Universität zu Köln

Steckelberg, Anna-Lena ORCID: 0000-0001-7988-9946 and Gehring, Niels H. ORCID: 0000-0001-7792-1164 (2014). Studying the composition of mRNPs in vitro using splicing-competent cell extracts. Methods, 65 (3). S. 342 - 350. SAN DIEGO: ACADEMIC PRESS INC ELSEVIER SCIENCE. ISSN 1095-9130

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Abstract

The correct processing and faithful decoding of mRNAs during gene expression depends on the interaction with RNA-binding proteins (RBPs). The association of RBPs with pre-mRNAs starts during transcription by RNA polymerase II and undergoes constant remodeling during pre-mRNA processing and later steps of genes expression. Recently developed high throughput methods enabled to define RBP binding sites in vivo and to identify a large number of novel RBPs in eukaryotic cells. However, the detailed characterization of RBP-RNA interactions as well as the analysis of functional RNPs is greatly facilitated by well-defined in vitro systems. Here, we describe a versatile method to study the assembly and splicing-dependent remodeling of mRNPs in vitro. This method employs splicing-competent whole cell extracts (WCE) generated from transfected human embryonic kidney (HEK) 293 cells. FLAG-tagged proteins present in the WCE are incorporated into mRNPs in vitro and afterwards used to immunoprecipitate substrate RNAs. We outline the principles of purifying in vitro assembled mRNPs and provide detailed protocols for the preparation and use of whole cell extracts. Alternative purification strategies and RNA substrates are discussed. (C) 2013 Elsevier Inc. All rights reserved.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Steckelberg, Anna-Lena UNSPECIFIED orcid.org/0000-0001-7988-9946 UNSPECIFIED Gehring, Niels H. UNSPECIFIED orcid.org/0000-0001-7792-1164 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-446267
DOI:	10.1016/j.ymeth.2013.08.033
Journal or Publication Title:	Methods
Volume:	65
Number:	3
Page Range:	S. 342 - 350
Date:	2014
Publisher:	ACADEMIC PRESS INC ELSEVIER SCIENCE
Place of Publication:	SAN DIEGO
ISSN:	1095-9130
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language EXON JUNCTION COMPLEX; NONSENSE-MEDIATED DECAY; MESSENGER-RNA EXPORT; CAP-BINDING COMPLEX; CYTOPLASMIC TRANSLATION; CORE COMPLEX; PROTEIN; EIF4AIII; REVEALS; RECRUITMENT Multiple languages Biochemical Research Methods; Biochemistry & Molecular Biology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/44626