Purification of cross-linked RNA-protein complexes by phenol-toluol extraction

Erika C Urdaneta; Carlos H Vieira-Vieira; Timon Hick; Hans-Herrmann Wessels; Davide Figini; Rebecca Moschall; Jan Medenbach; Uwe Ohler; Sander Granneman; Matthias Selbach; Benedikt M Beckmann

doi:10.1038/s41467-019-08942-3

Purification of cross-linked RNA-protein complexes by phenol-toluol extraction

Erika C Urdaneta, Carlos H Vieira-Vieira, Timon Hick, Hans-Herrmann Wessels, Davide Figini, Rebecca Moschall, Jan Medenbach, Uwe Ohler, Sander Granneman, Matthias Selbach, Benedikt M Beckmann

School of Biological Sciences

Research output: Contribution to journal › Article › peer-review

Abstract

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.

Original language	English
Article number	990
Pages (from-to)	1-17
Number of pages	17
Journal	Nature Communications
Volume	10
DOIs	https://doi.org/10.1038/s41467-019-08942-3
Publication status	Published - 1 Mar 2019

Keywords / Materials (for Non-textual outputs)

animals
base sequence
brain
HEK293 cells
humans
Mice
molecular biology
multiprotein complexes
phenol
proteome
proteomics
isolation & purification
RNA
RNA-binding proteins
ribonucleoproteins
salmonella typhimurium
sensitivity and specificity
Toluene
Inbred C57BL

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10.1038/s41467-019-08942-3Licence: Creative Commons: Attribution (CC-BY)

UrdanetaEtalNC2019PurificationOfVCross-LinkedRNA-ProteinComplexesByPhenol-ToluolFinal published version, 3.23 MBLicence: Creative Commons: Attribution (CC-BY)

Unravelling post-transcriptional regulatory networks in pathogenic S. aureus
Granneman, S. (Principal Investigator)
MRC
1/01/18 → 22/08/23
Project: Research

Purification of cross-linked RNA-protein complexes by phenol-toluol extraction
Urdaneta, E. C. (Data Collector), Vierira-Vieira, C. (Data Collector), Hick, T. (Data Collector), Wessels, H.-H. (Data Collector), Figini, D. (Data Collector), Moschall, R. (Data Collector), Medenbach, J. (Data Collector), Ohler, U. (Data Collector), Granneman, S. (Data Collector), Selbach, M. (Data Collector) & Beckmann, B. M. (Data Collector), PRIDE database hosted by European Bioinformatics Institute, EBI, 12 Mar 2019
http://doi.org/10.1038/s41467-019-08942-3 and one more link, https://www.ebi.ac.uk/pride/archive/projects/PXD009571 (show fewer)
Dataset

Sander Granneman
- School of Biological Sciences - Personal Chair of RNA Biochemistry
- Centre for Engineering Biology
Person: Academic: Research Active

Cite this

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title = "Purification of cross-linked RNA-protein complexes by phenol-toluol extraction",

abstract = "Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.",

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doi = "10.1038/s41467-019-08942-3",

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AU - Urdaneta, Erika C

AU - Vieira-Vieira, Carlos H

AU - Hick, Timon

AU - Wessels, Hans-Herrmann

AU - Figini, Davide

AU - Moschall, Rebecca

AU - Medenbach, Jan

AU - Ohler, Uwe

AU - Granneman, Sander

AU - Selbach, Matthias

AU - Beckmann, Benedikt M

PY - 2019/3/1

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N2 - Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.

AB - Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.

KW - animals

KW - base sequence

KW - brain

KW - HEK293 cells

KW - humans

KW - Mice

KW - molecular biology

KW - multiprotein complexes

KW - phenol

KW - proteome

KW - proteomics

KW - isolation & purification

KW - RNA

KW - RNA-binding proteins

KW - ribonucleoproteins

KW - salmonella typhimurium

KW - sensitivity and specificity

KW - Toluene

KW - Inbred C57BL

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DO - 10.1038/s41467-019-08942-3

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JO - Nature Communications

JF - Nature Communications

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ER -

Purification of cross-linked RNA-protein complexes by phenol-toluol extraction

Abstract

Keywords / Materials (for Non-textual outputs)

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Projects

Unravelling post-transcriptional regulatory networks in pathogenic S. aureus

Datasets

Purification of cross-linked RNA-protein complexes by phenol-toluol extraction

Profiles

Sander Granneman

Cite this