In Vivo Translation Studies of Plant Viral RNAs Using Reporter Genes

Daiki Matsuda1, Theo W. Dreher1

1 Oregon State University, Corvallis, Oregon
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16K.2
DOI:  10.1002/9780471729259.mc16k02s00
Online Posting Date:  October, 2005
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Abstract

This unit describes the analysis of the translation‐regulating properties of viral RNAs in cell‐based assays using a luciferase reporter system. Electroporation and polyethylene glycol–mediated introduction of luciferase reporter RNA into cowpea protoplasts are also presented. The Commentary section discusses employing the luciferase reporter system to study translational control by the 5′‐ and 3′‐untranslated regions of a viral mRNA. Inoculum RNA quality and half‐life after delivery to the protoplasts should be monitored closely for careful investigation of translational regulation elements.

Keywords: luciferase; protoplasts; electroporation; polyethylene glycol; mRNA; RNA stability; RNA functional half‐life

     
 
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Table of Contents

  • Basic Protocol 1: Expression of Luciferase from Reporter RNA Transcripts Introduced into Protoplasts by Electroporation
  • Alternate Protocol 1: PEG‐Mediated Protoplast Transfection of Reporter RNA Transcripts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Expression of Luciferase from Reporter RNA Transcripts Introduced into Protoplasts by Electroporation

  Materials
  • 8‐ to 10‐day‐old cowpea (Vigna unguiculata) seedlings, e.g., California Blackeye 46
  • 30% ethanol
  • Protoplast digestive enzyme solution (see recipe)
  • MMC buffer (see recipe)
  • Protoplast electroporation buffer (see recipe)
  • 5 mg/ml fluorescein diacetate in acetone (store indefinitely at –20°C)
  • In vitro–transcribed RNA (2 pmol/10 µl; see ) containing luciferase reporter gene, suspended in H 2O: chill on ice
  • Protoplast growth medium (see recipe)
  • Passive Lysis Buffer (Promega cat. no. E1941)
  • Luciferase assay reagent (see recipe)
  • Bradford dye‐binding protein assay reagent
  • BSA protein standards: dilute BSA in 1× Passive Lysis Buffer to produce stock concentrations of 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/ml
  • Whatman 3MM filter paper
  • Fine‐point tweezers
  • Petri dish, 15 cm diameter
  • Platform rotator
  • 30°C incubator
  • Miracloth (Calbiochem)
  • 50‐ml polypropylene conical tubes
  • Clinical centrifuge
  • Hemacytometer
  • UV/visible fluorescence microscope (unit 2.1)
  • Electroporation cuvettes (2‐mm gap), chilled on ice
  • Electroporation system with capacitance extender (CE) module (Bio‐Rad GenePulser Xcell)
  • Pasteur pipets, autoclaved (one per electroporation)
  • Refrigerated centrifuge
  • Standard 96‐well plate (optional)
  • Clear‐bottom black 96‐well plates (Costar #3631)
  • Microplate luminometer: e.g., Wallac Microbeta 1450 or Turner BioSystems Veritas (http//www.turnerdesigns.com)
  • Microplate spectrophotometer (e.g., Molecular Devices SpectraMax 250)
  • Additional reagents and equipment for electroporation of protoplasts (unit 16.4) and protein assay ( appendix 3A)
NOTE: Avoid exposure to ribonucleases in all procedures. Use gloved hands at all times, including when handling chemical ingredients of solutions, and make all solutions with MilliQ‐grade (Millipore) water. Alternatively, treat solutions with DEPC as described in Appendix 2A.

Alternate Protocol 1: PEG‐Mediated Protoplast Transfection of Reporter RNA Transcripts

  • PEG solution (see recipe)
  • 15‐ml round‐bottom glass or polypropylene tubes
  • Wide‐mouth 1000‐µl pipet tips
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Figures

Videos

Literature Cited

   Bradshaw, R.A., Brickey, W.W., and Walker, K.W. 1998. N‐terminal processing: The methionine aminopeptidase and N alpha‐acetyl transferase families. Trends Biochem. Sci. 23:263‐267.
   Brasier, A.R. and Fortin, J.J. 1995. Nonisotopic assays for reporter gene activity. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 9.7.12‐9.7.21. John Wiley & Sons, Hoboken, N.J.
   Dreher, T.W. 1999. Functions of the 3′‐untranslated regions of positive strand RNA viral genomes. Annu. Rev. Phytopathol. 37:151‐174.
   Fang, P., Spevak, C.C., Wu, C., and Sachs, M.S. 2004. A nascent polypeptide domain that can regulate translation elongation. Proc. Natl. Acad. Sci. U.S.A. 101:4059‐4064.
   Frolov, I. and Schlesinger, S. 1996. Translation of Sindbis virus mRNA: Analysis of sequences downstream of the initiating AUG codon that enhance translation. J. Virol. 70:1182‐1190.
   Gallie, D.R. 1991. The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev. 5:2108‐2116.
   Gallie, D.R. 2002. The 5′‐leader of tobacco mosaic virus promotes translation through enhanced recruitment of eIF4F. Nucleic Acids Res. 30:3401‐3411.
   Gallie, D.R. and Kobayashi, M. 1994. The role of the 3′‐untranslated region of non‐polyadenylated plant viral mRNAs in regulating translational efficiency. Gene 142:159‐165.
   Guo, L., Allen, E., and Miller, W.A. 2000. Structure and function of a cap‐independent translation element that functions in either the 3′ or the 5′ untranslated region. RNA 6:1808‐1820.
   Kozak, M. 1990. Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. Proc. Natl. Acad. Sci. U.S.A. 87:8301‐8305.
   Kozak, M. 2001. New ways of initiating translation in eukaryotes? Mol. Cell. Biol. 21:1899‐1907.
   Matsuda, D., Bauer, L., Tinnesand, K., and Dreher, T.W. 2004. Expression of the two nested overlapping reading frames of TYMV RNA is enhanced by a 5′‐cap and by 5′ and 3′ viral sequences. J. Virol. 78:9325‐9335.
   Qin, X. and Sarnow, P. 2004. Preferential translation of internal ribosome entry site‐containing mRNAs during the mitotic cycle in mammalian cells. J. Biol. Chem. 279:13721‐13728.
   Varshavsky, A. 1996. The N‐end rule: Functions, mysteries, uses. Proc. Natl. Acad. Sci. U.S.A. 93:121420‐121429.
Internet Resources
   http://www.promega.com
  The Promega Inc. Web site is a searchable resource with information on various aspects of the use of LUC as a reporter.
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