Chapter Eight - PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a Step-By-Step Protocol to the Transcriptome-Wide Identification of Binding Sites of RNA-Binding Proteins
Section snippets
Theory
Posttranscriptional regulation (PTR) of messenger RNAs (mRNAs) plays important roles in diverse cellular processes (Ambros, 2004; Halbeisen et al., 2008). The fates of mRNAs are determined predominantly by their interactions with RNA-binding proteins (RBPs) and noncoding, guide-RNA-containing ribonucleoprotein complexes (RNPs). Taken together, they form mRNA-containing ribonucleoprotein complexes (mRNPs). The RBPs influence the structure and interactions of the RNAs and play critical roles in
Equipment
Major equipment Radioisotope laboratory 365-nm UV-transilluminator Agarose gel electrophoresis equipment Protein electrophoresis equipment Equipment to cast and run 15 × 17 cm × 0.8 mm (or similar) polyacrylamide gels Balances (e.g., 0.1 mg–64 g and 0.1 g–4.2 kg) Heating block (90 °C) CO2 incubator for mammalian cell culture D-Tube Dialyzer Midi rack (EMD Biosciences, 71511-3) High-speed floor centrifuge (capable of at least 13 000 × g) Magnetic rack for 1.5-ml microcentrifuge tubes and 15-ml conical tubes
Materials
Reagents & Chemicals Appropriate cell culture medium and selection antibiotics 2-Mercaptoethanol (14.3 M; Sigma, M6250) Acetonitrile Agarose, electrophoresis grade (SeaKem LE Agarose, Lonza, 50004) Agarose, low melting (NuSieve GTG Agarose, Lonza, 50080) Ammonium persulfate (APS) Adenosine triphosphate (ATP) Bacterial Alkaline Phosphatase (Worthington Biochemical, LS006344) Bromophenol blue Bovine serum albumin, acetylated (BSA, acetylated; Ambion, AM2614) Calcium chloride (CaCl2·2H2O) Calf intestinal
Cells
Expand cells in an appropriate growth medium containing selection antibiotics as appropriate to maintain your stable cell line. We usually prepare lysates from 3 to 5 ml of wet cell pellet from crosslinked cells per experiment. This corresponds to 20–50 15-cm cell culture plates (for HEK293). However, if material is limiting, we have performed successful PAR-CLIPs experiments from < 0.5 ml of wet cell pellet (200 × 106 HEK293 cells (10 15-cm plates) will yield ~ 1 ml of wet cell pellet).
Grow cells
Overview
In this first step, the RNA-binding protein of interest is cross-linked to its bound mRNAs targets that incorporated the photoactivatable ribonucleoside into nascent transcripts during the labeling step (see also UV crosslinking of interacting RNA and protein in cultured cells). The cells are then collected and the resulting cell pellet will be used as the input for the following PAR-CLIP procedure.
Duration
About 2–3 h
Overview
The cell pellet obtained on day 1 will be lysed (see also Lysis of mammalian and Sf9 cells) in preparation for immunoprecipitation. Partial RNase T1 digestion of mRNAs facilitates the recovery of cross-linked mRNPs.
Duration
2 h
- 2.1
Thaw the cross-linked cell pellet on ice. Prepare the magnetic beads (see Step 3) while the pellet thaws. Resuspend the cell pellet in 3 cell pellet volumes of NP40 lysis buffer and incubate on ice for 10 min.
- 2.2
Clear the cell lysate by centrifugation at 13 000 × g for 15 min at 4 °C.
- 2.3
Overview
The antibody is conjugated to protein G magnetic beads to be used in the subsequent immunoprecipitation. Protein G is the optimal Ig-binding protein for anti-FLAG antibodies based on species and isotype. The choice of protein A versus protein G should be considered depending on the antibody used.
Duration
1.5 h
- 3.1
Transfer 10 μl of Protein G magnetic particles per ml cell lysate (typically ~ 100–150 μl of beads) to a 1.5-ml microtube. Put the magnetic rack on ice. Wash the beads twice with 1 ml of
Overview
The mRNA-RBP complex of choice is isolated from the lysate by immunoprecipitation. A second RNase T1 digestion ensures that only the RNA segment that was bound, crosslinked, and protected by the RBP is recovered and sequenced. This enables the precise definition of the binding sites.
Duration
2 h
- 4.1
Add 20 μl of freshly prepared antibody-conjugated magnetic beads per ml of the partially RNase T1-treated cell lysate from Step 2 and incubate in a 15-ml centrifuge tube on a rotating wheel for 1 h at 4 °C.
- 4.2
Collect
Overview
The RNAs crosslinked by the RBP of interest are radiolabeled using T4 PNK and [γ-32P]-ATP in order to visualize them by autoradiography after fractionation by SDS-PAGE (see also RNA Radiolabeling).
Duration
2 h
- 5.1
Add calf intestinal alkaline phosphatase (CIP from NEB) to a final concentration of 0.5 U μl− 1, and incubate the suspension for 10 min at 37 °C and mixing at 800 rpm.
- 5.2
Wash beads twice with 1 ml of phosphatase wash buffer.
- 5.3
Wash beads twice with polynucleotide kinase (PNK) buffer without DTT.
- 5.4
Resuspend
Overview
Size fractionation of the radiolabeled and cross-linked RNA protein complexes is achieved by SDS-PAGE (see One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)). The band corresponding to the expected mass of the protein will be excised and the cross-linked RNA protein complexes electroeluted. This step ensures that only the band corresponding to the correct RBP is isolated and additionally prevents any unbound but labeled RNA from further processing (see Fig. 8.7(a)).
Duration
4.5 h
- 6.1
Load 2
Overview
In this step, the recovered RBP is proteolyzed and the cross-linked RNA is recovered so that it can serve as the input material for subsequent ligation to adapters and Solexa sequencing.
Duration
3.5 h
- 7.1
Add 1 volume of 2× Proteinase K Buffer, followed by the addition of Proteinase K (Roche) to a final concentration of 1.2 mg ml− 1. Incubate at 55 °C for 30 min. If the volume per tube exceeds 800 μl at this stage, split the sample into two tubes.
- 7.2
Add 1 volume of acidic phenol/chloroform/isoamyl alcohol, vortex and
Overview
The recovered 5′-32P-phosphorylated RNA is now carried through a standard cDNA library preparation protocol, originally described for the cloning of small regulatory RNA (Hafner et al., 2008). As a first step, a preadenylated 3′-adapter is ligated using T4 Rnl2(1-249)K227Q ligase (see Fig. 8.7(b)).
Duration
Day 3: 30 min (+ overnight incubation)
Day 4: 4–5 h (highly dependent on required exposure time)
Day 5: 2 h
- 8.1
Prepare the following reaction mixture for ligating the 3′-adapter, multiplying the volumes by the
Overview
In this step, the 5′-adapter is joined to the 3′-ligated RNA to enable the cDNA synthesis in the next step (see Fig. 8.7(c)).
Duration
Day 5: about 5 h (highly dependent on required exposure time)
Day 6: 2 h
- 9.1
Prepare the following reaction mixture for the ligation of the 5′-adapter, multiplying the volumes by the number of ligation reactions to be performed plus two (for the positive control plus one extra to account for pipetting loss):
1 μl of 100 μM 5′-adapter
2 μl of 10× RNA ligase buffer with ATP
6 μl 50% DMSO
Overview
The RNA ligated to both sequencing adapters is reverse-transcribed and will be used for PCR in the subsequent step.
Duration
1.5 h
- 10.1
Prepare the following reaction mix (multiplied by the number of samples plus one for pipetting loss):
1.5-μl 0.1-M DTT
3-μl 5× First-strand buffer (Superscript)
4.2-μl 10× dNTPs
- 10.2
Denature the RNA by incubating the tube at 90 °C for 30 s and transfer the tube to a 50 °C thermomixer.
- 10.3
Add 8.7 μl of the reaction mix to each sample and incubate at 50 °C for 3 min. Add 0.75 μl of Superscript
Overview
This step concludes the PAR-CLIP protocol. To minimize the distortion of the cDNA library composition by excessive PCR and to recognize possible failure during reverse transcription leading to false positive PCR results, we monitor the accumulation of the PCR product during a pilot PCR. To determine the minimal cycle number, a small-scale trial PCR is performed before the final large-scale PCR. The PCR product is gel fractionated (see Agarose Gel Electrophoresis); the appropriately sized
Overview
To optimize crosslinking of protein to RNA, it is useful to determine the fraction of substitution of uridine by 4SU. This is especially necessary when changing cell growth conditions or cell type. Total RNA is isolated and enzymatically degraded to monomeric ribonucleosides, which are separated and quantified by HPLC analysis (Andrus and Kuimelis, 2001).
Duration
Cell labeling and harvesting: 16 h + 20 min
RNA isolation: about 2 h
Dephosphorylation and hydrolysis: 30 min + overnight incubation
Chromatography:
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