Dilshad H. Khan and James R. Davie (author for correspondence)

Dr. James R. Davie
Children's Hospital Research Institute of Manitoba
600A-715 McDermot Avenue
Winnipeg, Manitoba, R3E 3P4
Tel:  204-975-7732
Fax: 204.977.5691
jim.davie@umanitoba.ca

Abstract

Ribonucleoprotein immunoprecipitation (RIP) is an antibody based method to detect RNA-protein interactions in situ. In the assay, UV cross-linking is commonly used to preserve RNA-protein interactions for subsequent target identification. UV light is a zero length cross-linker, and thus identifies proteins directly bound to RNAs. Here, we describe a dual cross-linking RIP method that involves sequential protein-protein crosslinking step with a protein-protein cross-linker, followed by protein-RNA fixation by UV irradiation. In this way, proteins that indirectly bound to RNA can be analyzed.

Introduction

The research field of ‘RNA-protein interaction’ has witnessed rapid growth in recent years. The ribonucleoprotein (RNP) complexes are pivotal in regulation of gene expression. RNPs are formed by the interactions of RNAs with proteins through secondary or tertiary structures (Jones et al. 2001). Ribonucleoprotein immunoprecipitation (RIP) is an antibody-based technique to determine ‘RNA-protein interactions’. This technique aims to identify a transcript (RNAs) bound to ‘a protein of interest’ (RNPs) using a specific antibody. RNAs associated with the protein of interest can be isolated and analyzed by several biochemical techniques such as quantitative PCR, hybridization methods, microarray or high-throughput sequencing (Hosoda et al. 2006; Jonson et al. 2007; Sephton et al. 2011; Tenenbaum et al. 2002).

UV-irradiation is widely used in ribonucleoprotein immunoprecipitation protocol (Luo & Reed 2003; Reed & Chiara 1999; Ule et al. 2005). UV light is a zero-length cross-linker and helps to identify proteins that directly interact with RNA. Proteins, which are indirectly bound to RNAs, are not readily cross-linked by UV and thus cannot be detected. However, use of a proteinprotein cross-linker e.g. dithiobis (succinimidylpropionate) (DSP) to increase the spacer arm and capturing capability in combination with UV cross-linking, can be useful for characterization of proteins that indirectly bind with RNA. Here, we describe a dual cross-linking (DSP crosslinking prior to UV irradiation) RIP immunoprecipitation protocol in detail. An overview of the protocol is outlined in Figure 1. This protocol implies a technological advancement to the existing protocols and provides the advantage of capturing and identifying proteins bound directly or indirectly to RNAs. This method has been successfully employed for immunoprecipitation of complexes in which tested proteins such as lysine acetyltransferase (KAT) and histone deacetylases (HDAC) are not in direct contact with RNA of interest (Figure 2) (Khan et al. 2014). We analyzed the results obtained using UV cross-linked or the DSP-UV dual cross-linking RIP. As shown in the Figure 2, dual cross-linking RIP protocol significantly improves the immunoprecipitation efficiency of HDACs (HDAC1 and 2) and KAT (KAT2B) than UV cross-linking. In addition, our results demonstrated the indirect association of these proteins to myeloid cell leukemia sequence 1 (MCL1) transcripts in human colon cancer cell line, HCT116. Importantly, the sequential use of two cross-linking agents does not affect the recovery of protein that directly (UV-cross linking alone) binds to MCL1 transcript, such as the splicing protein, serine/arginine rich splicing factor 1 (SRSF1). Thus, this protocol represents an effective method for in situ detection of RNA bound proteins that are refractory to analysis by UV cross-linking. Moreover, use of dual cross-linking protocol may help in detection of additional RNA binding proteins. This holds promise for studies to advance our understanding of the functions of ‘larger RNA-protein’ complexes.

Materials

1. 1XPBS: Dissolve 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 and 0.24 g of KH2PO4 to approximately 800 mL of double-distilled water.
2. Adjust pH to 7.4 and then make the volume up to 1L with double-distilled water. Sterilize by autoclaving.
3. Dimethyl sulfoxide (DMSO) (Sigma) (filter-sterilized).
4. 1.0 mM DSP solution: Dissolve 4.04 mg of DSP (Pierce) in 0.500 mL of DMSO to prepare a 20 mM stock solution (prepare freshly). From the stock, make 1.0 mM solution in 1XPBS, just before use. Lysis buffer: All the stock solutions are prepared from analytical grade reagents dissolved in ultra-pure water. Add 20 mL of 1M Tris-HCl, pH 7.5 (20 mM), 25 mL of 4M NaCl (100 mM), 2.5 mL of 4M MgCl2 (10 mM) to approximately 800 mL of ultra-pure water. Then add 5 mL of 100 per cent NP-40 (0.5 per cent NP-40) and 5 mL of 100 per cent Triton X 100 (0.5 per cent Triton X 100) while stirring. Finally add 10 mL of 10 per cent SDS (0.1 per cent SDS). Adjust the volume up to 1L with ultra-pure water. Store at 4ºC. Supplement the lysis buffer with protease and phosphatase inhibitor cocktail (Roche) and RNasin (80 U/mL) (Promega) immediately before use.
5. RNase A/T1 (Ambion)
6. Protein A/G UltraLink resin (Pierce)
7. Yeast tRNA (Sigma)
8. TRIzol (Invitrogen)
9. Proteinase K (Invitrogen)
10. Ultra-pure water (Gibco)
11. RQ DNase I (Promega)
12. RNeasy Plus Mini Kit (Qiagen)
13. SuperScript III First-Strand Synthesis system (Invitrogen)

Methods

3.1 Dual cross-linking of tissue culture cells

1. Grow cells in recommended media in 150 mm plates ~70-80 per cent confluence and treat cells as per experimental requirements (see Note 1).
2. Rinse cells twice with 1X PBS to remove the traces of media.
3. Cross-link cells with DSP by adding 1.0 mM DSP solution into the plate and incubate for 30 min at room temperature with gentle rotation (see Note 2).
4. Take out the DSP solution, quickly rinse once with 1XPBS and cover the cells with minimal volume of 1XPBS (~2-3 mL) (see Note 3).
5. Irradiate the cells with UV light (400mJ/cm2), lid off of the plates and placing on ice in a tray, in an UV stratalinker 2400 (Stratagene).
6. Harvest cells in 1XPBS with cell scraper in a 15 mL tube. Centrifuge at 250 g for 5 min.
7. Wash the cell pellets twice with 1XPBS (see Note 4).

3.2 Preparation of beads

1. Wash the protein A/G UltraLink resin three times (5 min/wash) with ice-cold lysis buffer (500-1000 μL).
2. Prepare 50 per cent slurry of protein A/G UltraLink resin in ice-cold lysis buffer supplemented with protease and phosphatase inhibitors, RNasin and yeast tRNA (25 μg/mL).
3. Use 100-200 μL of 50 per cent slurry of protein A/G UltraLink resin for each immunoprecipitation reaction (see Note 5).

3.3 Cellular extract (cross-linked lysate) preparation

1. Lyse cells in appropriate volume (1.0-2.0 mL) of ice-cold lysis buffer (freshly add protease and phosphatase inhibitors) depending on pellet size. Leave on ice for 15-20 min.
2. Homogenize the cellular extract with sonication (Fisher Scientific Model 100 Sonic Dismembrator) at setting 2 (3X10 sec, each with 1 min intervals on ice).
3. Centrifuge at 17,000 g for 10 min at 4ºC to pellet the insoluble material. Treat the cellular extract with DNase I for 30 min at 37ºC as per manufacturer’s instructions.
4. Partially digest the cellular extract with diluted cocktails of RNase A/T1 at 37ºC at the final concentrations ranging from 1:1000 to 1:1000,000 for 10-40 min to obtain the RNA fragments of 200-500 bp long (with an average size of 300 bp) (see Note From each digested fraction, isolate RNA using RNeasy Plus Mini Kit according to the manufacturer’s instructions. Quantify RNA concentration by a Nanodrop and analyze RNA size by using preferably an Agilent 2100 Bioanalyzer instrument or by gel electrophoresis.
5. Add RNasin (80 U/mL) in the cellular extract (see Note 7).

3.4 Immunoprecipitation

1. Pre-clear the extract with 50 per cent slurry of protein A/G UltraLink resin (40μL/mL) and yeast tRNA (100 μg/mL) for 2 h with rotation at 4ºC. Quantify the concentration of the cellular extract.
2. Add the appropriate amount of antibody to the protein of interest (5-15 μg) to the cellular extract (1.0-2.0 mg), and, incubate overnight at 4ºC on a rotator. In parallel, perform a ‘negative control’ immunoprecipitation reaction to assess non-specific background level, with an equal amount of isotype-matched antibody. Save an aliquot of cellular extract to prepare ‘Input’ RNA fragments (see Note 8).
3. In the following day, add 100-200 μL of 50 per cent slurry of protein A/G UltraLink resin (supplemented with yeast tRNA, 100 μg/mL) and incubate for 2-3 h at 4ºC.
4. Collect the beads by quick spin and save the supernatant (immunodepleted fraction) to check the efficiency of immunoprecipitation by SDS-PAGE and immunoblotting analyses.
5. Wash the beads six times with the cold lysis buffer supplemented with RNasin (40 U/mL) (900 g, 5 min/wash) at 4ºC. To minimize background, take out the supernatant as much as possible during the washes. Save an aliquot of the beads for further analyses by SDS-PAGE and immunoblotting (see Note 9).

3.5 Purification and RNA extraction

1. After the final wash, resuspend the beads in lysis buffer (200-400 μL) supplemented with RNase inhibitor and treat with proteinase K (2.0 mg/mL) at 55ºC for 1 h, flicking the tube occasionally.
2. Extract the immunoprecipitated RNA and Input RNA using TRIzol as per manufacturer’s instructions and precipitate with ethanol at -80ºC for 3-4 h (to overnight).
3. Air-dry the RNA pellets and resuspend in an appropriate volume of ultra-pure water for subsequent analyses (see Note 10).
4. Digest the RNAs with DNase I at 37ºC for 1 h.
5. Purify the RNAs with RNeasy Plus Mini Kit according to manufacturer’s instructions and assess the quality and quantity by using a Nanodrop.

3.6 Reverse transcription of RNA and analysis

1. Perform the reverse transcription of immunoprecipitated RNA and Input RNA using oligo dT and random hexamer primers with SuperScript III reverse transcriptase following the manufacturer's specifications.
2. If target is known, use quantitative PCR to analyze; if target is unknown, use other downstream methods e.g. high throughput sequencing, depending on experimental requirements.

Notes

1. Three to four plates (150 mm plates) of tissue culture generate sufficient materials for immunoprecipitation reaction. However, the number of plates required for experiment may vary depending on the cell types and needs to be optimized.
2. Other cell permeable protein-protein cross-linkers with variable spacer arms can be used in place of DSP depending on experimental requirements. When using DSP as the cross-linker, do not add reducing agents to the buffers as DSP has a cleavable disulfide bond in its spacer arm.
3. A thin layer of liquid helps to prevent cells from drying out.
4. Cross-linked cells can be stored at -80ºC until further use.
5. The amount of beads required for each immunoprecipitation reaction needs to be determined for each antibody of interest.
6. The dilution of RNase A/T1 and the digestion time need to be determined empirically to obtain the desired RNA fragment size. A good starting point is to do a titration of the dilution of 1:1000, 1:2500, 1:5000, and 1:10000 per mL of cellular extracts for 10, 15, 20 and 30 min.
7. As the RNasin inhibitor does not inhibit RNase T1, it is advisable to use Ambion SUPERase RNase inhibitor which inhibits both RNase A and RNase T1.
8. For an efficient and successful immunoprecipitation reaction, the amount of antibody, beads, and the incubation time required need to be optimized for each antibody of interest.
9. Stringent washing of the beads is critical to reduce the background and may need to be optimized. Care should be taken to perform the washing steps as quickly as possible with caution of keeping the tubes on ice, in order to reduce degradation. Always use DNase RNase free tubes to set up the immunoprecipitation reactions.
10. To avoid over drying of the pellets, do not dry longer than 5 min. Over dried pellets become difficult to solubilize.

Acknowledgements

This research was supported by a Canadian Breast Cancer Foundation grant (to J.R.D.); Canada Research Chair (to J.R.D.); MHRC/CancerCare Manitoba studentship (to D.H.K.); Manitoba Next Generation Sequencing Platform was supported by generous funds from the Canadian Foundation for Innovation, Province of Manitoba, University of Manitoba Faculty of Medicine, Manitoba Health Research Council, CancerCare Manitoba Foundation, Manitoba Institute of Child Health, and Manitoba Institute of Cell Biology.

References

Hosoda,N., Lejeune,F., and Maquat,L.E. 2006. Evidence that poly(A) binding protein C1 binds nuclear pre-mRNA poly(A) tails. Mol. Cell Biol. 26(8): 3085-3097.

Jones,S., Daley,D.T., Luscombe,N.M., Berman,H.M., and Thornton,J.M. 2001. Protein-RNA interactions: a structural analysis. Nucleic Acids Res. 29(4): 943-954.

Jonson,L., Vikesaa,J., Krogh,A., Nielsen,L.K., Hansen,T., Borup,R., et al. 2007. Molecular composition of IMP1 ribonucleoprotein granules. Mol. Cell Proteomics. 6(5): 798-811.

Khan,D.H., Gonzalez,C., Cooper,C., Sun,J.M., Chen,H.Y., Healy,S., et al. 2014. RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing. Nucleic Acids Res. 42(3): 1656-1670.

Luo,M.J. and Reed,R. 2003. Identification of RNA binding proteins by UV cross-linking. Curr. Protoc. Mol. Biol. Chapter 27 Unit.

Reed,R. and Chiara,M.D. 1999. Identification of RNA-protein contacts within functional ribonucleoprotein complexes by RNA site-specific labeling and UV crosslinking. Methods 18(1): 3-12.

Sephton,C.F., Cenik,C., Kucukural,A., Dammer,E.B., Cenik,B., Han,Y., et al. 2011. Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes. J.Biol. Chem. 286(2): 1204-1215.

Tenenbaum,S.A., Lager,P.J., Carson,C.C., and Keene,J.D. 2002. Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays. Methods 26(2): 191-198.

Ule,J., Jensen,K., Mele,A., and Darnell,R.B. 2005. CLIP: a method for identifying protein-RNA interaction sites in living cells. Methods 37(4): 376-386.

Figure legend

Figure 1: Dual cross-linking ribonucleoprotein immunoprecipitation assay. The major steps of the protocol and the experimental timeline are shown.

Figure 2: SRSF1, HDAC1, HDAC2 and KAT2B are bound to MCL1 RNA. RNP complexes were isolated with anti-HDAC1, anti-HDAC2, anti-SRSF1, anti-KAT2B antibodies or isotype specific non-related IgG, from cycling HCT116 cells which were either UV cross-linked or DSP-UV dual cross-linked. Immunoprecipitation of MCL1 RNA associated with these proteins was validated by quantitative PCR using primers specific to MCL1 exons (E1, E2 and E3). The enrichment values are relative to the values obtained with IgG control, and are the mean of three independent experiments. The error bars represent standard deviation. The data were reproduced in part from Figure 6 of (Khan et al. 2014). Statistical analysis was performed using 2-tailed paired Student’s t-test. **P≤0.01, ***P≤0.001.