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Specific RNP capture with antisense LNA/DNA mixmers

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 transc...

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Autores principales: Rogell, Birgit, Fischer, Bernd, Rettel, Mandy, Krijgsveld, Jeroen, Castello, Alfredo, Hentze, Matthias W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513073/
https://www.ncbi.nlm.nih.gov/pubmed/28476952
http://dx.doi.org/10.1261/rna.060798.117
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author Rogell, Birgit
Fischer, Bernd
Rettel, Mandy
Krijgsveld, Jeroen
Castello, Alfredo
Hentze, Matthias W.
author_facet Rogell, Birgit
Fischer, Bernd
Rettel, Mandy
Krijgsveld, Jeroen
Castello, Alfredo
Hentze, Matthias W.
author_sort Rogell, Birgit
collection PubMed
description 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.
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spelling pubmed-55130732017-08-02 Specific RNP capture with antisense LNA/DNA mixmers Rogell, Birgit Fischer, Bernd Rettel, Mandy Krijgsveld, Jeroen Castello, Alfredo Hentze, Matthias W. RNA Method 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. Cold Spring Harbor Laboratory Press 2017-08 /pmc/articles/PMC5513073/ /pubmed/28476952 http://dx.doi.org/10.1261/rna.060798.117 Text en © 2017 Rogell et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article, published in RNA, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Method
Rogell, Birgit
Fischer, Bernd
Rettel, Mandy
Krijgsveld, Jeroen
Castello, Alfredo
Hentze, Matthias W.
Specific RNP capture with antisense LNA/DNA mixmers
title Specific RNP capture with antisense LNA/DNA mixmers
title_full Specific RNP capture with antisense LNA/DNA mixmers
title_fullStr Specific RNP capture with antisense LNA/DNA mixmers
title_full_unstemmed Specific RNP capture with antisense LNA/DNA mixmers
title_short Specific RNP capture with antisense LNA/DNA mixmers
title_sort specific rnp capture with antisense lna/dna mixmers
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513073/
https://www.ncbi.nlm.nih.gov/pubmed/28476952
http://dx.doi.org/10.1261/rna.060798.117
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