Immunoprecipitation of mRNA-protein complexes

Abstract

Immunoprecipitation of mRNA-protein complexes is a method that can be used to study RNA binding protein (RBP)-RNA interactions. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. It can also be used as a second, independent method to verify RBP-mRNA interactions discovered through more universal screening techniques. We describe the immunoprecipitation protocol in practical detail and discuss variations of the method as well as issues associated with it. The procedure takes three days to complete.

RNA-binding proteins regulate the expression of the immune activating ligand MICB

“…To investigate whether the identified RBPs can interact with MICB in living cells, we used RKO cells (also used in the RNA-AP) whole-cell lysates to perform RNP immunoprecipitation (RIP) experiments according to the protocol described by Peritz T et al 34 We successfully precipitated FUBP3, HuR, XRN2, CUGBP1, IGF2BP2 and hnRNP-M using specific antibodies. However, we were not able to pull down MATR3 effectively.…”

“…The assay was performed as described 34 . Briefly, RNA was purified using TRI reagent (T9424, Sigma-Aldrich), and the Protein A/G PLUS-Agarose beads (sc-2003, Santa Cruz) were used.…”

Early developmental alterations in GABAergic protein expression in fragile X knockout mice

“…To date only the δ subunit has been shown to interact directly with FMRP; however the studies examined changes in global gene expression (Miyashiro et al, 2003;Gantois et al, 2006;Dictenberg et al, 2008). Future studies that probe for specific FMRPGABAergic mRNA complexes using RNA-protein co-immunoprecipiation techniques (Peritz et al, 2006) could be useful in testing the validity of this theory.…”

Insulin-like Growth Factor-1 Increases Synthesis of Collagen Type I via Induction of the mRNA-binding Protein LARP6 Expression and Binding to the 5′ Stem-loop of COL1a1 and COL1a2 mRNA

“…Immunoprecipation of Ribonucleoprotein Complex-Messenger RNA-protein complexes have been immunoprecipitated as described (25)(26)(27). Briefly, mRNA-protein complexes were extracted from the cells using polysome lysis buffer (100 mM KCl, 5 mM MgCl 2 , 10 mM HEPES pH 7.0, 0.5% IGEPAL CA-630 (Sigma-Aldrich), 1 mM dithiothreitol, 100 units/ml RNase OUT (Invitrogen), and 0.2% Ribonucleoside Vanadyl Complex, protease inhibitor mixture (Halt Protease Inhibitor Cocktails, Thermo Scientific).…”