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Bio Protoc. 2016 Dec 20;6(24). pii: e2062. doi: 10.21769/BioProtoc.2062.

Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes.

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1
Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, Biomedical Research Center, Baltimore, USA.

Abstract

RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al., 2008). There are several methodologies available today to identify RNAs bound to specific RBPs; some detect only recombinant molecules in vitro, others detect recombinant and endogenous molecules, while others detect only endogenous molecules. Examples include systematic evolution of ligands by exponential enrichment (SELEX), biotinylated RNA pulldown assay, RNA immunoprecipitation (RIP) assay, electrophoretic mobility shift assay (EMSA), RNA footprinting analysis, and various UV crosslinking and immunoprecipitation (CLIP) methods such as CLIP, PAR-CLIP, and iCLIP (Popova et al., 2015). Here, we describe a simple and informative method to study and identify the RNA region of interaction between an RBP and its target transcript (Panda et al., 2014 and 2016). Its reproducibility and ease of use make this protocol a fast and useful method to identify interactions between RBPs and specific RNAs.

KEYWORDS:

Biotin pulldown; RNA-binding proteins; Ribonucleoprotein complex; Tagged RNA; in vitro transcription

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