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An oligonucleotide-based tandem RNA isolation procedure to recover eukaryotic mRNA-protein complexes

Iadevaia, Valentina, Matia-González, Ana M. and Gerber, André P. (2018) An oligonucleotide-based tandem RNA isolation procedure to recover eukaryotic mRNA-protein complexes Journal of Visualized Experiments (JoVE), 138, e58223.

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Abstract

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3'-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

Item Type: Article
Divisions : Faculty of Health and Medical Sciences > School of Biosciences and Medicine
Authors :
NameEmailORCID
Iadevaia, Valentinav.iadevaia@surrey.ac.uk
Matia-González, Ana M.
Gerber, André P.a.gerber@surrey.ac.uk
Date : 18 August 2018
Funders : Biotechnology and Biological Sciences Research Council (BBSRC)
DOI : 10.3791/58223
Copyright Disclaimer : Copyright © 2018 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License
Depositing User : Clive Harris
Date Deposited : 20 Jun 2018 09:59
Last Modified : 11 Dec 2018 11:24
URI: http://epubs.surrey.ac.uk/id/eprint/847082

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