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Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method
采用紫外线交联-免疫沉淀法(CLIP)分析RNA结合蛋白与其靶标RNA的体内互作   

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Oncogene
Apr 2016

 

Abstract

RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic PKM2 variant. Herein, we describe a step-by-step protocol of the ultraviolet (UV) light cross-linking and immunoprecipitation (CLIP) method to determine the direct binding of an RBP to specific regions of its target RNAs in adherent human cell lines.

Keywords: CLIP (CLIP), Protein-RNA interaction (蛋白质 - RNA相互作用), Protein-RNA-immunoprecipitation (蛋白质 - RNA免疫沉淀), RNA processing (RNA加工)

Background

While being transcribed in the nucleus, nascent RNAs are immediately assembled with trans-acting factors collectively named RNA binding proteins (RBPs). These factors interact directly with specific cis-acting regulatory sequences in RNA molecules, thus forming ribonucleoprotein (RNP) complexes (Dreyfuss et al., 2002; Singh et al., 2015). These complexes control co-transcriptional RNA processing events as well as post-transcriptional mechanisms involved in RNA metabolism, such as subcellular localization and translation. For instance, spliceosomal and cleavage/polyadenylation complex components recognize specific RNA elements in the pre-mRNA, permitting introns removal (Black, 2003) and coordination between 3’-end processing and transcription termination (Proudfoot, 2016). A large number of RBPs functions as splicing factors, by assisting recognition of constitutively and alternatively spliced exons by the spliceosome (Chen and Manley, 2009) or by improving usage of alternative polyadenylation signals (Tian and Manley, 2016). Likewise, RBP-mediated recognition of zip code localization elements allows transport and local translation of mRNA in the cytoplasm (Martin and Ephrussi, 2009).

Eukaryotic genomes encode a wide array of RBPs to fine-tune cell-specific gene expression programs in a time- and space-sensitive manner, thus contributing to tissue homeostasis. RNPs are highly dynamic structures, which remodel under the influence of specific cell signaling pathways that influence the fate of the RNA transcript (Naro and Sette, 2013; Fu and Ares, 2014). By precisely integrating co- and post-transcriptional RNA regulatory events, RBPs ensure the physiological adaptation in response to environmental constraints. It follows that the precise arrangement of RNP complexes must be highly coordinated and that deregulation of these complexes can be harmful for cells. Indeed, dysregulation of each aspect of RNA metabolism is involved in a large number of pathological conditions, such as neurodegenerative disease and cancer (Mayr and Bartel, 2009; Cooper et al., 2009; Silvera et al., 2010; Pagliarini et al., 2015). In cancer, aberrant alternative splicing regulation often yields splice variants that confer a selective advantage to the tumor, in terms of proliferation, metabolism, invasion, drug resistance and survival (David et al., 2010; Olshavsky et al., 2010; Paronetto et al., 2010; Valacca et al., 2010; Anczuków et al., 2012; Cappellari et al., 2014; Bielli et al., 2014; Calabretta et al., 2016). Moreover, specific splicing signatures correlate with cancer progression, and alteration of RBPs expression and/or of cis-regulatory elements can contribute to tumorigenesis (Cooper et al., 2009; Danan-Gotthold et al., 2015). High-throughput next-generation sequencing technologies now allow genome-wide identification of alternative splicing events associated with pathological processes (Chen and Weiss, 2015; Byron et al., 2016). Furthermore, they might help understanding the global complexity of RNA regulation and the correlation between binding sites for RBPs and the splicing outcome in health and disease (Wang and Burge, 2008). Thus, understanding alternative splicing changes in pathological conditions requires deciphering the regulatory network between RBPs and cis-regulatory elements and the identification of RBP binding sites is a key step in this direction.

In a recent study, we investigated the role of alternative splicing and RBPs in the acquisition of a drug-resistant (DR) phenotype in pancreatic ductal adenocarcinoma cells (PDAC) (Calabretta et al., 2016). We demonstrated that acquisition of the DR-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which is recruited to the PKM pre-mRNA and favors splicing of the oncogenic PKM2 variant. To investigate the recruitment of PTBP1 on PKM pre-mRNA in vivo, we used the UV cross-linking and immunoprecipitation (CLIP) experimental approach modified from Wang et al. (2009) protocol. Herein, we describe a step-by-step protocol to investigate the direct binding of a specific factor to its RNA target(s), which can be extended to most adherent human cell lines.

Materials and Reagents

  1. 100 mm dish
  2. PDAC cells or other adherent cells
  3. Cold PBS (Sigma-Aldrich, catalog number: D8537 )
  4. Liquid nitrogen
  5. Turbo DNAse (Thermo Fisher Scientific, AmbionTM, catalog number: AM2239 )
  6. Protein G Dynabeads (Thermo Fisher Scientific, NovexTM, catalog number: 10004D )
  7. RBP polyclonal hnRNP I antibody for immunoprecipitation (Santa Cruz Biotechnology, catalog number: sc-16547 )
  8. RNAse I (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2295 )
  9. Bradford solution (Bio-Rad Laboratories, catalog number: 500-0006 )
  10. Phenol:chloroform:isoamyl alcohol 25:24:1 (Sigma-Aldrich, catalog number: P3803 )
  11. Ethanol (VWR, catalog number: 20821.321 )
  12. Trizol (Thermo Fisher Scientific, AmbionTM, catalog number: 15596018 )
  13. Chloroform (Honeywell International, Riedel-de Haen, catalog number: 32211 )
  14. Isopropanol (CARLO ERBA Reagents, catalog number: 415156 )
  15. Agarose (Lonza, catalog number: 50004 )
  16. Water (Sigma-Aldrich, catalog number: W4502 )
  17. cDNA synthesis kit (Promega, catalog number: M1705 )
  18. Tris (VWR, catalog number: 0826 )
  19. Sodium chloride (NaCl) (VWR, catalog number: 27810.364 )
  20. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M2670 )
  21. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
  22. NP-40 (Alfa Aesar, Affymetrix/USB, catalog number: J19628 )
  23. Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750 )
  24. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  25. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
  26. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
  27. RNase inhibitor (Promega, catalog number: N2511 )
  28. EDTA (Sigma-Aldrich, catalog number: E5134 )
  29. Glycerol (Sigma-Aldrich, catalog number: G9012 )
  30. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  31. Bromphenol blue (Sigma-Aldrich, catalog number: 114391 )
  32. Proteinase K (Roche Molecular Systems, catalog number: 3115836001 )
  33. DEPC (Sigma-Aldrich, catalog number: D5758 )
  34. XQuantitative Real-Time PCR Kit (LightCycler®480 SYBR Green I Master) (Roche Molecular Systems, catalog number: 04887352001 )
  35. Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
  36. Sodium metavanadate (Sigma-Aldrich, catalog number: 72060 )
  37. Lysis buffer (see Recipes)
  38. High-salt buffer (see Recipes)
  39. 3 M sodium acetate (pH 5.2) (see Recipes)
  40. Laemmli buffer (2x) (see Recipes)
  41. Proteinase K buffer (see Recipes)

Equipment

  1. Centrifuge
  2. UV crosslinker (Uvitec, model: CL 508 )
  3. SDS-PAGE and PVDF/nitrocellulose transfer apparatus (Bio-Rad Laboratories)
  4. Denaturating agarose gel apparatus (Bio-Rad Laboratories)
  5. Magnetic stand (Thermo Fisher Scientific, Invitrogen)
  6. Thermoblock
  7. Quantitative Real-Time PCR (Roche Molecular Systems, model: LightCycler® 480 )
  8. Sonicator (Hielscher ultrasonics, model: UP200S )

Procedure

  1. Cell culture preparation and UV cross-linking
    1. Grow adherent PDAC cells until they reach 70-80% confluence. Usually, 5-10 x 106 cells are used for each immunoprecipitation (IP).
    2. Discard the medium and wash once with cold PBS. Add 4-5 ml of PBS in 100 mm cell culture Petri dish (~1 ml/cm2).
    3. Place the dish on ice without lid. Irradiate once with 400 mJ/cm2 (see Figure 1).

    Note: UV irradiation produces a covalent bound between RNA and protein that are in contact. The energy level to use depends on availability of aromatic acids. This covalent bound allows purification of RBP/RNA complex under stringent condition. The choice of UV irradiation should be set as the minimum irradiation that allows purification of a control RNA.


    Figure 1. UV cross-linking. UV cross-linking was performed to bind RNA covalently with proteins. During irradiation the dish is kept on ice to minimize heating.

    1. Remove the PBS and add 0.5 ml of lysis buffer into the 100 mm dish.
      Note: Do not exceed with lysis buffer.
    2. Alternatively, gently scrape off the cells in cold PBS. Centrifuge for 5 min at 300 x g, at 4 °C. Discard the supernatant, snap freeze the pellets in liquid nitrogen and store at -80 °C.

  2. Cell lysis
    1. Gently resuspend the cells in lysis buffer and transfer the suspended cells to a 1.5 ml centrifuge tube and sonicate on ice [Set: Amplitude (%) at 100 and Cycle at 1] (see Figure 2).
      Note: Cells tend to aggregate in lysis buffer. Sonication is required to completely dissolve cells aggregate. Usually 5 sec is enough.


      Figure 2. Cells sonication. Sonication was performed to dissolve cells aggregate. During sonication the tube is kept in ice to minimize protein denaturation.

    2. Incubate the cells for 10 min on ice.
    3. Add 5 µl per ml of sample of Turbo DNAse. Incubate for 3 min at 37 °C.
    4. Centrifuge at 15,000 x g at 4 °C for 3 min.
    5. Collect the supernatant (cell extract), dilute sample to 1 mg/ml in lysis buffer and store in ice.
      Note: Lysis buffer composition interferes with Bradford assay. No more than 1 µl of cell extract should be added to 1 ml of Bradford solution.

  3. RNA fragmentation and RBP immunoprecipitation
    For each IP use 10 µl of Protein G Dynabeads.
    1. Wash Protein G Dynabeads with 500 µl of lysis buffer and mixing the suspension. Place the tube containing the suspension in the magnetic stand and remove the supernatant. Repeat this step 3 times.
    2. Resuspend the Protein G Dynabeads in 100 µl of lysis buffer and add 3-5 µg of specific polyclonal antibody. Use IgG isotype as a negative control.
    3. Collect 3 aliquots of 10% of cell extract (100 µg) (Input) before adding RNase I.
      Note: Aliquots of inputs are required for (i) quantitative analysis of RBP binding (see steps D1a and F3), (ii) evaluation of RBP IP (see step C10a), and (iii) for evaluation of RNAse I fragmentation (see step D3, and Figure 4).
    4. Add 1 ml of cell extract (1 mg) to 100 µl Protein G Dynabeads (see step C2). Store on ice.
    5. Prepare a dilution of RNase I 1:1,000 in lysis buffer.
    6. Add to each IP 10 µl of RNase I 1:1,000.
    7. Rotate at 4 °C for 2 h.
    8. After incubation place the samples in the magnetic stand, collect supernatant aliquots (100 µl) from each IPs and discard the residual supernatant. Store aliquots on ice.
      Note: Aliquots are required for evaluation of RNAse I fragmentation (see step D3, and Figure 4).
    9. Wash IPs twice with 1 ml of high salt buffer and 2 times with 1 ml proteinase K buffer.
    10. Resuspend the Dynabeads in 100 µl of proteinase K buffer. Collect an aliquot of 10% of suspension (10 µl) from each IP. Add 10 µl of Laemmli buffer (2x) to the aliquots and boil aliquot for 5-10 min at 100 °C (see step C10a).
      Note: Aliquots are required for evaluation of RBP IP by Western-blot analysis (see C10a).
      1. Control of RBPs IP
        Run an SDS-PAGE following Western blot analysis using aliquots from step C2 (input) and aliquots from step C10 (IP) samples (Figure 3).


        Figure 3. Control of RBP immunoprecipitation in HEK293T cells. Western blot analysis of aliquotes (10%) from step C2 (Input) and step C10 (IPs).

  4. RNA isolation
    1. From IPs:
      1. RNA is eluted by adding 50 µg of proteinase K (2.5 µl of proteinase K solution) to 90 µl of the remaining IP suspensions (step C10).
      2. Incubate for 1 h at 55 °C.
      3. Place samples in the magnetic stand, collect the supernatants and add water up to 300 µl.
      4. Add at each supernatant 1 volume of phenol:chloroform:isoamilic alcohol (25:24:1).
      5. Mix vigorously for 10 sec. Incubate for 10 min at room temperature.
      6. Centrifuge at 15,000 x g for 10 min. Collect the supernatant.
      7. Add 1/10 volume of 3 M sodium acetate (pH 5.2).
      8. Mix and add 2.5 volume of ethanol absolute.
      9. Mix and incubate overnight at -20 °C.
      10. After incubation, centrifuge at 15,000 x g for 15 min.
      11. Discard the supernatant and wash the RNA pellet with 1 ml of 70% ethanol.
      12. Centrifuge at 15,000 x g for 10 min.
      13. Discard the supernatant and air dry the pellet.
    2. From Input:
      1. 50 µg of proteinase K (2.5 µl of proteinase K solution) was added to one of 10% input aliquot (step C2).
      2. Incubate for 1 h at 55 °C.
      3. RNA is recovered by adding 1 ml Trizol to the samples. Incubate for 5 min at RT.
      4. Add 200 µl of chloroform and mix vigorously.
      5. Centrifuge at 12,000 x g for 15 min.
      6. After centrifugation, collect half of aqueous phase (≈ 250 µl) to avoid DNA contamination.
        Note: In our experience, treatment of RNA input sample with DNAse is not sufficient to remove DNA contamination. We prevent DNA contamination by collecting only half of the aqueous phase and avoiding to touch the interphase layer during RNA isolation with Trizol reagent.
      7. Add 1 volume of 100% isopropanol. And incubate for 10 min at RT.
      8. Centrifuge at 12,000 x g for 15 min and discard the supernatant.
      9. Wash RNA pellet with 1 ml of 70% ethanol.
      10. Centrifuge at 12,000 x g for 10 min.
      11. Discard the supernatant and air dry the pellet.
    3. Control of RNAse I fragmentation
      1. Add 50 µg of proteinase K (2.5 µl of proteinase K solution) to one aliquot from step C2 and step C7.
      2. Incubate for 1 h at 55 °C. Add 1 volume of phenol:chloroform:isoamilic alcohol.
      3. Proceed as described in steps D1e-D1m.
    4. Resuspend the RNA in 20 µl of nuclease-free water. After quantification at 260 nm, load the samples on 1.5% denaturating agarose gel for evaluation of RNA fragmentation (Figure 4).


      Figure 4. Control of RNA fragmentation. Denaturating agarose gel (1.5%) showing RNA fragmentation in presence of different RNAse I dilutions (1:500 corresponds to 2 U/ml of RNase I), and in presence of different extract concentration (0.8 and 2 mg/ml, left and right panel respectively). The optimal RNA fragmentation is indicated (red box).

  5. Reverse transcription (cDNA synthesis)
    1. Resuspend the RNA pellets from steps D1m and D2k in the same volume of nuclease free water.
      Note: Volume of water depends on the cDNA synthesis kit used.
    2. Heat for 5 min at 55 °C and store on ice.
    3. Perform cDNA synthesis according to manufacturer’s instructions using half volume of RNA samples. Use the other half to perform cDNA synthesis reaction in absence of reverse transcriptase enzyme (negative control). cDNA synthesis has to be performed using random hexamer primers.
      Note: Negative control is required to evaluate DNA contamination in all samples.

  6. Real-time quantitative PCR (qRT-PCR)
    1. Design pairs of primer located in different regions of the pre-mRNA, including the region embedding the RBP binding site (Figure 5).
      Notes:
      1. Optimal PCR product length is 100 nt.
      2. If RBP binding site is not known, it is possible to identify the region in which this is embedded by using several primers pair designed to span the entire pre-mRNA.
    2. Perform qRT-PCR according to manufacturer’s instructions.
      Note: Quantification cycle (Cq) of negative control should be at least 6 cycles less than relative sample (i.e., Cq negative control = 36; Cq sample = 30).


      Figure 5. Schematic representation of primers position along the pre-mRNA for evaluation of RBP binding

Data analysis

Binding of RBP is reported as % of Input in different regions of the RNA target, using the comparative ∆Cq method as follow (Figure 6).

  1. Cq of Input (Cq5% input; 5%; see steps C2 and D2f) need to be adjusted to 100% (Cq100% Input) according to dilution: Cq100% Input = Cq5% input - [log2(DF)]
    Note: 5% input (see steps C2 and D2f) correspond to a dilution factor (DF) of 20. Moreover, Thus, Cq100% Input = Cq5% input - log2(10).
  2. Cq value of IPs (CqIP) is compared (∆Cq) to Cq value of 100% of Input (Cq100% input):
    ∆Cq = CqIP - Cq100% input
    Note: For IgG IP: ∆CqIgG = CqIgG IP - Cq100% input. For RBP IP: ∆CqRBP = CqRBP IP - Cq100% input.
  3. Association of RBP is reported as % input: % input = 100 x 2-∆Cq
    Note: For IgG: % InputIgG = 100 x 2-∆CqIgG. For RBP: % InputRBP = 100 x 2-∆CqRBP


    Figure 6. Data analysis of RBP association to RNA target. Using hypothetical Cq values, data analysis of RBP binding to the indicated pre-mRNA regions (Exon1-Intron1; Exon2-Intron2; Exon3-Intron3) is shown. * = % of Input is 5% see steps C2 and D2f; DF (dilution factor) = 20; R = replicate.

Notes

To ensure reproducibility, biological experiments are usually performed in replicates (triplicate). We also recommend to perform each CLIP IPs in duplicate (technical duplicates) to increase the reliability of each experiment (Figure 7). Statistical analysis is performed by t-test procedure.



Figure 7. Data analysis of RBP association to RNA target from three biological replicate experiments. Using hypothetical values, data analysis of RBP binding to pre-mRNA from three biological experiments is shown. By performing technical duplicates for each experiment is possible to eliminate value(s) that deviate from the others. In this example, the value highlighted in red clearly deviates from the other five. Thus, it is conceivable to eliminate it from further analysis, as it may result from a mistake.

Recipes

Note: Make sure to prepare all the following solution in DEPC-treated water.

  1. Lysis buffer
    50 mM Tris-HCl (pH 7.4)
    100 mM NaCl
    1 M MgCl2
    0.1 mM CaCl2
    1% NP-40
    0.5% sodium deoxycholate
    0.1% SDS
    Supplemented with fresh 1 mM DTT, 0.5 mM sodium metavanadate, protease inhibitor cocktail, RNase inhibitor (1 µl/ml)
  2. High-salt buffer
    50 mM Tris-HCl (pH 7.4)
    1 M NaCl
    1 mM EDTA
    1% NP-40
    0.5% sodium deoxycholate
    0.1% SDS
    Supplemented with fresh RNase inhibitor (0.5 µl/ml)
  3. Proteinase K buffer
    100 mM Tris-HCl (pH 7.4)
    50 mM NaCl
    10 mM EDTA
    Supplemented with fresh RNase inhibitor (0.5 µl/ml)
  4. 3 M sodium acetate (pH 5.2)
    For preparing this solution, please refer to (Iglesias et al., 2017, http://en.bio-protocol.org/e2180)
  5. Leammli buffer (2x)
    0.125 M Tris-HCl, pH 6.8
    20% glycerol
    4% SDS
    2% β-mercaptoethanol
    0.02% bromphenol blue
  6. Proteinase K solution (20 mg/ml)
    Dissolve 20 mg of lyophilized proteinase K in 1 ml of double distilled water

Acknowledgments

The CLIP method presented herein is a modified protocol from Wang et al. (2009). This method was employed to identify the binding sites of PTBP1 on the PKM pre-mRNA in Calabretta et al. (2016). The research in our laboratory was supported by the Associazione Italiana Ricerca sul Cancro (AIRC; IG18790), by Telethon Foundation (GGP14095) and by Italian Ministry of Health ‘Ricerca Finalizzata 2011’ (GR-2011-02348423) and '5x1000 Anno 2014' to Fondazione Santa Lucia.

References

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  9. Cooper, T. A., Wan, L. and Dreyfuss, G. (2009). RNA and disease. Cell 136(4): 777-793.
  10. Danan-Gotthold, M., Golan-Gerstl, R., Eisenberg, E., Meir, K., Karni, R. and Levanon, E. Y. (2015). Identification of recurrent regulated alternative splicing events across human solid tumors. Nucleic Acids Res 43(10): 5130-5144.
  11. David, C. J., Chen, M., Assanah, M., Canoll, P. and Manley, J. L. (2010). HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer. Nature 463(7279): 364-368.
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简介

RNA代谢在不同的组织和发育阶段被严格控制,其失调是癌症的分子特征之一。 通过直接结合特定的序列元件,RNA结合蛋白(RBP)在共转录和转录后调控事件中起关键作用。 我们最近证实,在胰腺癌细胞中,获得耐药(DR) - 表型取决于多聚嘧啶区结合蛋白(PTBP1)的上调,其又被引入丙酮酸激酶前mRNA并有利于剪接 致癌性PKM2变体。 在这里,我们描述了紫外(UV)光交联和免疫沉淀(CLIP)方法的逐步方案,以确定RBP与贴壁人细胞系中其目标RNA的特定区域的直接结合。

背景 在细胞核中转录时,新生的RNA立即与被称为RNA结合蛋白(RBP)的反式因子立即组装。这些因子直接与RNA分子中特定的顺式调控序列相互作用,从而形成核糖核蛋白(RNP)复合物(Dreyfuss et al。,2002; Singh等人2015)。这些复合物控制共转录RNA加工事件以及参与RNA代谢的转录后机制,如亚细胞定位和翻译。例如,剪接体和切割/多聚腺苷酸化复合物组分识别前mRNA中的特异性RNA元件,允许去除内含子(Black,2003)和3'-末端加工和转录终止之间的协调(Proudfoot,2016)。大量的RBP作为剪接因子,通过协助拼接体识别组成型和可变剪接的外显子(Chen和Manley,2009)或改进替代多聚腺苷酸化信号的使用(Tian和Manley,2016)。同样,RBP介导的邮政编码定位元件的识别允许mRNA在细胞质中的转运和局部翻译(Martin和Ephrussi,2009)。
 真核基因组编码大量RBP,以​​时间和空间敏感的方式微调细胞特异性基因表达程序,从而有助于组织体内平衡。 RNP是高度动态的结构,其在影响RNA转录物命运的特异性细胞信号通路的影响下重塑(Naro和Sette,2013; Fu和Ares,2014)。通过精确整合共转录和转录后RNA调控事件,RBP确保响应于环境约束的生理适应。因此,RNP复合物的精确布置必须高度协调,并且这些复合物的去调节对细胞是有害的。事实上,RNA代谢的各个方面的异常调节参与大量的病理状况,例如神经变性疾病和癌症(Mayr和Bartel,2009; Cooper等人,2009; Silvera et al。,2010; Pagliarini等人,2015)。在癌症中,异常的选择性剪接调节通常产生在增殖,代谢,入侵,耐药性和存活方面赋予肿瘤选择性优势的剪接变体(David等人,2010; Olshavsky 2010年; Paronetto等人,2010; Valacca等人,2010;Anczuków等人。 >,2012; Cappellari等人,2014; Bielli等人,2014; Calabretta等人,2016)。此外,特异性剪接特征与癌症进展相关,并且RBP表达和/或顺式调节元件的改变可能有助于肿瘤发生(Cooper et al。,2009; Danan -Gotthold 等,,2015)。高通量下一代测序技术现在允许全基因组鉴定与病理过程相关的可选剪接事件(Chen和Weiss,2015; Byron等人,2016)。此外,它们可能有助于理解RNA调节的全球复杂性以及RBP的结合位点与健康和疾病中的剪接结果之间的相关性(Wang and Burge,2008)。因此,理解病理条件下的选择性剪接变化需要破译RBP和顺式调节元件之间的调节网络,并且RBP结合位点的鉴定是这个方向的关键步骤。
 在最近的一项研究中,我们调查了选择性剪接和RBP在胰腺导管腺癌细胞(PDAC)中获得抗药性(DR)表型的作用(Calabretta等人,2016年) )。我们证明,DR-表型的获得依赖于多肽嘧啶结合蛋白(PTBP1)的上调,其被募集到PKM前mRNA并有利于致癌性PKM2变体的剪接。为了调查PTBP1在体外PKM pre-mRNA上的募集,我们使用了王等人修改的UV交联和免疫沉淀(CLIP)实验方法。(2009) ) 协议。在这里,我们描述了一个逐步的方案来研究特定因子与其RNA靶标的直接结合,这可以扩展到大多数贴壁的人类细胞系。

关键字:CLIP, 蛋白质 - RNA相互作用, 蛋白质 - RNA免疫沉淀, RNA加工

材料和试剂

  1. 100毫米盘
  2. PDAC细胞或其他粘附细胞
  3. 冷PBS(Sigma-Aldrich,目录号:D8537)
  4. 液氮
  5. Turbo DNAse(Thermo Fisher Scientific,Ambion TM ,目录号:AM2239)
  6. 蛋白G Dynabeads(Thermo Fisher Scientific,Novex TM,目录号:10004D)
  7. 用于免疫沉淀的RBP多克隆hnRNP I抗体(Santa Cruz Biotechnology,目录号:sc-16547)
  8. RNAse I(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2295)
  9. Bradford解决方案(Bio-Rad Laboratories,目录号:500-0006)
  10. 苯酚:氯仿:异戊醇25:24:1(Sigma-Aldrich,目录号:P3803)
  11. 乙醇(VWR,目录号:20821.321)
  12. Trizol(Thermo Fisher Scientific,Ambion TM ,目录号:15596018)
  13. 氯仿(Honeywell International,Riedel-de Haen,目录号:32211)
  14. 异丙醇(CARLO ERBA试剂,目录号:415156)
  15. 琼脂糖(Lonza,目录号:50004)
  16. 水(Sigma-Aldrich,目录号:W4502)
  17. cDNA合成试剂盒(Promega,目录号:M1705)
  18. Tris(VWR,目录号:0826)
  19. 氯化钠(NaCl)(VWR,目录号:27810.364)
  20. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M2670)
  21. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C1016)
  22. NP-40(Alfa Aesar,Affymetrix/USB,目录号:J19628)
  23. 脱氧胆酸钠(Sigma-Aldrich,目录号:D6750)
  24. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  25. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D9779)
  26. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)
  27. RNase抑制剂(Promega,目录号:N2511)
  28. EDTA(Sigma-Aldrich,目录号:E5134)
  29. 甘油(Sigma-Aldrich,目录号:G9012)
  30. β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  31. 溴酚蓝(Sigma-Aldrich,目录号:114391)
  32. 蛋白酶K(Roche Molecular Systems,目录号:3115836001)
  33. DEPC(Sigma-Aldrich,目录号:D5758)
  34. XQuantitative实时PCR试剂盒(LightCycler 480 SYBR Green I Master)(Roche Molecular Systems,目录号:04887352001)
  35. 乙酸钠(Sigma-Aldrich,目录号:S2889)
  36. 偏钒酸钠(Sigma-Aldrich,目录号:72060)
  37. 裂解缓冲液(见配方)
  38. 高盐缓冲液(见配方)
  39. 3 M醋酸钠(pH 5.2)(见配方)
  40. Laemmli缓冲液(2x)(参见食谱)
  41. 蛋白酶K缓冲液(参见食谱)

设备

  1. 离心机
  2. UV交联剂(Uvitec,型号:CL 508)
  3. SDS-PAGE和PVDF /硝酸纤维素转移装置(Bio-Rad Laboratories)
  4. 变性琼脂糖凝胶仪(Bio-Rad Laboratories)
  5. 磁性支架(Thermo Fisher Scientific,Invitrogen)
  6. Thermoblock
  7. 定量实时PCR(Roche Molecular Systems,型号:LightCycler 480)
  8. 超声波仪(Hielscher ultrasonics,型号:UP200S)

程序

  1. 细胞培养制备和UV交联
    1. 生长粘附的PDAC细胞,直到达到70-80%汇合。通常每个免疫沉淀(IP)使用5-10×10 6个细胞。
    2. 舍弃介质并用冷PBS洗一次。在100毫米细胞培养培养皿(约1毫升/厘米2)中加入4-5毫升PBS。
    3. 将盘放在没有盖子的冰上。以400 mJ/cm 2的速度辐射一次(参见图1)。

    注意:紫外线照射会产生与蛋白质接触的共价结合。使用的能量水平取决于芳香酸的可用性。这种共价结合允许在严格条件下纯化RBP/RNA复合物。紫外线照射的选择应设定为允许纯化对照RNA的最小照射。


    图1.紫外线交联进行UV交联以将蛋白质与共价RNA结合。在照射期间,将盘保持在冰上以最小化加热。

    1. 取出PBS,并加入0.5毫升裂解缓冲液至100毫米皿中。
      注意:不要超过裂解缓冲液。
    2. 或者,在冷PBS中轻轻刮掉细胞。在4℃下以300×g离心5分钟。丢弃上清液,将颗粒快速冻结在液氮中,并储存在-80°C
  2. 细胞裂解
    1. 将细胞轻轻地悬浮在裂解缓冲液中,并将悬浮细胞转移到1.5ml离心管中,并在冰上超声处理[Set:Amplitude(%)at 100 and Cycle at 1](见图2)。
      注意:细胞倾向于在裂解缓冲液中聚集。需要超声处理完成溶解细胞聚集体。通常5秒就够了


      图2.细胞超声处理进行超声处理以溶解细胞聚集体。超声处理时,将管保持在冰中,以使蛋白质变性最小化
    2. 在冰上孵育细胞10分钟。
    3. 每毫升Turbo DNAse样品中加入5μl。在37℃下孵育3分钟。
    4. 在4℃下以15,000 x g离心3分钟。
    5. 收集上清液(细胞提取物),稀释样品至裂解缓冲液中1 mg/ml,并储存在冰中。
      注意:裂解缓冲液组合物干扰了Bradford测定。不超过1微升的细胞提取物应加入1毫升Bradford溶液。

  3. RNA片段化和RBP免疫沉淀
    每个IP使用10μl蛋白G Dynabeads。
    1. 用500μl裂解缓冲液洗涤蛋白G Dynabeads并混合悬浮液。将含有悬浮液的管置于磁性支架上,取出上清液。重复此步骤3次。
    2. 将蛋白G Dynabeads重悬于100μl裂解缓冲液中,加入3-5μg特异性多克隆抗体。使用IgG同种型作为阴性对照。
    3. 在加入RNA酶I之前,收集10份细胞提取物(100μg)(Input)的3个等分试样 注意:(i)RBP结合的定量分析(参见步骤D1a和F3),(ii)评估RBP IP(参见步骤C10a)和(iii)评估RNA酶I片段(见步骤D3和图4)。
    4. 加入1 ml细胞提取物(1 mg)至100μl蛋白G Dynabeads(见步骤C2)。在冰上存放
    5. 在裂解缓冲液中制备RNA酶I 1:1,000稀释液。
    6. 加入每个IP 10μlRNAse I 1:1,000。
    7. 在4℃下旋转2小时。
    8. 孵育后将样品置于磁性支架中,从每个IP中收集上清液等分试样(100μl),并丢弃残留的上清液。在冰上储存等分试样。
      注意:评估RNA酶I片段时需要等分试样(见步骤D3和图4)。
    9. 用1ml高盐缓冲液洗涤IP两次,用1ml蛋白酶K缓冲液洗涤2次。
    10. 将Dynabeads重悬于100μl蛋白酶K缓冲液中。从每个IP收集10%悬浮液(10μl)的等分试样。加入10μlLaemmli缓冲液(2x)至等分试样,并在100°C下煮沸等份5-10分钟(见步骤C10a)。
      注意:通过蛋白质印迹分析评估RBP IP需要等分试样(参见C10a)。
      1. 控制RBP IP
        使用步骤C2(输入)等分试样和来自步骤C10(IP)样品的等分试样(图3)进行蛋白质印迹分析后进行SDS-PAGE。


        图3. HEK293T细胞中RBP免疫沉淀的控制。来自步骤C2(输入)和步骤C10(IP)的等分试样(10%)的Western印迹分析。

  4. RNA分离
    1. 来自知识产权
      1. 通过向90μl剩余的IP悬浮液中加入50μg蛋白酶K(2.5μl蛋白酶K溶液)来洗脱RNA(步骤C10)。
      2. 在55℃孵育1小时。
      3. 将样品置于磁性支架中,收集上清液,加水至300μl。
      4. 在每个上清液中加入1体积的苯酚:氯仿:异戊醇(25:24:1)
      5. 剧烈搅拌10秒。在室温下孵育10分钟。
      6. 以15,000 x g离心10分钟。收集上清液。
      7. 加入1/10体积的3M醋酸钠(pH5.2)
      8. 混合并加入2.5体积的绝对乙醇
      9. 在-20℃下混合并孵育过夜。
      10. 孵育后,以15,000 x g离心15分钟。
      11. 弃去上清液,用1ml 70%乙醇洗涤RNA沉淀。
      12. 以15,000 x g离心10分钟。
      13. 弃上清,空气干燥颗粒。
    2. 从输入:
      1. 将50μg蛋白酶K(2.5μl蛋白酶K溶液)加入到10%输入等分试样之一(步骤C2)中。
      2. 在55°C孵育1小时。
      3. 通过向样品中加入1ml Trizol来回收RNA。在室温下孵育5分钟。
      4. 加入200μl氯仿并剧烈混合。
      5. 以12,000 x g离心15分钟。
      6. 离心后,收集一半水相(≈250μl),以避免DNA污染。
        注意:根据我们的经验,用DNA酶处理RNA输入样品不足以清除DNA污染。我们通过收集仅一半的水相并避免在用Trizol试剂进行RNA分离期间接触相间层来防止DNA污染。
      7. 加入1体积的100%异丙醇。并在室温下孵育10分钟。
      8. 以12,000 x g离心15分钟,弃去上清液。
      9. 用1ml 70%乙醇洗涤RNA沉淀。
      10. 以12,000 x g离心10分钟。
      11. 弃上清,空气干燥颗粒。
    3. 控制RNA酶I片段化
      1. 将50μg蛋白酶K(2.5μl蛋白酶K溶液)加入到步骤C2和步骤C7中的一个等分试样中。
      2. 在55℃孵育1小时。加入1体积的苯酚:氯仿:异戊醇。
      3. 按照步骤D1e-D1m所述进行。
    4. 将RNA重悬在20μl无核酸酶的水中。在260nm定量后,将样品加载到1.5%变性琼脂糖凝胶上以评估RNA片段化(图4)。


      图4.控制RNA断裂。在不同的RNA酶I稀释液(1:500对应于2U/ml的RNA酶I)存在下,并且在不同提取物浓度(0.8和2mg/ml)存在下,使具有RNA片段化的变性琼脂糖凝胶(1.5%) ,左右面板)。指示最佳RNA断裂(红色框)。

  5. 逆转录(cDNA合成)
    1. 在相同体积的无核酸酶的水中重悬从步骤D1m和D2k的RNA颗粒。
      注意:水的体积取决于所用的cDNA合成试剂盒。
    2. 在55°C下加热5分钟并储存在冰上。
    3. 根据制造商的说明书使用半体积的RNA样品进行cDNA合成。使用另一半在不存在逆转录酶的情况下进行cDNA合成反应(阴性对照)。 cDNA合成必须使用随机六聚体引物进行。
      注意:需要负面控制来评估所有样品中的DNA污染。

  6. 实时定量PCR(qRT-PCR)
    1. 位于前mRNA的不同区域的引物的设计对,包括嵌入RBP结合位点的区域(图5)。
      注意:
      1. 最佳PCR产物长度为100nt。
      2. 如果RBP结合位点不是已知的,可以通过使用设计为跨越整个mRNA前体的几个引物对来鉴定嵌入其中的区域。
    2. 按照制造商的说明进行qRT-PCR。
      注意:阴性对照的定量循环(Cq)应该比相对样品少至少6个循环(即Cq阴性对照= 36; Cq样品= 30)。


      图5.用于评估RBP结合的引物前沿mRNA的位置示意图

数据分析

使用比较的ΔCq方法,RBP的结合报告为RNA靶标不同区域的输入百分比(图6)。

  1. 根据稀释度,输入Cq(Cq <5%输入<5%;参见步骤C2和D2f)需要调整为100%(Cq <100%Input ):Cq 100%输入 = Cq <5>输入 - [log 2 (DF)]
    注意:5%输入(见步骤C2和D2f)对应于20的稀释因子(DF)。此外,因此,Cq 100%输入 = Cq 5%输入 - 日志 子> (10)。
  2. 将IP(Cq IP )的Cq值与输入(Cq <100%输入)的100%的Cq值进行比较(ΔCq):
    ΔCq= Cq IP - Cq <100%输入
    注意:对于IgG IP:ΔCq IgG = Cq IgG IP - Cq 100%输入 。对于RBP IP:ΔCqRBP = Cq > 100%输入
  3. RBP的关联报告为%输入:%input = 100 x 2 -ΔCq
    注意:对于IgG:%输入 IgG = 100 x 2 -ΔCqIgG 。对于RBP:%输入 RBP = 100 x 2 -ΔCqRBP


    图6. RBP关联到RNA靶标的数据分析。 使用假设的Cq值,显示了与指定的前mRNA区域(Exon1-Intron1; Exon2-Intron2; Exon3-Intron3)结合的RBP的数据分析。 * =输入的百分比为5%,见步骤C2和D2f; DF(稀释因子)= 20; R =复制。

笔记

为了确保重复性,生物实验通常以重复(一式三份)进行。我们还建议重复执行每个CLIP IP(技术重复),以提高每个实验的可靠性(图7)。统计分析由测试程序执行。



图7.来自三个生物重复实验的RBP与RNA靶标的数据分析。使用假设值,显示了来自三个生物实验的RBP与前mRNA的结合的数据分析。通过执行每个实验的技术重复,可以消除与其他实验偏离的价值。在这个例子中,以红色突出显示的值明显偏离了其他五个。因此,可以将其从进一步分析中消除,这可能是由于错误而导致的。

食谱

注意:确保在DEPC处理过的水中准备所有以下溶液。

  1. 裂解缓冲液
    50mM Tris-HCl(pH7.4)
    100 mM NaCl
    1 M MgCl 2
    0.1mM CaCl 2
    1%NP-40
    0.5%脱氧胆酸钠
    0.1%SDS
    补充新鲜的1mM DTT,0.5mM偏钒酸钠,蛋白酶抑制剂混合物,RNase抑制剂(1μl/ml)
  2. 高盐缓冲液
    50mM Tris-HCl(pH7.4)
    1 M NaCl
    1 mM EDTA
    1%NP-40
    0.5%脱氧胆酸钠
    0.1%SDS
    补充新鲜的RNase抑制剂(0.5μl/ml)
  3. 蛋白酶K缓冲液
    100mM Tris-HCl(pH7.4)
    50 mM NaCl
    10 mM EDTA
    补充新鲜的RNase抑制剂(0.5μl/ml)
  4. 3 M醋酸钠(pH 5.2)
    有关准备此解决方案,请参阅(Iglesias et al。,2017, http://en.bio-protocol.org/e2180
  5. Leamli缓冲区(2x)
    0.125M Tris-HCl,pH6.8。
    20%甘油
    4%SDS
    2%β-巯基乙醇
    0.02%溴酚蓝
  6. 蛋白酶K溶液(20 mg/ml)
    将20毫克冻干蛋白酶K溶于1毫升双蒸水中

致谢

本文提出的CLIP方法是Wang等人的修改协议(2009)。采用该方法鉴定了Calabretta等人(2016)上的PKM前mRNA上PTBP1的结合位点。我们实验室的研究得到了Telethon基金会(GGP14095)和意大利卫生部"Ricerca Finalizzata 2011"(GR-2011-02348423)和"2010年4月5日"的意大利语Ricerca sul Cancro(AIRC; IG18790)的支持,到圣诞老人卢西亚Fondazione。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bielli, P. and Sette, C. (2017). Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method. Bio-protocol 7(10): e2274. DOI: 10.21769/BioProtoc.2274.
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