Monitoring mRNA Export

Abstract

Transport of mRNA from the nucleus to the cytoplasm is an essential process for gene expression in eukaryotic cells. In this unit, methods for monitoring nuclear mRNA export are described. Visualization of cellular mRNAs by fluorescence in situ hybridization with oligo(dT) probes is effectively applied to monitoring mRNA export from the nucleus in yeast and mammalian cells. In addition to the protocols for fluorescence in situ hybridization, this unit includes an alternate method that the authors have been developing for visual analysis of nuclear mRNA export in living mammalian cells by microinjection of fluorescently labeled pre-mRNA into the nuclei. Curr. Protoc. Cell Biol. 41:22.13.1-22.13.20. C 2008 by John Wiley & Sons, Inc.Keywords: mRNA r transport r in situ hybridization r microinjection r yeast r mammalian cells Supplement 41Current Protocols in Cell Biology poly(A) tails of mRNAs in the fixed cells. In the final stage of the experiment, the cells are washed with a series of sodium chloride/sodium citrate (SSC) solutions to remove unbound probe, then treated with avidin-FITC to visualize cellular mRNAs hybridized with the biotin-labeled probe. MaterialsS. pombe cells to be analyzed (e.g., wild-type strain 972 or temperature-sensitive export mutant strain ptr8; both strains available from Dr.

The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells

“…The RNA FISH method to visualize poly-adenylated mRNA using fluorescently labeled poly-(dT) 70 -TRITC (MWG) probes ( Dias et al, 2010 ) was described previously ( Tokunaga and Tani, 2008 ). Images of several optical sections were acquired on a Zeiss AxioImager Z1 microscope equipped with a CoolSNAP-HQ2 CCD camera (Photometrics) and AxioVision Rel.…”

Identification of cis- and trans-acting factors involved in the localization of MALAT-1 noncoding RNA to nuclear speckles

“…An Alexa Fluor 594-conjugated 29-O-alkyl antisense oligonucleotide probe (Gene Design, Inc.) was used to visualize U1 snRNA as described previously (Carmo-Fonseca et al 1991). Visualization of nuclear poly(A) RNA was carried out with an Alexa Fluor 594-conjugated oligonucleotide dT 34 probe (Gene Design, Inc.) as previously described (Tokunaga and Tani 2008).…”