In cells, protein-RNA binding is involved in a variety of physiological regulation and developmental processes. Direct protein-RNA interactions occur during mRNA capping, polyadenylation, shearing, nuclear translocation, translation initiation and degradation. At various stages of development, RNA-binding proteins can regulate gene expression by interacting with mRNA. This experiment is based on the "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Natural RNA library screening experiments for RNA sequences that bind specifically to proteins
Principle
In cells, protein-RNA binding is involved in a variety of physiological regulation and developmental processes. Direct protein-RNA interactions occur during mRNA capping, polyadenylation, shearing, nuclear translocation, translation initiation and degradation. At various stages of development, RNA-binding proteins can regulate gene expression by interacting with mRNA.
Materials and Instruments
mRNA Oligonucleotides Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Instrumental enzymes Dithiothreitol Nuclease-free Deionized water Potassium acetate PCI solution NT2 buffer KNET buffer
Microcentrifuge tubes and spiker tips Sephadex G-50 columns
1. mRNA (purchased from Clontech, or can be obtained by self-purification).
2. Instrumental enzymes: Superscript II reverse transcriptase and buffer (Life Technologies), E.coli RNase H (Life Technologies), Td Tase and buffer (Life Technologies), RNasin RNase inhibitor (Promega), Taq enzyme. RNase inhibitor (Promega), Taq enzyme.
3. Synthesize oligonucleotides (only an example is given here):
Forward primer [ oligo-dG ]: 5'-ACCAGGATCCTAATACGACTCACTATA [ G ] 11-3 '
Reverse primer [ oligo-dT ]: 5'-CATGGAATTCGGGATCC [ T ] 15-3 '
dNTP mixture: 10 mmol/L (10 mmol/L for dATP, dCTP, dGTP, and dUTP).
NTP mixture: 10 mmol/L (10 mmol/L for each of ATP, CTP, GTP, and UTP).
4. 0.1 mol/L dithiothreitol.
5. 0.5 ml or 1.5 ml microcentrifuge tubes without nuclease and spiker tips.
6. deionized nuclease-free water.
7. Sephadex G-50 column.
8. protein A-sepharose gel beads (P3391, Sigma).
9. 3 mol/L potassium acetate (pH 5.2), 100% and 70% ethanol, 7.5 mol/L ammonium acetate, 0.1 mol/L Na2EDTA (pH 8.0), 3 mol/L sodium acetate (pH 5.2).
10. PCI solution: phenol: chloroform: isoamyl alcohol (25 : 24 : 1, v/v), equilibrated with 10 mmol/L Tris-HCl ( pH 8.0), 1 mmol/L EDTA ( pH 8.0).
11. NT2 buffer: 50 mmol/L Tris-HCl (pH 7.4 ), 150 mmol/L NaCl, 0.05% NP-40, 1 mmol/L MgCl2.
12. KNET buffer: 20 mmol/L KCl, 80 mmol/L NaCl, 2 mmol/L EGTA, 50 mmol/L Tris-HCl (pH 7.4), 0.05% NP-40, 5 mg/ml mRNA, 1 mmol/L MgCl2, 2.5% poly(vinyl alcohol), 0.2% vanadyl ribonucleoside oxovanadium complex (VNBC). vanadyl ribonucleoside complex, VCR), 0.1 mg/ml bovine serum albumin, 0.5 mg/ml yeast tRNA, 10 mmoI/L DTT, and 80 U/ml RNasin. were prepared before use. 
II. Methods of operation
The experiment was carried out in 5 steps: firstly, the mRNA was reverse transcribed with oligo-dT primer; then the C-tail was synthesized at the end of the synthesized cDNA under the action of terminal deoxyribonucleic acid transferase; then PCR amplification was carried out, and the oligo-dG primer, which was paired with the C-tail, contained the promoter sequence of T7 RNA polymerase; then the cDNA obtained from the amplification was reverse transcribed to synthesize RNA; finally, the target protein was used to amplify RNA; and finally, the target protein was used to amplify RNA. The amplified cDNA was then reverse transcribed to synthesize RNA; finally, the target protein was used to screen the RNA library.
1. Reverse transcription of mRNA to synthesize cDNA.
(1) Add 1~5 μg of mRNA and 1 μl (0.5 μg) of oligo-dT primer to 13 μl of pre-treated water and mix gently.
(2) React the tube at 70 ℃ for 10 min, put it on ice for 1 min, centrifuge it instantly, and add 2 μl of 10X reverse transcriptase buffer, 1 μl of 10 mmol/L dNTP mixture, 2 μl of 0.1 mol/L DTT, and 1 μl of Superscript Ⅱ reverse transcriptase (200 U/μl) in a total volume of 20 μl.
(3) Gently mix and centrifuge the reaction tube and leave it at room temperature for 10 min.
(4) React the reaction tube at 42°C for 50 min. Incubate at 70°C for 15 min to terminate the reaction and transfer the reaction tube to ice.
(5) Collect the product by centrifugation, add 1 μl of E.coli RNase H (2 U/μl), and incubate at 37°C for 20 min.
(6) The reverse transcription product was passed through a Sephadex G-50 column to remove the residual unreacted nucleotides.
(7) Add 0.1 times the volume of 3 mol/L potassium acetate and ethanol precipitation of single-stranded DNA.
2. Add C-homodimerization tail to the end of cDNA library.
(1) Resuspend cDNA in water (3' end concentration is about 40 pmol/ml).
(2) Perform the terminal DNA transfer reaction in a 1.5 ml centrifuge tube: 10 μl of 5X TdT buffer, 25 μl of 100 μmol/L dCTP, cDNA, 48.5 μl of water, and 1.5 μl (15U) of TdTase; react at 37°C for 30 min.
(3) Place the reaction tube on ice and add 10 μl of 0.1 mol/L Na2EDTA (pH 8.0) to terminate the reaction.
(4) Add 60 μl PCI, mix thoroughly, and centrifuge at 15000 g for 5 min.
(5) Take the upper aqueous phase and pass it through a Sephadex G-50 column to remove the unreacted dCTP.
(6) Precipitate DNA with 0.5 times volume of 7.5 mol/L ammonium acetate and 2.5 times volume of ethanol.
(7) Centrifuge at 14000 g for 30 min at room temperature and carefully remove the supernatant.
(8) Resuspend cDNA in 50 μl of water and store at 20℃.
3. PCR amplification of cDNA library
(1) Reaction mixture: 41.5 μl water, 10 μl 10X PCR buffer, 2 μl dNTP mixture, 1 μl 0.1 μg/μl oligo-dG primer, 1 μl 0.1 μg/μl oligo-dT primer, 1 μl C-cohesionized cDNA, 0.5 μl 5 U/ml Taq enzyme. Reaction conditions: 94°C, 1 min; 50°C, 1 min; 72°C, 2 min; 25 cycles, then 72°C, 7 min.
(2) 1 μl 20U/μl Bam HI (or other endonuclease introduced during primer design to remove polymers that may be generated during the PCR process), react at 37°C for 1 h. The reaction conditions: 94°C, 1 min; 50°C, 1 min; 72°C, 2 min; 25 cycles, then 72°C, 7 min.
(3) PCI extraction, 0.1 times the volume of 3 mol/L sodium acetate, 2.5 times the volume of anhydrous ethanol in a dry ice/ethanol bath to precipitate DNA for 20 min.
(4) Centrifuge at 12000 g at 4℃ for 10 min, and carefully remove the supernatant.
(5) Wash the salt with 70% ethanol, centrifuge to remove the supernatant, dry and resuspend the nucleic acid with 15 μl of water.
(6) 1% agar gel electrophoresis, cut 200~800 bp DNA fragments.
(7) Purify the DNA recovered from the gel by PCI extraction, precipitation with 2.5 times volume of anhydrous ethanol (ice bath for 10 min), centrifugation at 12,000 g for 15 min at 4°C, and careful removal of the supernatant. 70% ethanol washed with salt, centrifuged to remove the supernatant, dried, and then resuspended the nucleic acid with 20 μl of DEPC-H2O.
4. cDNA library transcription to obtain 3' UTR library
(1) Reaction solution: 0.1 μg of PCR amplified cDNA library, 4 μl of 5X T7 or SP6 RNA polymerase buffer, 8 μl of NTP, 1 μl of 40 U/μl of RNasin, 1 μl of T7 RNA polymerase, and add DEPC-H2O to 20 μl. React at 37℃ for 10 min.
(2) Precipitate the RNA as in the previous section and wash the salt with 70% ethanol. Resuspend the obtained RNA sample in 20 μl of DEPC-H2O.
(3) Take 2 μl of 3% agarose gel for electrophoresis.
5. Screening of 3' UTR libraries with proteins
(1) Protein binding to 3' UTR library
① Take 1 mg of Protein A-sepharose gel beads and wash them 3 times with 1 ml of NT2 buffer in a 1.5 ml centrifuge tube.
① Take 1 mg of protein A-sepharose gel beads and wash them 3 times with 1 ml of NT2 buffer in a 1.5 ml centrifuge tube.
③ Mix for at least 10 min at 4°C.
③ Mix at least 10 min at 4°C. ④ Wash 3 times with 1 ml of NT2 buffer.
⑤ After the last wash, leave 100 μl of buffer, add 5 μl of 0.5 μg/ml purified protein, and mix at 4°C for at least 10 minutes.
⑥ Wash 3 times with 1 ml of NT2 buffer.
(vii) Resuspend protein A-sepharose gel beads/antiserum/protein in 100 μl of freshly prepared KNET buffer.
⑧ Add 4~5 μl of RNA.
⑨ Mix at room temperature for 5 min.
Wash 5 times with 1 ml of NT2 buffer.
(11) After the last wash, leave 100 μl of buffer and add 100 μl of water.
(12) 200 μl PCI extraction.
(13) Add 2 μl 1 mol/L MgCl2 (or 10 mmol/L tRNA ), 20 μl 3 mol/L sodium acetate, and 700 μl anhydrous ethanol to precipitate.
(14) Wash the salt with 70% ethanol and dry at room temperature.
(15) 14 μl DEPC-H2O resuspension.
(2) Reverse transcription screening to obtain 3'-UTR
① Add 1 μl of 0.1 μg/μl oligo-dT primer, 10X reverse transcription buffer, 1 μl of 10 mmol/L dNTP, 2 μl of 0.1 mol/L DTT, 1 μl of Superscript II, and transcriptase (200 U/μl) to 14 μl of DEPC-H2O resuspended RNA. Incubate at room temperature for 5 min, and react at 42℃ for 50 min.
The reaction was terminated at 72℃ for 15 min, and the reaction tube was transferred to ice.
(iii) Centrifuge the reaction tube, add 1 μl of 2 U/μl E.coli RNase H, and react at 37°C for 20 min.
The reaction product was passed through a Sephadex G-50 column twice to remove unreacted nucleotides.
⑤ 6 μl of the cDNA product was amplified by PCR (repeat PCR, reverse transcription and screening until final results were obtained).
If a high-rigor screening method is used, the binding screen can be performed only once. High-rigor binding screen conditions include increasing the salt concentration (up to 350 mmol/L) and increasing the concentration of urea in the rinse buffer (0.5-4 mol/L). In addition to saving time and labor, the most important advantage of using high stringency conditions for a primary binding screen is the reduction of errors introduced by PCR.
Isotope labeling can be added at the time of transcription to compare the isotopic activity of the 3'-UTR obtained from the same amount of screening with that of the initial 3'-UTR library that can be precipitated by the protein, and to determine whether the screen has yielded a specific protein-binding RNA or not. clones obtained from the screen can be cloned into PGEM or pSP64 plasmids by using the restriction enzyme sites introduced during PCR amplification. Single clones were extracted, sequenced and analyzed by BLAST.
The method described here is to screen for targets of RNA-binding proteins from a large library of naturally occurring mRNA 3'-UTRs. Care must be taken to correlate the results of the screen with biological function. If a protein is highly homologous to an RNA-binding protein with a known target RNA, consider directly testing whether this protein also has the ability to bind the same target RNA. If a certain pattern of sites is obtained from random RNA libraries, it can also be used to search for other target RNAs of known proteins according to the pattern. in vitro experiments to confirm protein-RNA interactions need to be further confirmed in vivo by in vivo methods (gradient centrifugation, immunoprecipitation coupled with Northern hybridization, RT-PCR, RNase protection method, etc.).