Transcriptome‐wide Identification of RNA‐binding Protein Binding Sites Using Photoactivatable‐Ribonucleoside‐Enhanced Crosslinking Immunoprecipitation (PAR‐CLIP)

Henrike Maatz1, Marcin Kolinski2, Norbert Hubner3, Markus Landthaler2

1 Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, 2 Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, 3 Charité Universitätsmedizin, Berlin
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 27.6
DOI:  10.1002/cpmb.35
Online Posting Date:  April, 2017
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RNA‐binding proteins (RBPs) mediate important co‐ and post‐transcriptional gene regulation by binding pre‐mRNA in a sequence‐ and/or structure‐specific manner. For a comprehensive understanding of RBP function, transcriptome‐wide mapping of the RNA‐binding sites is essential, and CLIP‐seq methods have been developed to elucidate protein/RNA interactions at high resolution. CLIP‐seq combines protein/RNA UV‐crosslinking with immunoprecipitation (CLIP) followed by high‐throughput sequencing of crosslinked RNA fragments. To overcome the limitations of low RNA‐protein crosslinking efficiency in standard CLIP‐seq, photoactivatable‐ribonucleoside‐enhanced CLIP (PAR‐CLIP) has been developed. Here, living cells or whole organisms are fed photo‐activatable nucleoside analogs that are incorporated into nascent RNA transcripts before UV treatment. This allows greater crosslinking efficiency at comparable radiation doses for enhanced RNA recovery and separation of crosslinked target RNA fragments from background RNA degradation products. Moreover, it facilitates the generation of specific UV‐induced mutations that mark the crosslinking nucleotide and allow transcriptome‐wide identification of RBP binding sites at single‐nucleotide resolution. © by 2017 John Wiley & Sons, Inc.

Keywords: CLIP; crosslinking immunoprecipitation; high‐throughput sequencing; PAR‐CLIP; RNA‐protein interaction

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Table of Contents

  • Introduction
  • Basic Protocol 1: Identification of RBP‐RNA Binding Sites at Single‐Nucleotide Resolution Using PAR‐CLIP
  • Support Protocol 1: Determination of Radionucleoside Incorporation into Nascent RNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Identification of RBP‐RNA Binding Sites at Single‐Nucleotide Resolution Using PAR‐CLIP

  • 19‐ and 35‐mer RNA size markers
  • T4 polynucleotide kinase (T4 PNK, New England Biolabs, cat. no. M0201)
  • PNK reaction buffer (New England Biolabs)
  • Cells of interest (e.g., HEK293 cells)
  • Appropriate cell culture medium and selection antibiotics
  • 1 M photoactivatable nucleoside: 4‐thiouridine (4SU, Sigma, T4509) or 6‐thioguanosine (6SG, Sigma, 858412) in dimethyl sulfoxide (DMSO)
  • Phosphate‐buffered saline (PBS)
  • Dynabeads Protein G (Thermo Fisher Scientific, 10003D)
  • NP40 lysis buffer (see recipe)
  • Antibody for immunoprecipitation, e.g., mouse monoclonal anti‐FLAG M2 (Sigma, F1804) for FLAG‐tagged RBPs
  • 100 U/μl RNase I (Ambion)
  • Turbo DNase (optional)
  • High‐salt wash buffer (see recipe)
  • 1 M dithiothreitol (DTT)
  • Triton X‐100
  • 5′‐[γ32P]ATP, 10 mCi/ml, 6000 Ci (222 TBq)/mmol (Perkin Elmer, NEG002Z500UC)
  • PNK buffer without DTT (see recipe)
  • 10 mM ATP
  • 1× SDS‐PAGE loading buffer (see recipe)
  • NuPAGE Novex 4‐12% Bis‐Tris Midi 1.0 gel (Invitrogen)
  • Protein ladder (e.g., 10‐250 kDa, Bio‐Rad, 161‐0374)
  • NuPAGE MOPS SDS running buffer (Invitrogen)
  • Nitrocellulose membrane
  • NuPAGE transfer buffer (Invitrogen)
  • Methanol
  • 20 mg/ml proteinase K stock solution (see recipe)
  • 2× proteinase K buffer (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol, pH 7.4
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M NaCl
  • Glycoblue (Ambion) or glycogen
  • 100% and 75% (v/v) ethanol
  • 10× RNA ligase buffer (New England Biolabs)
  • 24% (w/v) PEG8000 (New England Biolabs)
  • RNA and DNA oligonucleotides (Table 27.6.1):
    • 50 μM pre‐adenylated 3′ adapter 4 N‐RA3, 3InvdT
    • 50 μM 5′ adapter OR5‐4N
    • 5 μM reverse‐transcription primer (RTP)
    • 100 μM 5′ PCR primer RP1
    • 100 μM PCR index primers RPI1‐8
    • T4 RNA Ligase 2, truncated, K227Q (New England Biolabs)
    • 2× formamide loading buffer (see recipe)
    • 1× TBE buffer (see recipe)
    • 10× T4 RNA ligase buffer (New England Biolabs)
    • T4 RNA ligase 1 (New England Biolabs, M0204L)
    • 200 U/μl SuperScript III reverse transcriptase with 5× first‐strand buffer (Invitrogen, 18080‐044)
    • 2 mM dNTP solution (see recipe)
    • Phusion DNA polymerase and 5× Phusion buffer (New England Biolabs)
    • 6× DNA loading dye (see recipe)
    • Low‐melting‐temperature agarose (NuSieve GTG Agarose, Lonza, 50080)
    • Ethidium bromide
    • GeneRuler Low Range DNA Ladder (Thermo Fisher Scientific, SM1191)
    • Agarose, electrophoresis grade (SeaKem LE Agarose, Lonza, 50004)
    • Zymoclean Gel DNA Recovery Kit (Zymo Research)
    • Microspin columns (GE Healthcare)
    • 15‐cm culture dishes
    • UV crosslinker with 365‐nm light (e.g., Stratalinker 2400, Stratagene)
    • Cell scraper (Corning)
    • 15‐ and 50‐ml conical tubes (e.g., Falcon)
    • Benchtop centrifuge
    • 1.5‐ml microcentrifuge tubes, RNase free
    • Magnetic rack for 1.5‐ml microcentrifuge tubes and 15‐ml conical tubes (e.g., Dynal MPC‐S magnetic particle concentrator)
    • Rotating wheel
    • 37°C water bath
    • High speed floor centrifuge (capable of ≥20,000 × g) and tubes (e.g., 13 ml, Sarstedt, 55.518)
    • 5‐μm Supor membrane syringe filters (Pall Acrodisc)
    • 10‐ml syringes
    • Shaker
    • 50° and 95°C heat blocks or water baths
    • Plastic wrap
    • Phosphorimager and imaging plates (or regular X‐ray film and developer)
    • X‐ray exposure cassette
    • Scalpels or razor blades
    • Thermoshaker
    • 16°C cooling block or water bath
    • Thermocycler
    • Strips of 0.2‐ml PCR tubes (Thermo Fisher Scientific, AB0264)
    • UV transilluminator
    • Qubit fluorometer
    • DNA 1000 bioanalyzer
    • Additional reagents and equipment for SDS‐PAGE and immunoblotting of RNA‐protein complexes, denaturing urea PAGE of adapter‐ligated RNA, and agarose gel electrophoresis

Support Protocol 1: Determination of Radionucleoside Incorporation into Nascent RNA

  • 4SU‐ or 6SG‐labeled cells (see Basic Protocol 1, step 3)
  • Trypsin
  • Phosphate‐buffered saline (PBS)
  • Trizol
  • Chloroform
  • Isopropanol
  • 5 M NaCl
  • 75% (v/v) ethanol
  • Biotin‐HPDP
  • Dimethylformamide (DMF)
  • 1 M Tris·Cl, pH 7.4 (Promega, PR‐H5121)
  • 0.5 M EDTA
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • Isopropanol
  • Hyperbond N+ membrane (Amersham)
  • Blocking solution: PBS, pH 7.5, containing 10% SDS and 1 mM EDTA
  • Streptavidin–horseradish peroxidase (HRP; Pierce)
  • ECL detection reagent
  • 25‐G needle
  • 1.5‐ and 2‐ml microcentrifuge tubes, RNase free
  • Phase Lock Gel tubes
  • UV spectrophotometer
  • UV crosslinker
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Literature Cited

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