title: Analysis of mRNA nuclear export kinetics in mammalian cells by microinjection.
abstract: 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 oocytes and genetically tractable organisms such as yeast and the Drosophila derived S2 cell line, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly. 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 manner, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR). 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.
authors.affiliation: Department of Biochemistry, University of Toronto.
authors.forename: Serge
authors.initials: S
authors.lastname: Gueroussov
authors.forename: Stefan P
authors.initials: SP
authors.lastname: Tarnawsky
authors.forename: Xianying A
authors.initials: XA
authors.lastname: Cui
authors.forename: Kohila
authors.initials: K
authors.lastname: Mahadevan
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Palazzo
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D007700:Kinetics; D051379:Mice; D008845:Microinjections / Q000295:instrumentation / Q000379:methods*; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D004247:DNA