RIP-Chip Analysis: RNA-Binding Protein Immunoprecipitation-Microarray (Chip) Profiling

Abstract

Post-transcriptional regulation of gene expression plays an important role in complex cellular processes. Just like transcription factors regulate gene expression through combinatorial binding to multiple, physically dispersed cis elements, mRNA binding proteins can regulate the translation of functionally related gene products by coordinately binding to subsets of mRNAs. The networks of mRNA binding proteins that facilitate this fine-tuning of gene expression are poorly understood. By combining genomic technologies with standard molecular biology tools, we have helped pioneer the development of high-throughput technologies for the global analysis of subsets of mRNAs bound to RNA-binding proteins. This technique is termed RIP-Chip and stands for RNA-Binding Protein Immunoprecipitation-Microarray (Chip) Profiling. This approach is also referred to as "ribonomic profiling" and has revealed valuable information about the workings of mRNP networks in the cell and the regulation of gene expression. In this chapter, we describe the latest advances that we have made in the RIP-CHIP technology.

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition

“…IP of HEXIM1 from K562 cellular extracts followed by immunoblot (IB) analysis was performed as described (11,12). The RNA-binding protein IP assay (RIP) was performed according to the protocol described by Jain et al (58) with some modifications. Briefly, HEK293T cells transiently transfected with pDESTmycIG-F2BP3 were incubated 5 minutes in ice-cold polysome lysis buffer (10 mM Hepes, pH 7.0, 100 mM KCl, 5 mM MgCl 2 , 0.5% Nonidet P-40 [NP-40], 1 mM DTT) supplemented with EDTA-free protease inhibitor cocktail (Roche), 20 μM calpain inhibitor III, and 40 U/ml RNase inhibitor (Promega) and then stored at -80°C.…”

RNA regulons and the RNA-protein interaction network

“…These typically involve the affi nity purifi cation of an endogenous or epitope-tagged RBP, and the subsequent analysis of bound RNAs with DNA microarrays or high-throughput sequencing. In the following paragraphs, we briefl y outline some of the experimental procedures that have been developed in this fi eld, and we refer the reader to some recent reviews on this topic for more details (25,26 (27) . Thanks to its simplicity and robustness, this method has since been employed to determine the RNA targets of more than one hundred RBPs in mammalian cells, fl ies, worms, trypanosomes, and in yeast [a comprehensive list of RBP experiments is given in (28) ].…”

Post-transcriptional Regulation of Programmed Cell Death 4 (PDCD4) mRNA by the RNA-binding Proteins Human Antigen R (HuR) and T-cell Intracellular Antigen 1 (TIA1)

“…RNA Immunoprecipitation-RNA-IPs to assay endogenous HuR/PDCD4 mRNA interactions were performed using standard methods (58). Briefly, MCF-7 cells were grown to confluency in 100-mm dishes and rinsed twice with ice-cold PBS.…”