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RNA. 2017 Aug;23(8):1290-1302. doi: 10.1261/rna.060798.117. Epub 2017 May 5.

Specific RNP capture with antisense LNA/DNA mixmers.

Author information

1
European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany.
2
German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
3
Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom.

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.

KEYWORDS:

RNA interactome; RNA-binding proteins; RNA–protein interactions; ribosomal RNA; specific RNP capture

PMID:
28476952
PMCID:
PMC5513073
DOI:
10.1261/rna.060798.117
[Indexed for MEDLINE]
Free PMC Article

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