The spliceosome is a ribonucleoprotein body (RNP) composed of RNA and proteins, which removes introns from precursor mRNA during the splicing process of precursor mRNAs. snRNP, which is composed of snRNA and its binding proteins, plays an important role in the splicing process of precursor mRNAs. This experiment is from "RNA Lab Manual", edited by Xiaofei Zheng.
Operation method
Experiments on the isolation and analysis of pre-splitters and spliceosomes
Principle
The spliceosome is a ribonucleoprotein (RNP) composed of RNA and proteins, which removes introns from precursor mRNAs during splicing. snRNP is composed of snRNA and its binding protein, and plays an important role in the splicing process of precursor mRNA.
Materials and Instruments
PIP 10 Vector Nucleotide Pyrophosphatase RNasin T7 RNA Polymerase mRNA Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Transcription Buffer High Salt Buffer NaAc Ethanol TBE Electrophoresis Buffer Acrylamide Solution Ammonium Persulfate TEMED Denaturing Gel Sampling Buffer Gel Elution Buffer Creatine Phosphate Heparin Density Gradient Solution Amphoteric Electrolyte Solution SDS Gel Silver Stain Solution
Glass Plate Polyacrylamide Gel Electrophoresis Unit
1. In vitro transcription
(1) Template specifically digested for in vitro transcription (PIP 10 vector).
(2) Nucleotides: 10 mmol/L biotin-21-UTP, 10 μCi/μl [ α-32P ] UTP, 10 mmol/L UTP, 10 mmol/L (ATP, CTP, GTP).
(3) 12X transcription buffer: 480 mmol/L Tris-HCl (pH 8.1), 72 mmol/L MgCl2, 12 mmol/L spermidine, 60 mmol/L DTT, 0.2% Triton X-100, 960 g/ml polyethylene glycol (PEG).
(4) Pyrophosphatase (0.05 U/μl).
(5) RNasin (40 U/μl).
(6) T7 RNA polymerase.
(7) High salt buffer: 10 mmol/L Tris-HCl ( pH 7.5), 100 mmol/L LiCl, 10 mmol/L EDTA, 0.5% SDS, 7 mol/L urea.
(8) 3 mol/L NaAc ( pH 5.2).
(9) 100% and 80% ethanol.
2. Purification of precursor mRNA by denaturing gel and analysis of splicing products
(1) Glass plate (27 cm X 16.5 cm ) and polyacrylamide gel electrophoresis device.
(2) 5X TBE electrophoresis buffer: 446 mmol/L Tris, 445 mmol/L boric acid, 1 mmol/L EDTA.
(3) 15% acrylamide solution: 1X TBE, 14.5% acrylamide, 0.5% methylene bisacrylamide, 8 mol/L urea.
(4) 10% ammonium persulfate.
(5) TEMED.
(6) Denaturing Gel Sampling Buffer: 98% formamide, 10 mmol/L EDTA, 0.025% xylene blue, 0.025% bromophenol blue.
(7) Gel deconvolution buffer: 10 mmol/L Tris-HCl (pH 7.5), 0.5 mol/L ammonium acetate, 10 mmol/L magnesium acetate, 0.1 mmol/L EDTA, 0.1% SDS.
3. Splicing reaction
(1) PIP 10 precursor mRNA ( 0.13 mg/ml ).
(2) HeLa NE or DEAE II.
(3) 400 mmol/L KCl.
(4) 100 mmol/L MgCl2.
(5) 10 mmol/L ATP and 50 mmol/L phosphocreatine.
(6) 50 mg/ml heparin.
4. Glycerol gradient
(1) Density gradient solution: 15% or 30% (V/V) glycerol dissolved in 6.4 mmol/L HEPES-KOH (pH 7.9 ), 50 mmol/L KCl, 2 mmol/L MgCl2.
(2) Gradient Marker GA. 2994 ( Gibco-BRL, GrandIsland, NY, USA).
(3) Beckman homogeneous selective crystal (polyllomer ) polymerization centrifuge tube (14 mm X 95 mm).
5. Non-denaturing gel
(1) Glass plate ( 17 cm X 16.5 cm ).
(2) 30% ( m/V ) acrylamide dissolved in distilled water.
(3) 1% ( m/V ) methacrylamide dissolved in distilled water.
(4) 1 mol/L Tris (unbuffered, pH approximately 6.0).
(5) 1 mol/L Glycine (unbuffered, approx. 6.0 ).
(6) 10% ammonium persulfate.
(7) TEMED.
(8) Electrophoresis buffer: 50 mmol/L Tris, 50 mmol/L glycine.
6. Affinity purification
(1) Streptavidin magnetic beads (10 mg/ml).
(2) Bovine serum albumin.
(3) 5X binding buffer: 6.4 mmol/L HEPES-KOH ( pH 7.9), 50 mmol/L KCl, 12.5 mmol/L EDTA, 0.5% Triton X-100.
(4) 1 mol/L DTT.
(5) RNasin (40 U/μl).
(6) Wash buffer 1: 20 mmol/L Tris ( pH 7.9), 250 mmol/L NaCl.
(7) Wash buffer 2: 20 mmol/L Tris ( pH 7.9 ), 100 mmol/L NaCl.
(8) RNase A (0.2 mg/ml).
(9) Elution buffer: 20 mmol/L Tris-HCl ( pH 7.9), 20 mmol/L DTT, 2% SDS.
(10) Glycogen (20 mg/ml ).
(11) Acetone.
7. Bidirectional electrophoresis of proteins
(1) Amphoteric electrolyte solution: 22.4 mmol/L Tris-HCl, 17.6 mmol/L Tris base, 7.92 mol/L urea, 0.06% SDS, 1.76% amphoteric electrolyte of pH 3.0~10, 120 mmol/L DTT, 3.2% Triton X-100.
(2) Pre-made pH 3.0~10 amphoteric electrolyte IEF gel (1 mm X 18 cm) (ESA, Chelmsford, MA ).
(3) IEF electrophoresis buffer: 10 mmol/L phosphate anode solution, 100 mmol/L NaOH cathode solution.
(4) Gel equilibration buffer: 300 mmol/L Tris base, 75 mmol/L Tris-HCl, 50 mmol/L DTT, 3% SDS, 0.01% bromophenol blue.
(5) SDS gel: 373 mmol/L Tris ( pH 8.8), 9% acrylamide, 0.1% SDS, 0.02% ammonium persulfate, 0.005% TEMED.
(6) SDS electrophoresis buffer: 25 mmol/L Tris base, 192 mmol/L glycine, 0.1% SDS.
(7) Silver staining solution: 2% ( m/V) sodium citrate, 0.8% (m/V) ferrous sulfate, 0.1% ( m/V) silver nitrate. Wrap with tin foil and store at 4℃.
II. Methods of operation
1. Transcription
This 1 ml large-scale in vitro transcription reaction yields 100 μg of precursor mRNA after gel purification.
(1) To prevent DNA precipitation, the following components were added sequentially at room temperature: 83.3 μl 12X Transcription Buffer, 100 μl 100 mmol/L MgCl2, 200 μl (10 mmol/L ATP, CTP, GTP), 37.5 μl 10 mmol/L UTP, 37.5 μl 10 mmol/L Biotin-21-UTP, 30 μl 10 μCi/μl [ α-3-UTP ], 30 μl 10 μCi/μl [ α-3-UTP ], and 30 μl 10 mmol/L [ α-3-UTP ]. 30 μl 10 μCi/μl [ α-32P ]-UTP, 2 μl RNasin (40 U/μl), 10 μl pyrophosphatase (0.05 U/μl), 80 μl 1 mg/ml PIP 10 HindIII-digested plasmid (PIP 10), 22 μl T7 RNA polymerase, and finally, distilled water was added to 1 ml.
(2) Incubate for 3 h at 37℃.
(3) Add 1/10 volume of 3 mol/L sodium acetate (pH 5.2) and 2.5 volume of ethanol.
(4) Precipitate on dry ice for 20 min, then centrifuge at 4℃, 16000 g for 20 min. Wash the precipitate with cold 80% ethanol and air dry.
2. Denaturing gel purification of precursor mRNA and analysis of splicing products
(1) Clean and assemble the glass plate.
(2) Add 115 μl of 10% ammonium persulfate and 11.5 μl of TEMED to 21 ml of 15% acrylamide solution.
(3) Pour the mixture between the glass plates and insert the comb.
(4) Remove the comb after 1 h of gel polymerization.
(5) Dissolve RNA in denaturing gel sample buffer, heat at 100°C for 3 min to denature, and place on ice immediately until sample.
(6) Pre-electrophoresis at 60 V/cm for 15 min.
(7) Rinse the wells and apply the samples.
(8) Electrophoresis was carried out for about 2.5 h until both indicators were swept out of the gel.
(9) Remove the top glass plate and cover with plastic film. Place a fluorescent ruler on the gel to determine the position of the X-ray film.
(10) Expose the X-ray film to determine the position of the RNA transcript.
(11) Cut the RNA-containing band from the gel, crush it, add 500 μl of gel elution buffer, and shake at 4°C overnight or 37°C for several hours.
(12) Remove gel debris by disposable column centrifugation.
(13) Precipitate the RNA with ethanol as described in operation 1 "Transcription".
(14) Dissolve RNA in 500 μl of distilled water and store at -20℃.
3. Large-scale splicing reaction
(1) Mix the following ingredients: 56 μl of 100 mmol/L MgCl2, 119 μl of 400 mmol/L KCl, 280 μl of 10 mmol/L ATP, 50 mmoI/L phosphocreatine, 30 μl of 0.1 μg/μl of biotinylated precursor mRNA, 1.12 ml of NE or DEAE II, and finally 2.1 ml of distilled water.
(2) Incubate for 1 h at 30℃.
(3) Add 1/100 volume of heparin (5 mg/ml) and incubate at 30℃ for 5 min.
4. Glycerol gradient
(1) Rapidly fill three 13 ml glycerol density gradients before use. 7 ml of 30% glycerol gradient solution in the gradient labeled "heavy" tank and 7 ml of 15% glycerol gradient solution in the "light" tank.
(2) Spread 690 μl of clipping reaction solution on each gradient tube.
(3) Centrifuge at 40,000 r/min (285,000 g) for 8 h at 4°C with an SW-40Ti rotor.
(4) Carefully collect the fractions from top to bottom in 1 ml portions.
5. Non-denaturing gel
(1) Wash and assemble the glass plate.
(2) Mix 5.6 ml 30% acrylamide, 2 ml 1% flat-forked bisacrylamide, 2 ml 1 mol/L Tris, 2 ml 1 mol/L glycine, 28 ml water, 0.28 ml 10% ammonium persulfate, and 28 μl TEMED.
(3) Gels were perfused between glass plates and combs were inserted.
(4) After polymerization for 2~4 h, the comb was removed.
(5) Sample 20 μl of each glycerol gradient.
(6) Electrophoresis at 160 V (9.4 V/cm ).
(7) After electrophoresis, remove the gel and place it on a piece of Whatman 3 MM filter paper and vacuum dry at 80°C for 45 min.
(8) Radiation autoradiography was performed to show the splice complexes.
6. Affinity purification
(1) Pre-enclosed Chain Affinity Beads. Wash 4 times with 1X Binding Buffer containing 0.1% BSA for a total of over 2 hours.
(2) Merge the glycerol gradient containing the spliceosome.
(3) Add 1/5 volume of 5X Binding Buffer, 2 mmol/L DTT, 3.2 U/ml RNasin, and 64 μl/ml Streptavidin Magnetic Beads to the combined solution.
(4) Oscillate at 4℃ overnight.
(5) Collect the magnetic beads, add 10 ml of Wash Buffer 1 and shake at 4℃ for 15 min to wash the magnetic beads. Repeat the washing 3 times.
(6) Wash once with Wash Buffer 2.
(7) Resuspend with 0.5 ml Wash Buffer 2.
(8) Add 2 μl of 0.2 mg/ml RNase A and incubate at 30°C for 10 min.
(9) Recover and store the supernatant.
(10) Wash the beads 3 times with 0.5 ml elution buffer and combine the elution buffer with the stored supernatant to make a total of about 2 ml.
(11) Heat the eluate at 65°C for 5 min.
(12) Add 2 μl of 20 mg/ml glycogen and 4 times the volume of acetone.
(13) Incubate at room temperature for 10 min and centrifuge at 2800 g for 10 min at room temperature to collect the precipitated protein.
(14) Remove the supernatant and allow the precipitate to dry at room temperature for 15 min.
7. Bidirectional gel electrophoresis
(1) Assemble the InvestigatorTM 2-D Electrophoresis System (ESA, Chelmsford, MA, USA ) according to the operating instructions.
(2) Resuspend proteins affinity purified and precipitated with acetone in 15 μl Ampholyte ampholyte solution.
(3) Centrifuge at 16,000 g for 1 min and upsample the supernatant onto a precast isoelectric focusing (IEF) gel.
(4) 100 V electrophoresis for 1 h, 200 V electrophoresis for 1 h, 400 V for 24 h, 650 V for 1 h, 1000 V for 30 min, 1500 V for 10 min, and 2000 V for 5 min, for a total of approximately 11,250 V-h.
(5) The gel was squeezed into gel equilibration buffer and incubated for 5 min at room temperature.
(6) Spread the IEF gel onto an SDS gel and electrophoresed until the bromophenol blue was 1 cm from the bottom.
(7) Analyze the protein fractions on silver-stained gels. Before protein sequencing, the sample volume of protein can be increased to be detected by the method of Caulophylline blue or silver staining.