Specific RNP capture with antisense LNA/DNA mixmers

Rogell, B., Fischer, B., Rettel, M., Krijgsveld, J., Castello, A. and Hentze, M. W. (2017) Specific RNP capture with antisense LNA/DNA mixmers. RNA, 23(8), pp. 1290-1302. (doi: 10.1261/rna.060798.117) (PMID:28476952) (PMCID:PMC5513073)

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Abstract

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe “specific ribonucleoprotein (RNP) capture,” a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein–RNA interactions taking place at “zero distance.” Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Castello, Professor Alfredo
Authors: Rogell, B., Fischer, B., Rettel, M., Krijgsveld, J., Castello, A., and Hentze, M. W.
College/School:College of Medical Veterinary and Life Sciences > School of Infection & Immunity
College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
Journal Name:RNA
Publisher:Cold Spring Harbor Laboratory Press
ISSN:1355-8382
ISSN (Online):1469-9001
Published Online:05 May 2017
Copyright Holders:Copyright © 2017 Rogell et al.
First Published:First published in RNA 23(8): 1290=1302
Publisher Policy:Reproduced under a Creative Commons License

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