Monitoring mRNA Export

Kazuaki Tokunaga1, Tokio Tani1

1 Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kurokami, Kumamoto, Japan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.13
DOI:  10.1002/0471143030.cb2213s41
Online Posting Date:  December, 2008
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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. © 2008 by John Wiley & Sons, Inc.

Keywords: mRNA; transport; in situ hybridization; microinjection; yeast; mammalian cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Monitoring Nuclear mRNA Export by Fluorescence in situ Hybridization in Fission Yeast
  • Alternate Protocol 1: Monitoring Nuclear mRNA Export by Fluorescence In Situ Hybridization in Budding Yeast
  • Alternate Protocol 2: Monitoring Nuclear mRNA Export in Mammalian Cells by Fluorescence In Situ Hybridization
  • Basic Protocol 2: Monitoring Nuclear mRNA Export in a Living Mammalian Cell by Microinjection of Fluorescently Labeled Pre‐mRNA into the Nucleus
  • Support Protocol 1: Preparation of a Digoxigenin (or Biotin)–Labeled OLIGO(dT) Hybridization Probe
  • Support Protocol 2: Preparation of Fluorescently Labeled pre‐mRNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Monitoring Nuclear mRNA Export by Fluorescence in situ Hybridization in Fission Yeast

  Materials
  • S. pombe cells to be analyzed (e.g., wild‐type strain 972 or temperature‐sensitive export mutant strain ptr8; both strains available from Dr. Tokio Tani, Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Japan)
  • YE medium (see recipe)
  • 4% paraformaldehyde solution (see recipe; freshly prepared)
  • 0.1 M sodium phosphate buffer, pH 6.0 ( appendix 2A)
  • PEMS (see recipe), ice cold
  • Novozym (http://www.novozymes.com) or lysing enzyme from Trichoderma harzanum (Sigma, cat. no. L1412)
  • Zymolyase 100T (ICN Biomedicals)
  • 70%, 90%, and 100% ethanol
  • Prehybridization solution for yeast (see recipe)
  • Hybridization solution for yeast: 1 ng/µl biotin‐labeled oligo(dT) probe ( protocol 5) in prehybridization solution for yeast (see recipe); store at –20°C
  • 4× SSC (see recipe for 20×)
  • 2 µg/ml fluorescein isothiocyanate (FITC)–conjugated avidin (Roche, cat. no. 1975595) in 4× SSC/1% (w/v) RNase‐free BSA (prepare fresh)
  • 4× SSC/0.1% (v/v) Triton X‐100 (prepare from 0.5% Triton stock, see recipe)
  • 0.1 µg/ml DAPI solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Mounting medium (see recipe)
  • Clear nail polish
  • 30° (for wild‐type yeast) or 26°C (for temperature‐sensitive mutants) shaking incubator
  • Refrigerated microcentrifuge with swinging‐bucket rotor (e.g., Kubota model MF‐2036)
  • Phase‐contrast microscope
  • Coplin jars
  • Multi‐well slide coated with poly‐L‐lysine (see recipe)
  • Glass coverslips
  • Moist chamber: place several sheets of filter paper soaked with distilled H 2O in a petri dish and put two toothpicks on the paper; slide on the toothpicks and seal the dish with tape or Parafilm
  • 42°C air incubator
  • Epifluorescence microscope with FITC or DAPI filter set
  • Digital image acquisition system with a cooled CCD camera

Alternate Protocol 1: Monitoring Nuclear mRNA Export by Fluorescence In Situ Hybridization in Budding Yeast

  • S. cerevisiae cells to be analyzed
  • YPD medium (see recipe)
  • Sorbitol buffer (see recipe), ice cold
  • 2 mg/ml Zymolyase 100T
  • 2‐Mercaptoethanol

Alternate Protocol 2: Monitoring Nuclear mRNA Export in Mammalian Cells by Fluorescence In Situ Hybridization

  Materials
  • HeLa cells (ATCC no. CCL‐2) grown in a 100‐mm tissue culture dish
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Trypsin/EDTA (see recipe)
  • DMEM with 10% FBS (see recipe)
  • 3% paraformaldehyde (see recipe; freshly prepared)
  • 0.5% (v/v) Triton X‐100 (see recipe)
  • 1× and 2× SSC ( appendix 2A)
  • Hybridization solution for mammalian cells (see recipe)
  • Rubber cement
  • PBS‐BAG (see recipe)
  • Primary antibody: mouse anti‐digoxin antibody (Sigma, cat. no. D8156)
  • Secondary antibody: anti–mouse IgG antibody conjugated with fluorescein isothiocyanate (FITC)
  • 0.1 µg/µl DAPI solution (see recipe)
  • Mounting medium (see recipe)
  • Clear nail polish
  • Hemacytometer (unit 1.1)
  • 12‐mm glass coverslips, round or square
  • 35‐mm tissue culture dishes
  • Glass microscope slides
  • Moist chamber: place several sheets of filter paper soaked with distilled H 2O in a petri dish and put two toothpicks on the paper; slide on the toothpicks and seal the dish with tape or Parafilm
  • Forceps
  • Epifluorescence microscope with FITC or DAPI filter set
  • Digital image acquisition system with a cooled CCD camera
  • Additional reagents and equipment for counting cells using a hemacytometer (unit 1.1)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All cell culture incubations should be carried out in a 37°C, 5% CO 2 humidified incubator.

Basic Protocol 2: Monitoring Nuclear mRNA Export in a Living Mammalian Cell by Microinjection of Fluorescently Labeled Pre‐mRNA into the Nucleus

  Materials
  • HeLa cells (ATCC no. CCL‐2)
  • DMEM supplemented with 10% FBS, penicillin, and streptomycin (see appendix 2A)
  • Phenol red–free DMEM with 10% FBS (see recipe for DMEM with 10% FBS, but use phenol red–free DMEM)
  • 3 M sodium acetate, pH 5.3 ( appendix 2A)
  • 100% ethanol
  • Microinjection buffer (see recipe)
  • 1.5 mg/ml FITC‐dextran (see recipe)
  • Cy3‐labeled fushi tarazu pre‐mRNA ( protocol 6)
  • 3% paraformaldehyde solution (see recipe)
  • Mounting medium (see recipe)
  • Clear nail polish
  • 35‐mm glass‐bottom dishes
  • Humidified 5% CO 2 incubator
  • Refrigerated centrifuge with Kubota AF‐2536A rotor (or equivalent)
  • Microloader pipet tip (Eppendorf or equivalent)
  • Inverted epifluorescence microscope
  • Digital image acquisition system with a cooled CCD camera
  • Microscope stage incubator, 37°C, 5% CO 2 (optional)
  • Microinjection needle (Eppendorf Femtotips II or equivalent)
  • Microinjector and manipulator (Eppendorf 5170 microinjector or equivalent)
  • Additional reagents and equipment for trypsinizing HeLa cells and preparing cell suspension ( protocol 3, steps 1 to 7)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All cell culture incubations should be carried out in a 37°C, 5% CO 2 humidified incubator.

Support Protocol 1: Preparation of a Digoxigenin (or Biotin)–Labeled OLIGO(dT) Hybridization Probe

  Materials
  • DIG oligonucleotide 3′‐end labeling kit (Roche Applied Science)
  • 1 mM biotin‐16‐ddUTP (for preparation of biotin‐labeled probe)
  • STOP solution (see recipe)
  • 4 M LiCl
  • 100% ethanol, cold
  • 70% ethanol
  • TE buffer ( appendix 2A)
  • Microcentrifuge with, Kubota AF‐2536A rotor (or equivalent), 4°C
  • Vacuum desiccator

Support Protocol 2: Preparation of Fluorescently Labeled pre‐mRNA

  Materials
  • pSP64 (poly A)‐ftz plasmid (available from Dr. Tokio Tani, Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Japan)
  • Restriction enzyme EcoRI and high‐salt restriction buffer
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 7.0
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 100% ethanol, cold, and 70% ethanol
  • TE buffer ( appendix 2A)
  • 5× transcription buffer (see recipe)
  • 100 mM DTT
  • 10 mM ATP, UTP, GTP, and CTP
  • m7G cap analog (Promega, cat. no. P171B)
  • 15 U/µl SP6 RNA polymerase
  • 1 U/µl DNase I (RNase‐free grade)
  • RNase‐free H 2O (DEPC‐treated; appendix 2A)
  • RNA dye solution (see recipe)
  • 8% polyacrylamide/7 M urea gel (also see Brown et al., )
  • 1 µg/ml ethidium bromide
  • 10× labeling buffer (PanVera Corp.)
  • Label IT reagent (PanVera Corp.)
  • Microcentrifuge with Kubota AF2536A rotor (or equivalent), 4°C
  • Vacuum desiccator
  • MicroSpin G‐50 spin columns (GE Healthcare)
  • Spectrophotometer capable of reading at 260 nm
  • 90°C water bath or heat block
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ), extraction of DNA from agarose gels (Moore et al., ), and denaturing polyacrylamide electrophoresis of RNA (Brown et al., )
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Figures

  •   FigureFigure 22.13.1 Fluorescence in situ hybridization with oligo(dT) probe in S. pombe. The wild‐type strain 972 (“WT,” upper panels) and the mRNA export mutant ptr8 (lower panels, Mizuki et al., ) were grown at 26°C and either maintained at 26°C or shifted to 37°C for 60 min. The cells were fixed and analyzed by fluorescence in situ hybridization with the biotin‐labeled oligo(dT)50 probe. Hybridized signals were detected by FITC‐conjugated avidin. The FITC columns show the distribution of mRNA in the respective cells. The DAPI columns show staining of DNA of the cells in the same field.

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Literature Cited

Literature Cited
   Azad, A.K., Tani, T., Shiki, N., Tsuneyoshi, S., Urushiyama, S., and Ohshima, Y. 1997. Isolation and molecular characterization of mRNA transport mutants in Schizosaccharomyces pombe. Mol. Biol. Cell 8:825‐841.
   Brown, T., Mackey, K., and Du, T. 2004. Analysis of RNA by northern and slot‐blot hybridization. Curr. Protoc. Mol. Biol. 67:4.9.1‐4.9.19.
   Groner, B. and Phillips, S.L. 1975. Polyadenylate metabolism in the nuclei and cytoplasm of Saccharomyces cerevisiae. J. Biol. Chem. 250:5640‐5646.
   Heath, C.V., Copeland, C.S., Amberg, D.C., Del Priore, V., Snyder, M., and Cole, C.N. 1995. Nuclear pore complex clustering and nuclear accumulation of poly (A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene. J. Cell Biol. 131:1677‐1697.
   Herrick, D., Parker, R., and Jacobson, A. 1990. Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae. Mol. Cell. Biol. 10:2269‐2284.
   Jarmolowski, A., Boelens, W.C., Izaurralde, E., and Mattaj, I.W. 1994. Nuclear export of different class of RNA is mediated by specific factors. J. Cell Biol. 124:627‐635.
   Lamond, A. and Spector, D.L. 2003. Nuclear speckles: A model for nuclear organelles. Nat. Rev. Mol. Cell Biol. 4:605‐635.
   Mizuki, F., Namiki, T., Sato, H., Furukawa, H., Matsusaka, T., Ohshima, Y., Ishibashi, R., Andoh, T., and Tani, T. 2007. Participation of XPB/Ptr8p, a component of TFIIH, in nucleocytoplasmic transport of mRNA in fission yeast. Gene Cells 12:35‐47.
   Moore, D., Dowhan, D., Chory, J., and Ribaudo, R.K. 2002. Isolation and purification of large DNA restriction fragments from agarose gels. Curr. Protoc. Mol. Biol. 59:2.6.1‐2.6.12.
   Rio, D.C. 1988. Accurate and efficient pre‐mRNA splicing in Drosophila cell‐free extracts. Proc. Natl. Acad. Sci. U.S.A. 85:2904‐2908.
   Shibata, T., Matsuoka, Y., and Yoneda, Y. 2002. Nucleocytoplasmic transport of proteins and poly (A)+ RNA in reconstituted Tpr‐less nuclei in living mammalian cells. Genes Cells 7:421‐434.
   Strässer, K. and Hurt, E. 1999. Nuclear RNA export in yeast. FEBS Lett. 452:77‐81.
   Tokunaga, K., Shibuya, T., Ishihama, Y., Tadakuma, H., Ide, M., Yoshida, M., Funatsu, T., Ohshima, Y., and Tani, T. 2006. Nucleocytoplasmic transport of fluorescent mRNA in living mammalian cells: Nuclear mRNA export is coupled to ongoing gene transcription. Genes Cells 11:305‐317.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
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