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UPF1 RNA Immunoprecipitation from Mini-μ Construct–expressing Cells
Mini-μ 载体-表达细胞中的UPF1 RNA 免疫沉淀   

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Abstract

UPF1, an RNA helicase and a core factor of nonsense-mediated mRNA decay (NMD), interacts with RNA independently of the sequence context. To investigate the influence of translation on the association of UPF1 with specific reporter transcripts, UPF1 RNA immunoprecipitations (RIPs) are performed from Hela cells that either express a normally translated immunoglobulin-µ (Ig-µ) reporter (mini µ) or a version with a stable stem loop in the 5' UTR (SL mini µ) that efficiently inhibit translation initiation (Zund et al., 2013). Both the cloning of the SL mini µ reporter construct and the UPF1 RIP experiment are described in detail.

Keywords: RNA-IP(RNA-IP), RIP(RIP), NMD(NMD), Nonsense-mediated mRNA decay(无义介导的mRNA衰变), RNA helicase UPF1(RNA解旋酶Upf1)

Materials and Reagents

  1. Hela cells
  2. XL-10 Gold Ultracompetent Cells (Agilent Technologies, Stratagene, catalog number: 200315 )
  3. Dulbecco’s Modified Eagle Media (DMEM) (powder, high glucose) (Life Technologies, catalog number: 52100-039 )
  4. MQ-water (pure water from the ELGA system)
  5. Fetal Calf Serum (FCS) (BioConcept, Amimed, catalog number: 2-01F30-I )
  6. Penicillin-Streptomycin solution (P/S) (1 unit/ml) (BioConcept, Amimed, catalog number: 4-01F00-H )
  7. Trypsin-EDTA (T/E) (BioConcept, Amimed, catalog number: 5-51F00-H )
  8. DreamFectTM (OZ Biosciences, catalog number: DF45000 )
  9. KpnI (New England Biolabs, catalog number: R0142S )
  10. SL Oligo 1 (Microsynth AG)
    Oligo sequence: 5'-CGGGTTCCGTCCAAGCACTGTTGAAGCAGGAAACCCGGGTTGCTAGTCGATCGACTAG-CAACCCGGGTTTCCTGCTTCAACAGTGCTTGGACGGAACCCCGATCGTAC-3'
  11. SL Oligo 2 (Microsynth AG)
    Oligo sequence: 5'-GATCGGGGTTCCGTCCAAGCACTGTTGAAGCAGGAAACCCGGGTTGCTAGTCGATCG-ACTAGCAACCCGGGTTTCCTGCTTCAACAGTGCTTGGACGGAACCCGGTAC-3'
  12. T4 DNA Ligase (New England Biolabs, catalog number: M0202S )
  13. T4 Polynucleotide Kinase (PNK) (New England Biolabs, catalog number: M0201L )
  14. Alkaline phosphatase calf intestinal (CIAP) (Promega Corporation, catalog number: M182A )
  15. 100x HaltTM Protease Inhibitor Cocktail (Thermo Fisher Scientific, catalog number: 1861279 )
  16. RiboLock RI RNase Inhibitor (40 U/µl) (Thermo Fisher Scientific, catalog number: EO0381 )
  17. RNase I (cloned, 100 U/µl) (Life Technologies, Ambion®, catalog number: AM2294 )
  18. Turbo DNase (Life Technologies, Ambion®, catalog number: AM2238 )
  19. Glycogen for molecular biology (Roche Diagnostics, catalog number: 10 901 393 001 )
  20. Pre-stained Protein Ladder (broad range) (New England Biolabs, catalog number: P7710S )
  21. Goat anti-UPF1 Antibody (G-α-RENT1) (Bethyl Laboratories, catalog number: A300-038A )
  22. Goat anti-rabbit IgG (polyclonal) (Bio-Rad Laboratories, catalog number: 172-1053 )
  23. Rabbit anti-actin (polyclonal) (Sigma-Aldrich, catalog number: A5050 )
  24. AffiniPure Goat Anti-mouse IgM (µ chain specific) (Jackson ImmunoResearch Laboratories, catalog number: 115-005-020 )
  25. IRDye 800CW Donkey anti-Rabbit (LI-COR, catalog number: 926-32213 )
  26. IRDye 800CW Donkey anti-Goat (LI-COR, catalog number: 926-32214 )
  27. Dynabeads® Protein G (Life Technologies, catalog number: 10004D )
  28. Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, catalog number: A9282 )
  29. Affinity Script Multi-Temp Reverse Transcriptase (Agilent, catalog number: 600105 )
  30. Random hexamer primers (Microsynth AG)
  31. Brilliant III Ultra-Fast qPCR Master mix (Agilent, catalog number: 600880 )
  32. Chloroform
  33. Isopropanol
  34. DMEM-/- (see Recipes)
  35. DMEM+/+ (see Recipes)
  36. Phosphate-bufferd saline (PBS) (pH 7.4) (see Recipes)
  37. Hypotonic gentle lysis buffer (pH 7.5) (RNase-free) (see Recipes)
  38. Wash buffer (pH 7.5) (RNase-free) (see Recipes)
  39. Net-2 buffer (pH 7.5) (RNase-free) (see Recipes)
  40. Hybridization buffer (pH 7.5) (see Recipes)
  41. 2x SDS loading buffer (pH 6.8) (see Recipes)
  42. TRI-reagent (see Recipes)
  43. Tris buffered saline (pH 7.6) (TBS) (see Recipes)
  44. TBS-Tween milk (see Recipes)
  45. Bjerrum transfer buffer (see Recipes)
  46. DEPC treated water/buffer (see Recipes)
  47. Turbo DNase mix (see Recipes)

Equipment

  1. Pure water system: PURELAB Priama (Prima 7) and PURELABULTRA (Ultra Genetic) (ELGA LabWater)
  2. CO2 incubator (BINDER GmbH, model: 9140-0047 )
  3. Clear-viewTM Snap-Cap microtubes (1.5 ml, natural, low retention) (Sigma-Aldrich, catalog number: T4816-250EA )
  4. Multiply®-Pro 0.2Ml Biosphere® (Sarstedt AG, catalog number: 72.727 )
  5. Filter Tips (10 µl, 20 µl and 200 µl) (Axon Lab AG, catalog numbers: AL60X10 , AL60X20 , AL60X200 )
  6. Filter Tips (1,250 µl) (Greiner Bio-One GmbH, catalog number: 7.750.261 )
  7. CountessTM automated cell counter (Life Technologies, model: C10227 )
  8. CountessTM cell counting chamber slides (Life Technologies, catalog number: C10283 )
  9. 15 cm tissue culture dishes (TPP Techno Plastic Products, catalog number: 93150 )
  10. GP Millipore express® PLUS Membrane (0.22 µm) (500 ml Funnel, 45 mm Neck Size) (EMD Millipore, catalog number: SCGPT05RE )
  11. NanoDrop 2000 (Thermo Fisher Scientific)
  12. Heat block, Thermomixer® compact (Eppendorf)
  13. DynaMagTM-2 magnet (Life Technologies, catalog number: 12321D )
  14. Eppendorf centrifuge 5415R with rotor F45-24-11 (Eppendorf, catalog numbers: 022621459 and 022636502 )
  15. Lab cycler gradient equipped with Thermoblock 96 (SensoQuest GmbH, models: 011-101 and 012-103 )
  16. Blotting paper (ALBET Lab Science, catalog number: BP 002 46579 )
  17. Corbett Rotor-Gene® 6000 (QIAGEN)
  18. Corbett CAS-1200 (QIAGEN)
  19. SE260 Mighty Small II Deluxe Mini vertical electrophoresis unit (Hoefer, model: SE260-10A-1.5 )
  20. Optitran BA-S 85 reinforced nitrocellulose membrane (GE Healthcare, Whatman, catalog number: 10 439 196 )
  21. Semi-dry transfer unit with built-in power supply TE77XP (Hoefer, model: TE77XP)
  22. Odyssey® infrared imaging system (LI-COR)

Procedure

  1. Cloning of the p-β SL mini µ WT uA1 plasmid
    To generate the p-β SL mini µ WT uA1 plasmid, a double-stranded oligonucleotide encoding a stable stem-loop structure is introduced into the KpnI site of p-β mini µ WT uA1 plasmid (Yepiskoposyan et al., 2011).
    1. Open the p-β mini µ WT uA1 vector by KpnI restriction digestion
      1. Digest 4.2 µg p-β mini µ WT uA1 plasmid with 36 U Kpn I in a total volume of 40 µl according to the manufaturer’s protocol at 37 °C for 1 h.
      2. Add 5 µl 10x CIP Buffer and 5 µl CIP (1 U/µl) and incubate at 37 °C for 30 min to dephosphorylate the open vector.
      3. Run the digested vector on 1% agarose gel, excise the DNA band and isolated the vector using the Wizard SV Gel PCR Clean-Up System.
    2. Anneal SL Oligo 1 and SL Oligo 2
      1. Mix 1 µl SL oligo 1 (100 µM), 1 µl SL oligo 2 (100 µM) and 48 µl hybridization buffer.
      2. Heat the hybridization mix to 90 °C and slowly cool down to 40 °C (in a heat block).
      3. To phosphorylate the double stranded oligonucleotide, combine 2 µl of the hybridization mix, 24 µl MQ-water, 3 µl 10x T4 DNA ligase buffer, 1 µl T4 PNK and incubate at 37 °C for 30 min.
      4. Inactivate the kinase at 70 °C for 15 min.
    3. Ligate 50 ng of the open p-β mini µ WT uA1 vector with 2 µl of the double-stranded oligonucleotide in 1x T4 DNA ligase buffer with 1 µl of T4 DNA ligase (400 U/µl) in a total volume of 15 µl at room temperature for 2.5 h.
    4. Transform the 5 µl of ligation mix into 100 µl XL10-Gold ultracompetent cells.
    5. The presence of the sequence encoding the stem-loop preceding the mini µ open reading frame is verified by sequencing.
  2. Transfect hela cells either with p-β SL mini µ WT uA1 or p-β mini µ WT uA1 plasmid
    1. Hela cells are cultivated in DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 10% FCS (DMEM+/+: DMEM with antibiotics and FCS).
    2. (Day 1) Seed 3 x 106 hela cells into each of two 15 cm diameter dishes to have 60% confluent cells the next day.
    3. (Day 2) Cells were transfected either with 5 µg p-β mini µ WT uA1 or 5 µg p-β SL mini µ WT uA1 plasmid using 40 µl DreamFectTM according the manufacturer’s protocol.
    4. (Day 3) Split the cells from one plate into two 15 cm diameter dishes.
    5. (Day 4) Harvest the cells.
      1. Wash the cells with 25 ml PBS.
      2. To detach the cells they are incubated with 4 ml trypsin-EDTA (T/E) at 37 °C for 10 min.
      3. Add 9 ml DMEM+/+ and resuspend the cells.
      4. Count the cells using the Countess™ automated cell counter according to the manufacturer’s protocol.
  3. Lyse the cells
    1. 1.2 x 107 cells are lysed on ice in 1.2 ml hypotonic gentle lysis buffer supplemented with 40 U/ml RiboLock RI RNase inhibitor for 20 min.
    2. Clear the lysate by centrifugation in a microcentrifuge (16,000 x g, 4 °C, 15 min).
  4. Take protein and RNA input samples
    1. Protein input samples are prepared by mixture of 50 µl lysate with 50 µl SDS loading buffer, cooked at 90 °C for 5 min and stored at -20 °C.
    2. Prior to the extraction of input RNA, 100 µl cell lysates are supplemented with 2.5 µl RiboLock RI RNase inhibitor and treated with 20 U/ml TURBO DNase at 37 °C for 5 min. Subsequently, the RNA was extracted with TRI reagent according the following TrIzol protocol.
      1. Mix 100 µl TURBO DNase treated cell lysate with 900 µl TRI reagent, vortex.
      2. Incubate at room temperature for 5 min.
      3. Add 180 ml chloroform to each sample and vortex for 15 s.
      4. Incubate at RT for 5 min.
      5. Centrifuge the samples (12,000 x g, 4 °C, 15 min), lift 350 µl of the aqueous phase and transfer into a fresh test tube.
      6. Add 2 µl glycogen, 450 µl isopropanol, vortex and incubate at -20 °C for 30 min.
      7. Precipitate the RNA by centrifugation (12,000 x g, 4 °C, 10 min).
      8. Discard the supernatant and wash the pellet with 1 ml 70% EtOH.
      9. Spin the samples (12,000 x g, 4 °C, 10 min), discard the supernatant and air dry the pellet.
      10. Resolve the RNA in 60 µl MQ-water.
      11. Measure the RNA concentration on the NanoDrop 2000. A ratio of OD260/OD230 < 2 is indicative for phenol contamination and demands for an additional EtOH precipitation of the samples.
  5. UPF1 RNA immunopurification (RIP)
    1. Adapt the NaCl concentration to 150 mM by the addition of 52.5 µl 3 M NaCl to the remaining 1,050 µl cell lysates expressing either the mini µ or SL mini µ constructs.
    2. 500 µl of each lysate is incubated head-over-tail at 4 °C for 90 min with 3.7 µg goat anti-RENT1 and goat anti-rabbit IgG antibody, respectively (for RIP incubation procedure see Table 1).
    3. Preparation of the Dynabeads® protein G beads.
      1. 128 µl Dynabeads® protein G beads are washed with 1 ml wash buffer.
      2. Beads are washed with 1 ml hypotonic gentle lysis buffer.
      3. Beads are equilibrated in hypotonic gentle lysis buffer supplemented with 1% (w/v) BSA and 0.1% yeast tRNA at 4 °C for 1 h (to minimize unspecific binding).
      4. Wash the beads twice with 1 ml hypotonic gentle lysis buffer.
    4. The Dynabeads® are equally distributed on the four RIP samples and incubated head over tail at 4 °C for 90 min (Table 1).

            Table 1. UPF1 RIP incubation schema


    5. Precipitates were washed six times with 1 ml Net-2 buffer.
    6. During the last wash step the beads are separated.
      1. One-third of the beads are incubated with 40 µl 2x SDS loading buffer at 90 °C for 5 min in order to elute the protein for subsequent western blot analysis.
      2. Two-thirds of the beads are dissolved in 50 µl Turbo DNase mix and DNase treated at 37 °C for 10 min. To isolate the RNA 900 µl TRI reagent is added to the samples and treated according to the Trizol protocol (see 4b). The RNA is dissolved in 34.5 µl MQ-water, reverse transcribed and analyzed by RT-qPCR.
  6. Quantitative real-time reverse-transcription PCR
    1. The entire recovered RNA of the UPF1 RIPs, or 1 µg of the input RNA samples are reverse transcribed in a total volume of 50 µl containing 1x StrataScript RT buffer, 0.1 mM DTT, 0.4 mM dNTPs, 300 ng random hexamer primers and 1 µl of StrataScript Multi-Temp reverse transcriptase according to the manufacturere’s protocol.
    2. Twelve microliters of the reverse transcription reactions were amplified with Brilliant III Ultra-Fast qPCR Master mix in the Corbett Rotor-Gene 6000 (Pipetting was done using the Corbett CAS-1200 robot.). The primers and TaqMan probes to measure SMG5 and mini µ mRNA levels are described elsewhere (Yepiskoposyan et al., 2011) (see also Table 2). The levels of SMG5 mRNA, which efficiently associate with UPF1 (Yepiskoposyan et al., 2011) are measured to normalize for variable RIP efficiencies in different samples.

      Table 2. TaqMan probes


  7. Western blotting
    1. For protein analysis, 2 x 105 cells and one-third of the RIP samples are separated on a 10% SDS-PAGE using the Mighty Small II running chamber SE260. The proteins are transferred to Optitran BA-S 85 reinforced nitrocellulose membrane using a semi dry blotter and Bjerrum transfer buffer.
    2. After blocking in TBS-Tween milk, the membrane is probed in the blocking buffer with 1:3,000-diluted polyclonal goat anti-RENT1 antibody to assess the specificity of the UPF1 RIP experiment, 1:500-diluted AffiniPure goat anti-mouse IgM, µ chain-specific antibody to compare mini µ expression levels either from p-β SL mini µ WT uA1 or p-β mini µ WT uA1 plasmid or 1:5,000-diluted polyclonal rabbit anti-actin antibody to be used as a loading control.
      1. The membrane is probed with 1:10,000-diluted donkey anti-rabbit IRDye 800CW or donkey anti-goat IRDye 800CW antibodies.
      2. The membrane is scanned on an Odyssey infrared imager.


    Figure 1. Stem loop-induced stranscript specific translation inhibition. a. Representation of the stem loop structure introduced in the KpnI site (green palindromic sequence) preciding the mini µ open reading frame (red boxed sequence) of the Ig-µ reporter transcript mini µ. b. Schematic representation of mini µ and SL mini µ reporter transcripts. The translation-inhibing stable stem loop in the 5' UTR of SL mini µ is shown. The CDS is represented as a white box flanked by the 5' and 3' UTRs. Gray bars, exon-exon junctions; black line, position of the TaqMan assay used for real-time qPCR with reverse transcription (RT-qPCR) (adapted from Reference 1)

Recipes

Note: All buffers used in the RIP protocol have to be sterile and RNase-free. Therefore, all buffers are prepared using MQ-water and if possible DEPC treated. Duran bottles, glassware and spatulas are baked at 180 °C for 2 h. Polycarbonate or polystyrene materials (e.g. magnetic stirrers…) are soaked in 3% hydrogen peroxide or 2 M NaOH for 10 min and extensively rinsed with DEPC-treated water. If a buffer cannot be DEPC treated (e.g. Tris buffers) prepare the buffer in DEPC or MQ-water and filter it with a 0.22 µm filter.

  1. DMEM-/- (per 900 ml)
    12 g DMEM/F12 powder
    2.48 g NaHCO3
    Make up to 900 ml with ddH2O
    Adjust pH to 7.2 with 32% HCl or 10 M NaOH
    Sterilize per filtration with bottle top filter (0.22 µm)
  2. DMEM+/+ (per 500 ml)
    Supply 450 ml DMEM-/- with 50 ml FCS and 5 ml P/S
  3. Phosphate-bufferd saline (PBS)
    137 mM NaCl
    10 mM Na2HPO4
    2.7 mM KCl
    2 mM KH2PO4
    Adjust pH to 7.4 with HCl
  4. Hypotonic gentle lysis buffer (RNase-free)
    10 mM Tris-HCl (pH 7.5)
    10 mM NaCl
    2 mM EDTA
    0.5% (v/v) Triton X-100
    Prior to usage supplement with 1x HaltTM protease inhibitor cocktail
  5. Wash buffer (RNase-free)
    50 mM Tris-HCl (pH 7.5)
    150 mM NaCl
    0.05% (v/v) NP-40
    Prior to usage supplement with 1x HaltTM protease inhibitor cocktail
  6. Net-2 buffer (RNase-free)
    150 mM NaCl
    50 mM Tris-HCl (pH 7.5)
    0.1% (v/v) Triton-X-100
    Prior to usage supplement with 1x HaltTM protease inhibitor cocktail
  7. Hybridization buffer
    100 mM KOAc
    30 mM HEPES-KOH (pH 7.5)
    2 mM MgOAc
  8. 2x SDS loading buffer
    200 mM DTT
    120 mM Tris-HCl (pH 6.8)
    0.44% (w/v) SDS
    20% (v/v) glycerol
    0.25% (w/v) bromophenol blue
  9. TRI-reagent
    800 mM guanidine thiocyanate
    400 mM ammonium thiocyanate
    100 mM sodium acetate
    38% (v/v) phenol
    5% (v/v) glycerol
    0.1% (w/v) 8-quinolinol (pH 5.0)
  10. Tris buffered saline (TBS)
    137 mM NaCl
    20 mM Tris-HCl (pH 7.6)
  11. TBS-Tween milk
    5% (w/v) milk powder, fat-free
    0.1% (v/v) Tween-20
    Make up to 250 ml using TBST
  12. Bjerrum transfer buffer
    48 mM Tris base
    39 mM glycin
    0.1% (w/v) SDS
    20 (v/v) MeOH
  13. DEPC treated water/buffer
    0.1% (v/v) DEPC
    Stir over night at 4 °C
    Autoclave twice in order to inactivate DEPC
  14. Turbo DNase mix
    1x Turbo DNase buffer
    0.1 U/µl Turbo DNase
    1 U/µl RiboLock RNase inhibitor

Acknowledgments

We thank L. Maquat (University of Rochester, Rochester, New York, USA) for providing their protocol for RIP coupled with RNase-H cleavage, and S. Rufener (Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland) for help with the RIPs. This work was supported by grants to Oliver Mühlemann from the European Research Council (StG 207419), the Swiss National Science Foundation (31003A-127614 and 31003A-143717) and the canton of Bern.

References

  1. Yepiskoposyan, H., Aeschimann, F., Nilsson, D., Okoniewski, M. and Mühlemann, O. (2011). Autoregulation of the nonsense-mediated mRNA decay pathway in human cells. RNA 17(12): 2108-2118. 
  2. nd, D., Gruber, A. R., Zavolan, M. and Mühlemann, O. (2013). Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3' UTRs. Nat Struct Mol Biol 20(8): 936-943.

简介

UPF1,RNA解旋酶和无义介导的mRNA衰变(NMD)的核心因素,独立于序列上下文与RNA相互作用。 为了研究翻译对UPF1与特异性报告基因转录物的关联的影响,从表达正常翻译的免疫球蛋白-μ(Ig-μ)报道分子(微型μ)的Hela细胞或其中的一个版本进行UPF1RNA免疫沉淀(RIP) 在5'UTR(SL miniμ)中的稳定茎环,其有效抑制翻译起始(Zund等人,2013)。 详细描述了SL miniμ报告构建体的克隆和UPF1 RIP实验

关键字:RNA-IP, RIP, NMD, 无义介导的mRNA衰变, RNA解旋酶Upf1

材料和试剂

  1. 赫拉细胞
  2. XL-10 Gold Ultracompetent Cells(Agilent Technologies,Stratagene,目录号:200315)
  3. Dulbecco改良的Eagle培养基(DMEM)(粉末,高葡萄糖)(Life Technologies,目录号:52100-039)
  4. MQ-水(来自ELGA系统的纯水)
  5. 胎牛血清(FCS)(BioConcept,Amimed,目录号:2-01F30-I)
  6. 青霉素 - 链霉素溶液(P/S)(1单位/ml)(BioConcept,Amimed,目录号:4-01F00-H)
  7. 胰蛋白酶-EDTA(T/E)(BioConcept,Amimed,目录号:5-51F00-H)
  8. DreamFect TM (OZ Biosciences,目录号:DF45000)
  9. KpnI(New England Biolabs,目录号:R0142S)
  10. SL Oligo 1(Microsynth AG)
    寡核苷酸序列: 5'-CGGGTTCCGTCCAAGCACTGTTGAAGCAGGAAACCCGGGTTGCTAGTCGATCGACTAG-CAACCCGGGTTTCCTGCTTCAACAGTGCTTGGACGGAACCCCGATCGTAC-3'
  11. SL Oligo 2(Microsynth AG)
    寡核苷酸序列:
  12. T4 DNA连接酶(New England Biolabs,目录号:M0202S)
  13. T4多核苷酸激酶(PNK)(New England Biolabs,目录号:M0201L)
  14. 碱性磷酸酶小牛肠(CIAP)(Promega Corporation,目录号:M182A)
  15. 100x Halt TM 蛋白酶抑制剂混合物(Thermo Fisher Scientific,目录号:1861279)
  16. RiboLock RI RNA酶抑制剂(40U /μl)(Thermo Fisher Scientific,目录号:EO0381)
  17. RNA酶I(克隆的,100U /μl)(Life Technologies,Ambion ,目录号:AM2294)
  18. Turbo DNase(Life Technologies,Ambion ,目录号:AM2238)
  19. 分子生物学糖原(Roche Diagnostics,目录号:10 901 393 001)
  20. 预染色的蛋白质梯(宽范围)(New England Biolabs,目录号:P7710S)
  21. 山羊抗UPF1抗体(G-α-RENT1)(Bethyl Laboratories,目录号:A300-038A)
  22. 山羊抗兔IgG(多克隆)(Bio-Rad Laboratories,目录号:172-1053)
  23. 兔抗肌动蛋白(多克隆)(Sigma-Aldrich,目录号:A5050)
  24. AffiniPure山羊抗小鼠IgM(μ链特异性)(Jackson ImmunoResearch Laboratories,目录号:115-005-020)
  25. IRDye 800CW驴抗兔(LI-COR,目录号:926-32213)
  26. IRDye 800CW驴抗山羊(LI-COR,目录号:926-32214)
  27. Dynabeads Protein G(Life Technologies,目录号:10004D)
  28. Wizard SV Gel and PCR Clean-Up System(Promega Corporation,目录号:A9282)
  29. Affinity Script Multi-Temp Reverse Transcriptase(Agilent,目录号:600105)
  30. 随机六聚体引物(Microsynth AG)
  31. Brilliant III超快速qPCR主混合物(Agilent,目录号:600880)
  32. 氯仿
  33. 异丙醇
  34. DMEM -/- (见配方)
  35. DMEM +/+(见配方)
  36. 磷酸盐缓冲盐水(PBS)(pH 7.4)(见Recipes)
  37. 低渗温和裂解缓冲液(pH 7.5)(无RNase)(见配方)
  38. 洗涤缓冲液(pH 7.5)(无RNase)(见配方)
  39. Net-2缓冲液(pH 7.5)(无RNase)(见配方)
  40. 杂交缓冲液(pH 7.5)(参见配方)
  41. 2x SDS上样缓冲液(pH 6.8)(参见配方)
  42. TRI试剂(参见配方)
  43. Tris缓冲盐水(pH 7.6)(TBS)(参见Recipes)
  44. TBS-Tween牛奶(见配方)
  45. Bjerrum传输缓冲区(请参阅配方)
  46. DEPC处理的水/缓冲液(见配方)
  47. Turbo DNase mix(参见配方)

设备

  1. 纯水系统:PURELAB Priama(Prima 7)和PURELAB ULTRA (Ultra Genetic)(ELGA LabWater)
  2. CO 2培养箱(BINDER GmbH,型号:9140-0047)
  3. Clear-view TM Snap-Cap微管(1.5ml,天然,低保留)(Sigma-Aldrich,目录号:T4816-250EA)
  4. 乘以 -Pro 0.2Ml Biosphere (Sarstedt AG,目录号:72.727)
  5. 过滤嘴(10μl,20μl和200μl)(Axon Lab AG,目录号:AL60X10,AL60X20,AL60X200)
  6. 过滤嘴(1,250μl)(Greiner Bio-One GmbH,目录号:7.750.261)
  7. Countess TM 自动化细胞计数器(Life Technologies,型号:C10227)
  8. (Life Technologies,目录号:C10283)
  9. 15cm组织培养皿(TPP Techno Plastic Products,目录号:93150)
  10. GP Millipore express(0.22μm)(500ml漏斗,45mm颈部尺寸)(EMD Millipore,目录号:SCGPT05RE)
  11. NanoDrop 2000(Thermo Fisher Scientific)
  12. 热块,Thermomixer ® compact(Eppendorf)
  13. DynaMag TM -2磁体(Life Technologies,目录号:12321D)
  14. 带有转子F45-24-11的Eppendorf离心机5415R(Eppendorf,目录号:022621459和022636502)
  15. Lab循环仪梯度,配备有Thermoblock 96(SensoQuest GmbH,型号:011-101和012-103)
  16. 印花纸(ALBET Lab Science,目录号:BP 002 46579)
  17. Corbett Rotor-Gene ® 6000(QIAGEN)
  18. Corbett CAS-1200(QIAGEN)
  19. SE260 Mighty Small II豪华迷你垂直电泳装置(Hoefer,型号:SE260-10A-1.5)
  20. 带有转子F45-24-11的Eppendorf离心机5415R(Eppendorf,目录号:022621459和022636502)
  21. Lab循环仪梯度,配备有Thermoblock 96(SensoQuest GmbH,型号:011-101和012-103)
  22. 印花纸(ALBET Lab Science,目录号:BP 002 46579)
  23. Corbett Rotor-Gene ® 6000(QIAGEN)
  24. Corbett CAS-1200(QIAGEN)
  25. SE260 Mighty Small II豪华迷你垂直电泳装置(Hoefer,型号:SE260-10A-1.5)
  26. ... 通过KpnI限制性消化打开p-β微型μWT uA1载体
    1. 按照制造商的方案,在37℃下,将总量为40μl的Digest4.2μgp-β微型μWT uA1质粒与36U Kpn I在37℃下孵育1小时。
    2. 加入5μl10x CIP缓冲液和5μlCIP(1 U /μl),并在37°C孵育30分钟,以去磷酸化开放的载体。
    3. 在1%琼脂糖凝胶上运行消化的载体,切除DNA条带,并使用Wizard SV凝胶PCR清理系统分离载体。
  27. Anneal SL Oligo 1和SL Oligo 2
    1. 混合1μlSL oligo 1(100μM),1μlSL oligo 2(100μM)和48μl杂交缓冲液。
    2. 将杂交混合物加热至90℃,并缓慢冷却至40℃(在加热块中)
    3. 为了磷酸化双链寡核苷酸,将2μl杂交混合物,24μlMQ-水,3μl10×T4 DNA连接酶缓冲液,1μlT4 PNK并在37℃孵育30分钟。
    4. 在70°C灭活激酶15分钟
  28. 用1μlT4 DNA连接酶(400U /μl)在1×T4 DNA连接酶缓冲液中的50μl的开放p-β微型μWT uA1载体与2μl双链寡核苷酸在总体积15μl中 室温下2.5小时
  29. 将5μl连接混合物转化到100μlXL10-Gold超活性细胞中
  30. 通过测序验证编码微型μ开放读框之前的茎环的序列的存在
  • 用p-βSL miniμWT uA1或p-βminiμWT uA1质粒转染hela细胞
    1. Hela细胞在补充有100U/ml青霉素,100μg/ml链霉素和10%FCS(DMEM +/+:具有抗生素和FCS的DMEM)的DMEM中培养。
    2. (第1天)将3×10 6个hela细胞种入两个15cm直径的皿中的每一个中,第二天使60%汇合的细胞生长。
    3. (第2天)使用40μlDreamFect TM根据制造商的方案用5μgp-β微型μWT uA1或5μgp-βSL微型μWT uA1质粒转染细胞。
    4. (第3天)将细胞从一块板分成两个15cm直径的皿
    5. (第4天)收获细胞。
      1. 用25 ml PBS洗涤细胞
      2. 为了分离细胞,将它们与4ml胰蛋白酶-EDTA(T/E)在37℃温育10分钟。
      3. 加入9ml DMEM +/+并重悬细胞
      4. 使用Countess™自动细胞计数器根据制造商的协议计数细胞
  • 裂解细胞
    1. 将1.2×10 7个细胞在冰上在补充有40U/ml RiboLock RI RNA酶抑制剂的1.2ml低渗温和裂解缓冲液中裂解20分钟。
    2. 通过在微量离心机(16,000×g,4℃,15分钟)中离心来清除裂解物。
  • 取蛋白和RNA输入样品
    1. 蛋白质输入样品通过将50μl裂解物与50μlSDS上样缓冲液混合,在90℃下烹饪5分钟并储存在-20℃下制备。
    2. 在提取输入RNA之前,将100μl细胞裂解物用2.5μlRiboLock RI RNA酶抑制剂补充并用20U/ml TURBO DNase在37℃处理5分钟。 随后,根据以下TrIzol方案用TRI试剂提取RNA。
      1. 混合100微升TURBO DNase处理的细胞裂解物与900微升TRI试剂,涡旋
      2. 在室温下孵育5分钟
      3. 向每个样品中加入180ml氯仿并涡旋15秒
      4. 在室温下孵育5分钟。
      5. 离心样品(12,000×g,4℃,15分钟),提起350μl水相,并转移到新鲜试管中。
      6. 加入2μl糖原,450μl异丙醇,涡旋并在-20℃下孵育30分钟
      7. 通过离心(12,000×g,4℃,10分钟)沉淀RNA。
      8. 弃去上清液并用1ml 70%EtOH洗涤沉淀
      9. 旋转样品(12,000×g,4℃,10分钟),弃去上清液并将沉淀物风干。
      10. 在60μlMQ-水中溶解RNA
      11. 测量NanoDrop 2000上的RNA浓度。OD 260与OD 230之比为: 2表示苯酚污染和需要额外的EtOH沉淀样品
  • UPF1 RNA免疫纯化(RIP)
    1. 通过向剩余的1,050μl表达miniμ或SL miniμ构建体的细胞裂解物中加入52.5μl3MNaCl使NaCl浓度适应150mM。
    2. 使用3.7μg山羊抗RENT1和山羊抗兔IgG抗体(对于RIP温育程序,参见表1),将500μl每种裂解物在4℃下头尾孵育90分钟。
    3. 制备Dynabeads 蛋白G珠。
      1. 用1ml洗涤缓冲液洗涤128μlDynabeads 蛋白G珠子
      2. 珠子用1ml低渗温和裂解缓冲液洗涤
      3. 珠子在补充有1%(w/v)BSA和0.1%酵母tRNA的低渗温和裂解缓冲液中在4℃下平衡1小时(以最小化非特异性结合)。
      4. 用1ml低渗温和裂解缓冲液洗涤珠子两次
    4. Dynabeads 均匀地分布在4个RIP样品上,并在4℃下尾部孵育90分钟(表1)。

            表1. UPF1 RIP孵化模式


    5. 沉淀物用1ml Net-2缓冲液洗涤6次
    6. 在最后的洗涤步骤中,分离珠子。
      1. 将三分之一的珠子与40μl2x SDS上样缓冲液在90℃温育5分钟,以洗脱蛋白质,用于随后的蛋白质印迹分析。
      2. 将三分之二的珠溶解在50μlTurbo DNA酶混合物中,并且DNA酶在37℃处理10分钟。 为了分离RNA,将900μlTRI试剂加入样品中并根据Trizol方案处理(参见4b)。 将RNA溶解在34.5μlMQ-水中,反转录并通过RT-qPCR分析
  • 定量实时逆转录PCR
    1. 在含有1×StrataScript RT缓冲液,0.1mM DTT,0.4mM dNTP,300ng随机六聚体引物的总体积50μl中反转录全部回收的UPF1 RIP的RNA或1μg输入RNA样品,并将1μl StrataScript Multi-Temp逆转录酶。
    2. 使用Corbett Rotor-Gene 6000中的Brilliant III Ultra-Fast qPCR Master混合物扩增12微升的逆转录反应(使用Corbett CAS-1200机器人完成移液)。用于测量SMG5和微小mRNA水平的引物和TaqMan探针在别处描述(Yepiskoposyan等人,2011)(也见表2)。测量与UPF1有效结合的SMG5 mRNA的水平(Yepiskoposyan等人,2011),以对不同样品中的可变RIP效率进行归一化。

      表2. TaqMan探针


  • 免疫印迹
    1. 对于蛋白质分析,使用Mighty Small II流动室SE260在10%SDS-PAGE上分离2×10 5个细胞和三分之一的RIP样品。将蛋白质转移至Optitran BA-S 85增强的硝酸纤维素膜使用半干印迹和Bjerrum转移缓冲液。
    2. 在TBS-Tween乳中封闭后,用1:3,000稀释的多克隆山羊抗RENT1抗体在封闭缓冲液中探测膜,以评估UPF1 RIP实验,1:500稀释的AffiniPure山羊抗小鼠IgM的特异性, μ链特异性抗体,以比较来自p-βSL miniμWT uA1或p-βminiμWT uA1质粒或1:5,000稀释的多克隆兔抗肌动蛋白抗体的微型μ表达水平以用作加载对照。
      1. 用1:10,000稀释的驴抗兔IRDye 800CW或驴抗山羊IRDye 800CW抗体探测该膜。
      2. 在Odyssey红外成像仪上扫描膜


    图1.茎环诱导的转录物特异性翻译抑制。在KpnI位点(绿色回文序列)中引入的茎环结构的表示预制微型μ开放阅读框(红色 盒式序列)的Ig-μ报道转录物微型。 b。迷你μ和SL迷你μ报告基因转录物的示意图。显示了SL miniμ的5'UTR中的翻译抑制稳定茎环。 CDS表示为侧翼为5'和3'UTR的白盒。灰色条,外显子 - 外显子连接;黑线,用于具有逆转录的实时qPCR(RT-qPCR)(改编自参考文献1)的TaqMan测定的位置
  • 食谱

    注意:RIP协议中使用的所有缓冲区都必须是无菌的,无RNase的。因此,所有缓冲液使用MQ-水制备,如果可能的话用DEPC处理。 Duran瓶,玻璃器皿和铲子在180℃烘烤2小时。将聚碳酸酯或聚苯乙烯材料(例如磁力搅拌器)在3%过氧化氢或2M NaOH中浸泡10分钟,并用DEPC处理的水充分冲洗。如果缓冲液不能被DEPC处理(例如Tris缓冲液),则在DEPC或MQ-水中制备缓冲液,并用0.22μm过滤器过滤。

    1. DMEM -/- (每900ml) 12克DMEM/F12粉末
      2.48g NaHCO 3水溶液 用ddH 2 O·
      补充至900ml 用32%HCl或10M NaOH将pH调节至7.2 用瓶顶过滤器(0.22μm)过滤灭菌
    2. DMEM +/+(每500ml) 供应450毫升DMEM -/- 与50毫升FCS和5毫升P/S
    3. 磷酸盐缓冲盐水(PBS)
      137 mM NaCl 10mM Na 2 HPO 4
      2.7 mM KCl
      2mM KH 2 PO 4 sub/
      用HCl
      调节pH至7.4
    4. 低渗温和裂解缓冲液(不含RNase)
      10mM Tris-HCl(pH7.5) 10mM NaCl 2mM EDTA 0.5%(v/v)Triton X-100 在使用之前补充1x Halt TM 蛋白酶抑制剂混合物
    5. 洗涤缓冲液(无RNase)
      50mM Tris-HCl(pH7.5) 150mM NaCl 0.05%(v/v)NP-40
      在使用之前补充1x Halt TM 蛋白酶抑制剂混合物
    6. Net-2缓冲区(无RNase)
      150mM NaCl 50mM Tris-HCl(pH7.5) 0.1%(v/v)Triton-X-100 在使用之前补充1x Halt TM 蛋白酶抑制剂混合物
    7. 杂交缓冲区
      0.5%(v/v)Triton X-100 在使用之前补充1x Halt TM 蛋白酶抑制剂混合物
    8. 洗涤缓冲液(无RNase)
      50mM Tris-HCl(pH7.5) 150mM NaCl 0.05%(v/v)NP-40
      在使用之前补充1x Halt TM 蛋白酶抑制剂混合物
    9. Net-2缓冲区(无RNase)
      150mM NaCl 50mM Tris-HCl(pH7.5) 0.1%(v/v)Triton-X-100 在使用之前补充1x Halt TM 蛋白酶抑制剂混合物
    10. 杂交缓冲区
      ... 0.1% (w/v) 8-quinolinol (pH 5.0)
    11. Tris buffered saline (TBS)
      137 mM NaCl
      20 mM Tris-HCl (pH 7.6)
    12. TBS-Tween milk
      5% (w/v) milk powder, fat-free
      0.1% (v/v) Tween-20
      Make up to 250 ml using TBST
    13. Bjerrum transfer buffer
      48 mM Tris base
      39 mM glycin
      0.1% (w/v) SDS
      20 (v/v) MeOH
    14. DEPC treated water/buffer
      0.1% (v/v) DEPC
      Stir over night at 4 °C
      Autoclave twice in order to inactivate DEPC
    15. Turbo DNase mix
      1x Turbo DNase buffer
      0.1 U/µl Turbo DNase
      1 U/µl RiboLock RNase inhibitor

    Acknowledgments

    We thank L. Maquat (University of Rochester, Rochester, New York, USA) for providing their protocol for RIP coupled with RNase-H cleavage, and S. Rufener (Department of Chemistry and Biochemistry, University of Bern, Bern, 瑞士)帮助RIPs。 这项工作得到了来自欧洲研究委员会(StG 207419),瑞士国家科学基金会(31003A-127614和31003A-143717)和伯尔尼州的OliverMühlemann的资助。

    参考文献

    1. Yepiskoposyan,H.,Aeschimann,F.,Nilsson,D.,Okoniewski,M。和Mühlemann,O.(2011)。 人类细胞中无义介导的mRNA衰减途径的自动调节 RNA 17(12):2108-2118。 
    2. nd,D.,Gruber,A. R.,Zavolan,M. andMühlemann,O.(2013)。 翻译依赖于UPF1从编码序列的置换导致其在3'UTR的富集。 Nat Struct Mol Biol 20(8):936-943。
    • English
    • 中文翻译
    免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
    Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
    引用:Zünd, D. and Mühlemann, O. (2014). UPF1 RNA Immunoprecipitation from Mini-μ Construct–expressing Cells. Bio-protocol 4(7): e1086. DOI: 10.21769/BioProtoc.1086.
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