migrate with the bro- mophenol blue dye front. 4. Disassemble the apparatus, taking care with the bottom buffer tank (containing unincorporated radionucleotides), and separate the plates using a thin spatula or razor blade. Cover the gel/plate with plastic wrap. 32 P labeled RNA can be de- tected by direct autoradiography as detailed in step 5, whereas unlabeled RNA is detected by UV shadowing as described in step 6. 5. Stick several fluorescent markers (see Note 14) around the edge of the gel and autoradiograph briefly (generally 1–5 min) to locate the intact RNA species. Mark the band corresponding to the full-length RNA on the autoradiograph, align the film with the gel using the image of the fluorescent markers, and secure firmly with tape. Cut out the band using a sterile scalpel, remove the plastic wrap from the gel slice, and transfer the gel slice to a 1.5-mL sterile screw-capped Eppendorf tube. The gel can be reautoradiographed to confirm that the correct band has been cut. 6. To UV shadow, turn the plastic wrap covered plate over. Carefully remove the other gel plate and cover the gel with plastic wrap. Place the plastic wrapped gel on a fluorescent thin-layer chromatography plate or an intensifying screen. The RNA can be visualized using a hand-held UV light set at a short wavelength (254 nm). The RNA band will be seen as a dark purplish band and is cut using a sterile scalpel. Remove the plastic wrap from the gel slice and place the gel slice in a 1.5-mL sterile screw-capped Eppen-dorf tube. 7. Elute the RNA from the gel slice by the addition of 400 µL of TE/SDS and incu- bate for 2–4 h at room temperature on a rotator. Remove the supernatant to a fresh screw-capped tube and store. Add an additional 400 µL of TE/SDS elution buffer to the gel slice and elute for an additional 2–4 h. 8. Extract the supernatants with equal volumes of phenol/chloroform and chloro- form as described in Subheading 3.1. 9. Precipitate the RNA by the addition of 12.5 µL of 5 M NaCl and 2.5 vol of etha- nol as described in Subheading 3.1. Resuspend the RNA in a small volume of DEPC-treated H 2 O. The cpm of labeled RNA can be quickly estimated by Ceren- kov counting the aqueous solution in a scintillation counter. 4. Notes 1. When linearizing the transcription vector it is often preferable to use a double digest; this minimizes copurifying undigested plasmid that can give rise to high molecular weight RNAs. In addition, Schenborn and Mierindorf (7) have reported the production of complementary RNA from templates that have 3' overhanging ends (e.g., those produced by KpnI, PstI, and SacI). To avoid this problem it is best to choose restriction endonucleases that give a blunt 8 Clarke end or a 5' overhang. Mellits et al. (6) have also reported the presence of low levels of double-stranded RNAs (dsRNAs) that are produced during in vitro transcription with bacteriophage RNA polymerases. Their presence may com- plicate analysis of proteins with dsRNA binding motifs (dsRBMs) (5). In this case sequential purification through denaturing and native polyacrylamide gels is required for their complete removal. 2. The following promoter sequences should be added to the PCR primer that is located upstream of the sequence to be transcribed. The core promoter sequence is in the bold type-face. T3 — 5'-GCATGCAATTAACCCTCACTAAAGGG-3' T7 — 5'-GCATGCTAATACGACTCACTATAGGG-3' SP6 — 5'-GCATGCATTTAGGTGACACTATAGAA-3' The first six nucleotides downstream of the promoter are important for tran- scription efficiency (4). This is especially true for the first three nucleotides, GGG for T3 and T7 and GAA for SP6, that should not be changed. 3. All stock solutions or reaction buffers should be made with DEPC-treated double- distilled H 2 O and sterilized by autoclaving or filtration. 4. It is not always necessary to gel purify templates generated by PCR, provided the PCR reaction gives a single product. However, before transcription we gener- ally remove the PCR primers using one of a number of commercially available PCR “clean-up” kits. 5. The transcription reaction should be set up at room temperature, as the reaction buffer contains spermidine which will precipitate the template DNA at low temperatures. 6. The yield of RNA is somewhat template dependent and is also affected by the choice of polymerase, the incubation temperature, and incubation time. Two hours at 37°C is suggested as a starting point and will usually give a yield suffi- cient for most purposes. The yield of transcription can be improved by increasing either the incubation time or temperature (up to 40°C) or both (if a large yield is crucial; see ref. 3 for further details). 7. During polyacrylamide gel purification some template DNA may copurify with the RNA; therefore DNase digestion is absolutely necessary prior to gel purification. 8. The yields of RNA can be estimated by measuring absorbance at A 260 (1OD 260 = 40 µg/mL). Unincorporated nucleotides may coprecipitate with the full-length RNA and will influence the A 260 . If accurate quantitation is required it is best to either gel purify the full-length RNA or remove unincorporated nucleotides by gel filtration through Sephadex G-25 or G-50. Alternatively, two sequential precipita- tions with 1/10 vol of 5 M NH 4 OAc and 3 vol of ethanol will eliminate most of the unincorporated nucleotides. If radioactive nucleotides are included, incorporation can be estimated by trichloroacetic acid precipitation and scintillation counting. 9. Agarose minigels are usually run under nondenaturing conditions. RNAs ana- lyzed in this way will occasionally give multiple bands, probably due to the single RNA species adopting a number of different structural conformations. These can usually be resolved to a single band by heating the RNA sample to 68°C and quenching on ice prior to analysis. If there is still concern, the RNA should be Labeling and Purification of RNA 9 analyzed on denaturing urea polyacrylamide gels or denaturing formaldehyde agarose gels. If prematurely terminated RNA products are detected, the most likely cause is the presence of sequences resembling factor-independent tran- scription termination sequences. These consist of a GC-rich hairpin loop followed by a poly(U) tract. In most cases sufficient full-length RNA can be still be recov- ered by gel purification. If larger yields are required, premature termination can sometimes be overcome by increasing nucleotide concentrations or lowering the incubation temperature while increasing the reaction time (8). 10. RNAs can either be trace labeled for gel purification purposes or labeled at high specific activity for other uses. The concentration of nonradioactive limiting nucleotide will depend on the required specific activity. The greater the concen- tration of unlabeled limiting nucleotide the lower the specific activity of the RNA. For trace labeling full concentrations of all nucleotides and 1–10 µCi of an [_- 32 P]NTP are used. For very high specific activities the nonradioactive nucleotide should be omitted. At nucleotide concentrations <5 µM the yield of full-length RNA will decline dramatically (especially true for longer RNAs). Generally the final concentration of limiting nucleotide (nonradioactive plus radioactive) should be between 10 and 50 µM. The specific activity of the labeled nucleotide should be around 800 Ci/mmol; higher specific activity label is generally less concentrated, requiring the addition of unlabeled nucleotide to get the concentra- tion above 10 µM. If the limiting nucleotide is found within approximately the first 10 nucleotides of the substrate there may be problems with premature termi- nation. This can be avoided by increasing the limiting nucleotide concentration or by using a nucleotide not found in the first 10 nucleotides. We have found this is occasionally a problem with [_- 32 P]CTP and T3 bacteriophage RNA poly- merase and can also be a problem with [_- 32 P]UTP (9). Another problem with limiting nucleotide concentrations is premature termination of transcription that is template dependent. This can be overcome by increasing nucleotide concentrations or by lowering the incubation temperature, but increasing the reaction time (8). 11. This method can also be used for nonisotopic labeling of RNA. Biotin-14-CTP, biotin-16-UTP, digoxigenin-11-CTP, and fluorescein-12-UTP can all be incor- porated into RNA by in vitro transcription with bacteriophage RNA polymerases. 12. T4 polynucleotide kinase requires single-stranded 5' ends for efficient labeling and does not work efficiently on recessed 5' ends. To avoid this problem the RNA should be denatured prior to labeling. If 5' labeling is still a problem it is possible to “cap” in vitro transcripts using [_- 32 P]GTP and guanylyltransferase (GTP transferase) (10). 13. T4 RNA ligase prefers single-stranded 3' termini, and prior denaturation of the RNA may improve labeling. If necessary the efficiency of 3' labeling can also be improved by increasing the time of incubation to a maximum of 96 h. 14. After being marked with an ordinary pen and briefly exposed to light, the fluo- rescent labels will give a unique image on the autoradiograph (within 1–2 min). An alternative is to cut the fluorescent label into unique shapes that can be easily identified. [...]... (1998) Uses of site-specifically modified RNAs constructed by RNA ligation, in RNA–Protein Interactions: A Practical Approach (Smith, C., ed.), Academic, New York, pp 75–108 2 Zimmermann, R A., Gait, M J and Moore, M J (1998) Incorporation of modified nucleotides into RNA for studies on RNA structure, function and intermolecular interactions, in Modification and Editing of RNA: The Alteration of RNA Structure... A and Cech, T R (1993) Tertiary interactions with the internal guide sequence mediate docking of the P1 helix into the catalytic core of the Tetrahymena ribozyme Biochemistry 32, 13,593–13,604 28 Moore, M J and Sharp, P A (1993) The stereochemistry of pre-mRNA splicing: evidence for two active sites in the spliceosome Nature 365, 364–368 RNA–Protein Crosslinking 21 3 RNA–Protein Crosslinking with Photoreactive... chemically reactive species upon irradiation The yield of From: Methods in Molecular Biology, Vol 118: RNA-Protein Interaction Protocols Edited by: S Haynes © Humana Press Inc., Totowa, NJ 21 22 Hanna, Bentsen, Lucido, and Sapre Fig 1 Nucleotide analogs for analysis of protein–nucleic acid interactions Four ribonucleoside triphosphate analogs are described 5-azido(phenacylthio)-uridine-5'triphosphate... 3.3 for RNA–protein crosslinking 3.3 Crosslinking of RNA to Protein (Fig 2B) 1 Using the optimal UTP or CTP concentration determined in the previous section for protein–RNA crosslinking, repeat steps 1–9 of Subheading 3.1 Resuspend the RNA in distilled, deionized, autoclaved water or the buffer appropriate for 28 2 3 4 5 6 7 8 9 10 11 Hanna, Bentsen, Lucido, and Sapre the specific RNA–protein interaction. .. analysis of protein-RNA Interactions Nucleic Acids Res 73, 1231–1238 RNA–Protein Crosslinking 33 5 Hanna, M M., Zhang, Y., Reidling, J C., Thomas, M J., and Jou, J (1993) Synthesis and characterization of a new photocrosslinking CTP analog and its use in photoaffinity labeling E coli and T7 RNA polymerases Nucleic Acids Res 21, 2073–2079 6 Liu, K and Hanna, M M (1995) NusA interferes with interactions between... efficient for single-stranded RNA In our experience, MB and UV crosslinking are excellent complementary approaches that detect distinct arrays of RNA–protein interactions (see, e.g., Fig 2) We originally demonstrated that MB crosslinking could be used to detect interactions of dsRNA with either purified proteins or with dsRNA binding proteins in crude cell extracts (12) However, various other applications... various control experiments had to be carried out to verify the functional specificity of the interaction Both the proteins detected, U5 116kDa and the unidentified 65-kDa protein, were undetectable by UV crosslinking Thus these examples show that MB crosslinking can be harnessed to extend the range of RNA–protein interactions that are accessible to analysis by crosslinking Finally, it is worth noting that... 1992 (5), RNA ligations were performed either chemically or with T4 RNA ligase Although both of these approaches have advantages in particuFrom: Methods in Molecular Biology, Vol 118: RNA-Protein Interaction Protocols Edited by: S Haynes © Humana Press Inc., Totowa, NJ 11 12 Moore lar situations, both also have important limitations Chemical coupling with cyanogen bromide or water-soluble carbodiimides... RNAbinding protein (at a concentration appropriate for that interaction) to the RNA (see Note 13) (6,7) Split the reaction in half and place one half in a 1-mL colorless polystyrene microcentri-fuge tube Place the polystyrene tube 1.5 cm above the UV light source and irradiate for 2 min at room temperature, unless the temperature must be controlled for the interaction being examined Keep the other half of the... Sapre added, only those mercapto groups that are accessible (analogs on surface) will be modified and have the ability to crosslink This approach allows one to distinguish between interactions with the surface binding sites and interactions with proteins that occur when the RNA is buried within a complex 12 The Sephadex G-50 column is a size-exclusion column The large RNA will flow through the column, . Biology RNA–Protein Interaction Protocols Edited by Susan R. Haynes VOLUME 118 HUMANA PRESS HUMANA PRESS Methods in Molecular Biology Edited by Susan R. Haynes RNA–Protein Interaction Protocols Labeling. Haynes RNA–Protein Interaction Protocols Labeling and Purification of RNA 1 1 From: Methods in Molecular Biology, Vol. 118: RNA-Protein Interaction Protocols Edited by: S. Haynes © Humana Press Inc., Totowa, NJ 1 Labeling and Purification. microinjection, or transfection. The RNA can be end-labeled for structural analysis or for examining RNA–protein interactions. Alternatively, the RNA can be internally labeled during transcrip- tion and