Approaches for the Identification and Characterization of RNA–Protein Interactions - Biophysical Approaches to Translational Control of Gene Expression - page 201

10.3 Immunoprecipitation of RNA-Protein Complexes

and Global Analyses of Target Interactions
(e.g., RIP-CHIP)

10.3.1 Introduction

Oftentimes the need arises to take a more global look at mRNAs that are associated
with a particular protein factor. This can be approached by a variety of techniques
including RIP-CHIP and CLIP analyses (e.g., HITS-CLIP, PAR-CLIP, etc.). All of
these approaches rely upon the specific immunoprecipitation of RNA-protein com-
plexes. RIP-CHIP involves the precipitation of intact mRNAs bound to proteins fol-
lowed by analysis of bound RNAs by either microarray or deep sequencing
(Tenenbaum et al. 2002 ; Jain et al. 2011 ; Galgano and Gerber 2011 ; Lee et al. 2010 ) .
HITS-CLIP (Darnell 2010 ; Jensen and Darnell 2008 ) and PAR-CLIP (Hafner et al.
2010 ) rely on the immunoprecipitation of RNAse-treated UV cross-linked RNA-
protein complexes and analysis of the small RNA fragments by deep sequencing. An
attractive feature of PAR-CLIP is that RNAs are modified using 4-thio uridine in
cells prior to UV cross-linking. In addition to increasing the efficiency of cross-
linking, cross-linked U residues get converted to C residues during the RT step,
allowing one to pinpoint the exact site of RNA-protein interaction. A universally
applicable RIP-CHIP protocol is presented below. Please note that a number of recent
excellent reviews have focused on the “CLIP” strategies (e.g., Kishore et al. 2011 ) .

10.3.2 Specialized Materials

1. Tissues/cells of interest.
2. Antibody speci fi c for protein of interest and a nonspeci fi c antibody.
3. Antibody binding matrix (Protein G or protein A Sepharose beads)
4. Glycogen (20 mg/mL) (Fermentas).
5. RNAse OUT (Fermantas).
6. Dnase 1—RNase-free (Fermantas).
7. Micro-spin columns (Pierce).
8. Random hexamers (IDT).
9. Reverse transcriptase (Im Prom-II™).
10. Protease inhibitor cocktail (Roche).

Buffers :

1 . RNP lysis buffer ( RLB ): 100 mM KCl, 5 mM MgCl 2 , 10 mM HEPES, pH 7.0,
0.5% NP-40; To be added at the time of use ; 1 mM DTT, 100 units/mL RNase
OUT, 0.2% vanadyl ribonucleoside complexes, 0.2 mM PMSF.
2 . NT2 buffer : 50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 0.05% Nonidet
P-40.