Universität zu Köln

Castello, Alfredo ORCID: 0000-0002-1499-4662, Frese, Christian K., Fischer, Bernd ORCID: 0000-0001-9437-2099, Jarvelin, Aino I., Horos, Rastislav ORCID: 0000-0001-7730-9557, Alleaume, Anne-Marie, Foehr, Sophia ORCID: 0000-0001-5148-7945, Curk, Tomaz ORCID: 0000-0003-4888-7256, Krijgsveld, Jeroen and Hentze, Matthias W. (2017). Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap. Nat. Protoc., 12 (12). S. 2447 - 2465. LONDON: NATURE PUBLISHING GROUP. ISSN 1750-2799

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Abstract

RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supernatant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Castello, Alfredo UNSPECIFIED orcid.org/0000-0002-1499-4662 UNSPECIFIED Frese, Christian K. UNSPECIFIED UNSPECIFIED UNSPECIFIED Fischer, Bernd UNSPECIFIED orcid.org/0000-0001-9437-2099 UNSPECIFIED Jarvelin, Aino I. UNSPECIFIED UNSPECIFIED UNSPECIFIED Horos, Rastislav UNSPECIFIED orcid.org/0000-0001-7730-9557 UNSPECIFIED Alleaume, Anne-Marie UNSPECIFIED UNSPECIFIED UNSPECIFIED Foehr, Sophia UNSPECIFIED orcid.org/0000-0001-5148-7945 UNSPECIFIED Curk, Tomaz UNSPECIFIED orcid.org/0000-0003-4888-7256 UNSPECIFIED Krijgsveld, Jeroen UNSPECIFIED UNSPECIFIED UNSPECIFIED Hentze, Matthias W. UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-210344
DOI:	10.1038/nprot.2017.106
Journal or Publication Title:	Nat. Protoc.
Volume:	12
Number:	12
Page Range:	S. 2447 - 2465
Date:	2017
Publisher:	NATURE PUBLISHING GROUP
Place of Publication:	LONDON
ISSN:	1750-2799
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language BOUND PROTEOME; MASS-SPECTROMETRY; CROSS-LINKING; FRACTIONATION; EXPRESSION; ENRICHMENT; PEPTIDES; CAPTURE; YEAST Multiple languages Biochemical Research Methods Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/21034