Materials and Reagents

100 mm dish
PDAC cells or other adherent cells
Cold PBS (Sigma-Aldrich, catalog number: D8537 )
Liquid nitrogen
Turbo DNAse (Thermo Fisher Scientific, Ambion^TM, catalog number: AM2239 )
Protein G Dynabeads (Thermo Fisher Scientific, Novex^TM, catalog number: 10004D )
RBP polyclonal hnRNP I antibody for immunoprecipitation (Santa Cruz Biotechnology, catalog number: sc-16547 )
RNAse I (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM2295 )
Bradford solution (Bio-Rad Laboratories, catalog number: 500-0006 )
Phenol:chloroform:isoamyl alcohol 25:24:1 (Sigma-Aldrich, catalog number: P3803 )
Ethanol (VWR, catalog number: 20821.321 )
Trizol (Thermo Fisher Scientific, Ambion^TM, catalog number: 15596018 )
Chloroform (Honeywell International, Riedel-de Haen, catalog number: 32211 )
Isopropanol (CARLO ERBA Reagents, catalog number: 415156 )
Agarose (Lonza, catalog number: 50004 )
Water (Sigma-Aldrich, catalog number: W4502 )
cDNA synthesis kit (Promega, catalog number: M1705 )
Tris (VWR, catalog number: 0826 )
Sodium chloride (NaCl) (VWR, catalog number: 27810.364 )
Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M2670 )
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C1016 )
NP-40 (Alfa Aesar, Affymetrix/USB, catalog number: J19628 )
Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
RNase inhibitor (Promega, catalog number: N2511 )
EDTA (Sigma-Aldrich, catalog number: E5134 )
Glycerol (Sigma-Aldrich, catalog number: G9012 )
β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
Bromphenol blue (Sigma-Aldrich, catalog number: 114391 )
Proteinase K (Roche Molecular Systems, catalog number: 3115836001 )
DEPC (Sigma-Aldrich, catalog number: D5758 )
XQuantitative Real-Time PCR Kit (LightCycler^®480 SYBR Green I Master) (Roche Molecular Systems, catalog number: 04887352001 )
Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
Sodium metavanadate (Sigma-Aldrich, catalog number: 72060 )
Lysis buffer (see Recipes)
High-salt buffer (see Recipes)
3 M sodium acetate (pH 5.2) (see Recipes)
Laemmli buffer (2x) (see Recipes)
Proteinase K buffer (see Recipes)

Equipment

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

Procedure

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.
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.
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).
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).
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.
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).

Cq of Input (Cq_{5% input}; 5%; see steps C2 and D2f) need to be adjusted to 100% (Cq_{100% Input}) according to dilution: Cq_{100% Input} = Cq_{5% input} - [log₂(DF)]
Note: 5% input (see steps C2 and D2f) correspond to a dilution factor (DF) of 20. Moreover, Thus, Cq_{100% Input} = Cq_{5% input} - log₂(10).
Cq value of IPs (Cq_IP) is compared (∆Cq) to Cq value of 100% of Input (Cq_{100% input}):
∆Cq = Cq_IP- Cq_{100% input}
Note: For IgG IP: ∆Cq_IgG = Cq_{IgG IP} - Cq_{100% input}. For RBP IP: ∆Cq_RBP= Cq_{RBP IP} - Cq_{100% input}.
Association of RBP is reported as % input: % input = 100 x 2^-∆CqNote: For IgG: % Input_IgG= 100 x 2^-∆CqIgG. For RBP: % Input_RBP = 100 x 2^-∆CqRBPFigure 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.

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)
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)
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)
3 M sodium acetate (pH 5.2)
For preparing this solution, please refer to (Iglesias et al., 2017, http://en.bio-protocol.org/e2180)
Leammli buffer (2x)
0.125 M Tris-HCl, pH 6.8
20% glycerol
4% SDS
2% β-mercaptoethanol
0.02% bromphenol blue
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

Anczuków, O., Rosenberg, A. Z., Akerman, M., Das, S., Zhan, L., Karni, R., Muthuswamy, S. K. and Krainer, A. R. (2012). The splicing factor SRSF1 regulates apoptosis and proliferation to promote mammary epithelial cell transformation. Nat Struct Mol Biol 19(2): 220-228.
Bielli, P., Busa, R., Di Stasi, S. M., Munoz, M. J., Botti, F., Kornblihtt, A. R. and Sette, C. (2014). The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis. EMBO Rep 15(4): 419-427.
Black, D. L. (2003). Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72: 291-336.
Byron, S. A., Van Keuren-Jensen, K. R., Engelthaler, D. M., Carpten, J. D. and Craig, D. W. (2016). Translating RNA sequencing into clinical diagnostics: opportunities and challenges. Nat Rev Genet 17(5): 257-271.
Calabretta, S., Bielli, P., Passacantilli, I., Pilozzi, E., Fendrich, V., Capurso, G., Fave, G. D. and Sette, C. (2016). Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells. Oncogene 35(16): 2031-2039.
Cappellari, M., Bielli, P., Paronetto, M. P., Ciccosanti, F., Fimia, G. M., Saarikettu, J., Silvennoinen, O. and Sette, C. (2014). The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells. Oncogene 33(29): 3794-3802.
Chen, J. and Weiss, W. A. (2015). Alternative splicing in cancer: implications for biology and therapy. Oncogene 34(1): 1-14.
Chen, M. and Manley, J. L. (2009). Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat Rev Mol Cell Biol 10(11): 741-754.
Cooper, T. A., Wan, L. and Dreyfuss, G. (2009). RNA and disease. Cell 136(4): 777-793.
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.
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.
Dreyfuss, G., Kim, V. N. and Kataoka, N. (2002). Messenger-RNA-binding proteins and the messages they carry. Nat Rev Mol Cell Biol 3(3): 195-205.
Fu, X. D. and Ares, M., Jr. (2014). Context-dependent control of alternative splicing by RNA-binding proteins. Nat Rev Genet 15(10): 689-701.
Iglesias, R., Citores, L. and Ferreras, J. M. (2017). Ribosomal RNA N-glycosylase activity assay of ribosome-inactivating proteins. Bio-protocol 7(6): e2180.
Martin, K. C. and Ephrussi, A. (2009). mRNA localization: gene expression in the spatial dimension. Cell 136(4): 719-730.
Mayr, C. and Bartel, D. P. (2009). Widespread shortening of 3’UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 138(4): 673-684.
Naro, C. and Sette, C. (2013). Phosphorylation-mediated regulation of alternative splicing in cancer. Int J Cell Biol 2013: 151839.
Olshavsky, N. A., Comstock, C. E., Schiewer, M. J., Augello, M. A., Hyslop, T., Sette, C., Zhang, J., Parysek, L. M. and Knudsen, K. E. (2010). Identification of ASF/SF2 as a critical, allele-specific effector of the cyclin D1b oncogene. Cancer Res 70(10): 3975-3984.
Pagliarini, V., Naro, C. and Sette, C. (2015). Splicing regulation: A molecular device to enhance cancer cell adaptation. Biomed Res Int 2015: 543067.
Paronetto, M. P., Cappellari, M., Busa, R., Pedrotti, S., Vitali, R., Comstock, C., Hyslop, T., Knudsen, K. E. and Sette, C. (2010). Alternative splicing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68. Cancer Res 70(1): 229-239.
Proudfoot, N. J. (2016). Transcriptional termination in mammals: Stopping the RNA polymerase II juggernaut. Science 352(6291): aad9926.
Silvera, D., Formenti, S. C. and Schneider, R. J. (2010). Translational control in cancer. Nat Rev Cancer 10(4): 254-266.
Singh, G., Pratt, G., Yeo, G. W. and Moore, M. J. (2015). The clothes make the mRNA: Past and present trends in mRNPfashion. Annu Rev Biochem 84: 325-354.
Tian, B. and Manley, J. L. (2016). Alternative polyadenylation of mRNA precursors. Nat Rev Mol Cell Biol.
Valacca, C., Bonomi, S., Buratti, E., Pedrotti, S., Baralle, F. E., Sette, C., Ghigna, C. and Biamonti, G. (2010). Sam68 regulates EMT through alternative splicing-activated nonsense-mediated mRNA decay of the SF2/ASF proto-oncogene. J Cell Biol 191(1):87-99.
Wang, Z. and Burge, C. B. (2008). Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA 14(5): 802-813.
Wang, Z., Tollervey, J., Briese, M., Turner, D. and Ule, J. (2009). CLIP: construction of cDNA libraries for high-throughput sequencing from RNAs cross-linked to proteins in vivo. Methods 48(3): 287-293.