Analysis of RNA Export Using Xenopus Oocytes

Deborah Duricka1, Katharine S. Ullman1

1 University of Utah, Salt Lake City, Utah
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.14
DOI:  10.1002/0471143030.cb1114s10
Online Posting Date:  May, 2001
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Abstract

This unit describes a procedure for monitoring RNA export in Xenopus oocytes. The technique involves synthesizing labeled RNA in vitro and microinjecting the RNA into oocyte nuclei. Following incubation the oocytes are dissected into nuclear and cytoplasmic fractions. These samples are then processed for RNA analysis, allowing the extent of export to be quantitatively assessed.

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of RNA Export in Microinjected Xenopus Oocytes
  • Support Protocol 1: Preparation of Xenopus Oocytes for Microinjection
  • Support Protocol 2: Preparation of RNA for Injection
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of RNA Export in Microinjected Xenopus Oocytes

  Materials
  • RNA sample (see protocol 3)
  • Xenopus oocytes (see protocol 2)
  • Light mineral oil (e.g., Fisher Scientific)
  • RNA harvest buffer (see recipe)
  • 4 mg/ml proteinase K solution: 1:4 (v/v) 20 mg/ml proteinase K (e.g., Ambion, Worthington) in RNA harvest buffer, prepared fresh
  • 5:1 (v/v) acid phenol/chloroform, pH 4.5 (Ambion)
  • 20 mg/ml glycogen or seeDNA (Amersham Pharmacia Biotech)
  • RNA sample buffer (see recipe)
  • Glass capillary tubes, 1‐mm o.d., 0.75‐mm i.d., 10‐cm length (e.g., Sutter Instruments)
  • Needle‐pulling apparatus (e.g., Sutter Instruments micropipet puller, model P‐87)
  • Pico‐injector, basic model (e.g., Model PL1‐100, Medical Systems Corp.)
  • Nitrogen
  • Dumont no. 3 and Dumont SS forceps (Fine Science Tools), including one pair of no. 3 forceps set aside for use with oil
  • Disecting microscope (e.g., MZ7.5, Leica) with optional cold stage (e.g., Labworks # Equipment Service, Inc.; also requires circulating cold bath), 4°C
  • 18°C incubator
  • Disposable transfer pipets (e.g., Fisher Scientific)
  • Whatman 3MM filter paper, cut into ∼0.5‐in × 3‐in strips
  • Mid‐sized polystyrene weighing dishes
  • 1‐ml syringes
  • 27.5‐G needles
  • 100‐µl glass capillary tubes with 25‐µl microcaps (e.g., Drummond Scientific)
  • Additional reagents and equipment for phenol/chloroform extraction, ethanol precipitation ( appendix 3A), and polyacrylamide gel electrophoresis of RNA (unit 6.1)

Support Protocol 1: Preparation of Xenopus Oocytes for Microinjection

  Materials
  • Female Xenopus laevis (e.g., Nasco, Xenopus I, Xenopus Express)
  • 0.05% (w/v) benzocaine (Sigma) in H 2O from 10% (w/v) stock in ethanol, stored at 4°C.
  • MBS (see recipe), 4°C
  • Toothed and Dumont no. 3 forceps (Fine Science Tools)
  • Surgical scissors
  • Sutures, Vicryl 4.0 (Ethicon) with attached needle
  • Hemostat
  • Recovery tank with lid
  • 1‐ml syringes
  • 18‐G needles bent into triangular loops
  • Disposable transfer pipets (e.g., Fisher)
  • 18°C incubator

Support Protocol 2: Preparation of RNA for Injection

  Materials
  • 5 mM each ATP, CTP, UTP
  • 20 U/µl SP6 or T7 RNA polymerase and 10× buffer
  • DEPC‐treated H 2O ( appendix 2A)
  • 250 to 500 ng/µl DNA template, linearized ( appendix 3A)
  • 1 mM GTP
  • 5 mM m7 GpppG (for capped analogs only)
  • 3000 Ci/mmol [32P]GTP
  • 40 U/µl RNase inhibitor (e.g., RNasin, Promega)
  • 5 M ammonium acetate
  • 20 mg/ml glycogen or seeDNA (Amersham Pharmacia Biotech)
  • 70% and 100% (v/v) ethanol
  • Blue dextran (avg. mol. wt. 2,000 kDa; e.g., Sigma‐Aldrich) or rhodamine B dextran (mol. wt. 70 kDa; e.g., Molecular Probes)
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Figures

Videos

Literature Cited

Literature Cited
   Bataille, N., Helser, T., and Fried, H.M. 1990. Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus oocyte nucleus: A generalized, facilitated process. J. Cell Biol. 111:1571‐1582.
   Bonner, W.M. 1975. Protein migration into nuclei. I. Frog oocyte nuclei in vivo accumulate microinjected histones, allow entry to small proteins, and exclude large proteins. J. Cell Biol. 64:421‐430.
   Feldherr, C.M., Kallenbacj, E., and Schultz, N. 1984. Movement of a karyophilic protein through the nuclear pores of oocytes. J. Cell Biol. 99:2216‐2222.
   Feldherr, C., Akin, D., and Moore, M.S. 1998. The nuclear import factor p10 regulates the functional size of the nuclear pore complex during oogenesis. J. Cell Sci. 111:1889‐1896.
   Fischer, U., Huber, J., Boelens, W.C., Mattaj, I.W., and Luhrmann, R. 1995. The HIV‐1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82:475‐483.
   Gruter, P., Tabernero, C., von Kobbe, C., Schmitt, C., Saavedra, C., Bachi, A., Wilm, M., Felber, B.K., and Izaurralde, E. 1998. TAP, the human homolog of Mex67p, mediates CTE‐dependent RNA export from the nucleus. Mol. Cell 1:649‐659.
   Guddat, U., Bakken, A.H., and Pieler, T. 1990. Protein‐mediated nuclear export of RNA: 5S rRNA containing small RNPs in Xenopus oocytes. Cell 60:619‐628.
   Jarmolowski, A., Boelens, W.C., Izaurralde, E., and Mattaj, I.W. 1994. Nuclear export of different classes of RNA is mediated by a specific factor. J. Cell Biol. 124:627‐635.
   Kutay, U., Lipowsky, G., Izaurralde, E., Bischoff, F.R., Schwarzmaier, P., Hartmann, E., and Gorlich, D. 1998. Identification of a tRNA‐specific nuclear export receptor. Mol. Cell 1:359‐369.
   Lund, E. and Paine, P.L. 1990. Nonaqueous isolation of transcriptionally active nuclei from Xenopus oocytes. Methods Enzymol. 181:36‐43.
   Luo, M.J. and Reed, R. 1999. Splicing is required for rapid and efficient mRNA export in metazoans. Proc. Natl. Acad. Sci. U.S.A. 96:14937‐14942.
   Pokrywka, N.J. and Goldfarb, D.S. 1995. Nuclear export pathways of tRNA and 40S ribosomes include both common and specific intermediates. J. Biol. Chem. 270:3619‐3624.
   Powers, M.A., Forbes, D.J., Dahlberg, J.E., and Lund, E. 1997. The vertebrate GLFG nucleoporin, Nup98, is an essential component of multiple RNA export pathways. J. Cell Biol. 136:241‐250.
   Sive, H.L., Grainger, R.M., and Harland, R.M., eds. 2000. Early Development of Xenopus Laevis: A Laboratory Manual, pp. 283‐284. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Smith, L.D., Xu, W., and Varnold, R.L. 1991. Oogenesis and oocyte maturation. Methods Cell Biol. 36:45‐60.
   Stutz, F., Izaurralde, E., Mattaj, I.W., and Rosbash, M. 1996. A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus. Mol. Cell Biol. 16:7144‐7150.
   Terns, M.P. and Goldfarb, D.S. 1998. Nuclear transport of RNAs in microinjected Xenopus oocytes. Methods Cell Biol. 53:559‐589.
   Ullman, K.S., Shah, S., Powers, M.A., and Forbes, D.J. 1999. The nucleoporin nup153 plays a critical role in multiple types of nuclear export. Mol. Biol. Cell 10:649‐664.
Key References
   Lund and Paine 1990. See above.
  Describes the specific steps and advantages of dissecting oocytes under oil.
   Terns and Goldfarb, 1998. See above.
  Covers many technical aspects of oocyte microinjections, including a detailed description and illustration of suggested equipment.
Internet Resources
   http://oacu.od.nih.gov/ARAC/oocyte.htm
  This site contains Animal Research Advisory Committee guidelines for oocyte harvest from Xenopus laevis.
   http://froglab.biology.utah.edu/Oogenesis/oogenesis.html
  This site contains a summary of the different stages of oocyte maturation.
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