iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

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The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

Date Published: 4/30/2011, Issue 50; doi: 10.3791/2638

Keywords: Cellular Biology, Issue 50, RNA biochemistry, transcriptome, systems biology, RNA-binding protein

Konig, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., et al. iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. J. Vis. Exp. (50), e2638, doi:10.3791/2638 (2011).

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation¹. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs².

Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)^3-5. These approaches were prone to identifying indirect or non-physiological interactions⁶. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced^7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)^9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking^11,12.

Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide¹³. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)¹⁴. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation¹⁴.

1. UV cross-linking of tissue culture cells

1. Remove the media and add 6 ml ice-cold PBS to cells grown in a 10 cm plate (enough for three experiments).
2. Remove lid and place on ice. Irradiate once with 150 mJ/cm² at 254 nm.
3. Harvest the cells by scraping with a cell lifter.
4. Transfer 2 ml cell suspension to each of three microtubes. Spin at top speed for 10 sec at 4°C to pellet cells, then remove supernatant.
5. Snap-freeze the cell pellets on dry ice and store at -80°C until use.

2. Bead preparation

1. Add 100 μl of protein A Dynabeads (Dynal, 100.02) per experiment to a fresh microtube (Use protein G Dynabeads for a mouse or goat antibodies).
2. Wash beads 2x with lysis buffer (50 mM Tris-HCl, pH 7.4; 100 mM NaCl; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate; 1/100 protease inhibitor cocktail III, Calbiochem).
3. Resuspend beads in 100 μl lysis buffer with 2-10 μg antibody.
4. Rotate tubes at room temperature for 30-60 min.
5. Wash 3x with 900 μl lysis buffer and leave in the last wash until ready to proceed to step 4.1.

3. Cell lysis and partial RNA digestion

1. Resuspend the cell pellet in 1 ml lysis buffer and transfer to 1.5 ml microtubes.
2. Prepare a 1/500 dilution of RNase I (Ambion, AM2295). Add 10 μl RNase I dilution as well as 2 μl Turbo DNase to the cell lysate (1/500 RNase I dilutions [low RNase] are used for library preparation; 1/50 dilutions [high RNase] are necessary to control for antibody specificity).
3. Incubate the samples for exactly 3 min at 37°C, shaking at 1,100 rpm. Immediately transfer to ice.
4. Spin at 4°C and 22,000 g for 20 min to clear the lysate. Carefully collect the supernatant (leave about 50 μl lysate with the pellet).

4. Immunoprecipitation

1. Remove the wash buffer from the beads (from step 2.5), then add the cell lysate (from step 3.4).
2. Rotate the samples for 2 h at 4°C.
3. Discard the supernatant and wash the beads 2x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate).
4. Wash 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl₂; 0.2% Tween-20).

5. Dephosphorylation of RNA 3'ends

1. Discard the supernatant and resuspend the beads in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mMTris-HCl, pH 6.5; 50mMMgCl₂ 25mMdithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]).
2. Incubate for 20 min at 37°C.
3. Add 500 μl wash buffer and wash 1x with high-salt buffer.
4. Wash 2x with wash buffer.

6. Linker ligation to RNA 3' ends

1. Carefully remove the supernatant and resuspend the beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mMTris-HCl; 40m MM gCl₂; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]).
2. Incubate overnight at 16°C.
3. Add 500 μl wash buffer and then wash 2x with 1 ml high-salt buffer.
4. Wash 2x with 1 ml wash buffer and leave in 1 ml of the second wash.

7. RNA 5' end labelling

1. Remove the supernatant and resuspend the beads in 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl ³²P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water).
2. Incubate for 5 min at 37°C.
3. Remove the hot PNK mix and resuspend the beads in 20 μl 1x Nupage loading buffer (Invitrogen).
4. Incubate on a thermomixer at 70°C for 10 min.
5. Immediately place on a magnet to precipitate the empty beads and load the supernatant on the gel (see step 8).

8. SDS-PAGE and membrane transfer

1. Load the samples on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) according to the manufacturer's instructions. Use 0.5 l of 1x MOPS running buffer (Invitrogen). Also load 5 μl of a pre-stained protein size marker (for example PAGE ruler plus, Fermentas, SM1811).
2. Run the gel for 50 min at 180 V.
3. Remove the gel front and discard as solid waste (contains free radioactive ATP).
4. Transfer the protein-RNA complexes from the gel to a nitrocellulose membrane using the Novex wet transfer apparatus according to the manufacturer's instructions (Invitrogen, transfer 1 h at 30 V).
5. After the transfer, rinse the membrane in PBS buffer, then wrap it in saran wrap and expose it to a Fuji film at -80°C (place a fluorescent sticker next to the membrane to later align the film and the membrane; perform exposures for 30 min, 1h and over night).

9. RNA isolation

1. Isolate the protein-RNA complexes from the low-RNase experiment using the autoradiograph from step 8.5 as a mask. Cut this piece of membrane into several small slices and place them into a 1.5 ml microtube.
2. Add 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001) to the membrane pieces. Incubate shaking at 1,100 rpm for 20 min at 37°C.
3. Add 200 μl of PKurea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) and incubate for 20 min at 37°C.
4. Collect the solution and add it together with 400 μl of RNA phenol/chloroform (Ambion, 9722) to a 2 ml Phase Lock Gel Heavy tube (713-2536, VWR).
5. Incubate for 5 min at 30°C, shaking at 1,100 rpm. Separate the phases by spinning for 5 min at 13,000 rpm at room temperature.
6. Transfer the aqueous layer into a new tube (be careful not to touch the gel with the pipette). Add 0.5 μl glycoblue (Ambion, 9510) and 40 μl 3 M sodium acetate pH 5.5 and mix. Then add 1 ml 100% ethanol, mix again and precipitate over night at -20°C.

10. Reverse transcription

1. Spin for 20 min at 15,000 rpm and 4°C. Remove the supernatant and wash the pellet with 0.5 ml 80% ethanol.
2. Resuspend the pellet in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5pmol/μl]; 0.5 μl dNTP mix [10mM]). For each experiment or replicate, use a different Rclip primer containing individual barcode sequences (see 14).
3. Incubate for 5 min at 70°C before cooling to 25°C.
4. Add 2.75 μl RT mix (2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen]).
5. Incubate 5 min at 25°C, 20 min at 42°C, 40 min at 50°C and 5 min at 80°C before cooling to 4°C.
6. Add 90 μl TE buffer, 0.5 μl glycoblue and 10 μl sodium acetate pH 5.5 and mix. Then add 250 μl 100% ethanol, mix again and precipitate over night at -20°C.

11. Gel purification of cDNA

1. Spin down and wash the samples (see 10.1), then resuspend the pellets in 6 μl of water.
2. Add 6 μl 2x TBE-urea loading buffer (Invitrogen). Heat samples to 80°C for 3 min directly before loading.
3. Load the samples on a precast 6% TBE-urea gel (Invitrogen) and run for 40 min at 180 V as described by the manufacturer. Also load a low molecular weight marker for subsequent cutting (see below).
4. Cut three bands at 120-200 nt (high), 85-120 nt (medium) and 70-85