Universität zu Köln

Gerbracht, Jennifer V. ORCID: 0000-0002-3377-4649, Boehm, Volker ORCID: 0000-0001-7588-9842 and Gehring, Niels H. (2017). Plasmid transfection influences the readout of nonsense-mediated mRNA decay reporter assays in human cells. Sci Rep, 7. LONDON: NATURE PUBLISHING GROUP. ISSN 2045-2322

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Abstract

Messenger RNA (mRNA) turnover is a crucial and highly regulated step of gene expression in mammalian cells. This includes mRNA surveillance pathways such as nonsense-mediated mRNA decay (NMD), which assesses the fidelity of transcripts and eliminates mRNAs containing a premature translation termination codon (PTC). When studying mRNA degradation pathways, reporter mRNAs are commonly expressed in cultivated cells. Traditionally, the molecular mechanism of NMD has been characterized using pairs of reporter constructs that express the same mRNA with (PTC-containing mRNA) or without (wild-type mRNA) a PTC. Cell lines stably expressing an NMD reporter have been reported to yield very robust and highly reproducible results, but establishing the cell lines can be very time-consuming. Therefore, transient transfection of such reporter constructs is frequently used and allows analysis of many samples within a short period of time. However, the behavior of transiently and stably transfected NMD constructs has not been systematically compared so far. Here, we report that not all commonly used human cell lines degrade NMD targets following transient transfection. Furthermore, the degradation efficiency of NMD substrates can depend on the manner of transfection within the same cell line. This has substantial implications for the interpretation of NMD assays based on transient transfections.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Gerbracht, Jennifer V. UNSPECIFIED orcid.org/0000-0002-3377-4649 UNSPECIFIED Boehm, Volker UNSPECIFIED orcid.org/0000-0001-7588-9842 UNSPECIFIED Gehring, Niels H. UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-217994
DOI:	10.1038/s41598-017-10847-4
Journal or Publication Title:	Sci Rep
Volume:	7
Date:	2017
Publisher:	NATURE PUBLISHING GROUP
Place of Publication:	LONDON
ISSN:	2045-2322
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 DEGRADATION; TERMINATION; MECHANISMS Multiple languages Multidisciplinary Sciences Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/21799