Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila de...

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Main Authors:	Gueroussov, Serge, Tarnawsky, Stefan P., Cui, Xianying A., Mahadevan, Kohila, Palazzo, Alexander F.
Format:	Online
Language:	English
Published:	MyJove Corporation 2010
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159668/

id	pubmed-3159668
recordtype	oai_dc
spelling	pubmed-31596682011-09-19 Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection Gueroussov, Serge Tarnawsky, Stefan P. Cui, Xianying A. Mahadevan, Kohila Palazzo, Alexander F. Cellular Biology In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data. MyJove Corporation 2010-12-04 /pmc/articles/PMC3159668/ /pubmed/21178962 http://dx.doi.org/10.3791/2387 Text en Copyright © 2010, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Gueroussov, Serge Tarnawsky, Stefan P. Cui, Xianying A. Mahadevan, Kohila Palazzo, Alexander F.
spellingShingle	Gueroussov, Serge Tarnawsky, Stefan P. Cui, Xianying A. Mahadevan, Kohila Palazzo, Alexander F. Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
author_facet	Gueroussov, Serge Tarnawsky, Stefan P. Cui, Xianying A. Mahadevan, Kohila Palazzo, Alexander F.
author_sort	Gueroussov, Serge
title	Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
title_short	Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
title_full	Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
title_fullStr	Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
title_full_unstemmed	Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection
title_sort	analysis of mrna nuclear export kinetics in mammalian cells by microinjection
description	In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.
publisher	MyJove Corporation
publishDate	2010
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159668/
_version_	1611471762867879936

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

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