Purification of Translating Ribosomes and Associated mRNAs from Soybean (Glycine max)

Norma A. Castro‐Guerrero1, Yaya Cui1, David G. Mendoza‐Cozatl1

1 Division of Plant Sciences, C.S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20011
Online Posting Date:  May, 2016
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Cell identity and function are largely determined by specific gene expression patterns and ultimately by the proteome. Current high‐throughput sequencing technologies offer the possibility of quantifying gene expression at high resolution, with minimum input and without the constraints of array‐based systems, such as the need for specific probes. In addition, techniques are now available to capture genes that are actively being translated. These techniques use either density gradients or epitope‐based immunoprecipitation to purify translating ribosomes and associated mRNAs (i.e., translatomes). More recently, the combination of tissue‐specific promoters driving epitope‐tagged ribosomes with high‐throughput sequencing has allowed the identification of genes and networks unique to specific cell types. Translatome analyses have the potential to unravel genetic programs and cellular responses to environmental stresses at cell‐specific resolution. This unit describes steps for the use of epitope‐based immunoprecipitation to purify translating ribosomes from soybean and the recovery of mRNA for downstream applications such as gene expression analysis. © 2016 by John Wiley & Sons, Inc.

Keywords: monosomes; polysomes; Ribo‐Seq; ribosome immunoprecipitation; TRAP; translatome

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Immunopurification of Translating Ribosomes
  • Alternate Protocol 1: Immunopurification of FLAG‐Tagged Ribosomes Using Anti‐FLAG Magnetic Beads
  • Alternate Protocol 2: RNA Recovery Using Chaotropic Agents
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Immunopurification of Translating Ribosomes

  • Frozen plant tissue
  • Liquid nitrogen
  • Extraction buffer (see recipe), ice cold
  • EZ‐View anti‐FLAG agarose beads (50% slurry, Sigma‐Aldrich, cat. no. F2426‐1ML)
  • Wash buffer (see recipe), ice cold
  • Elution buffer (see recipe)
  • Trizol (ThermoFisher, cat. no. 15596018)
  • Chloroform
  • Ethanol, molecular biology grade
  • Mortar and pestle (per sample)
  • 1.5‐ml microcentrifuge tubes
  • 15‐ and 50‐ml polypropylene tubes
  • Standard rocking shaker (Labquake or similar)
  • 50‐ml round‐bottom polycarbonate centrifuge tubes
  • Sterile cheesecloth or 25‐ml syringe with fiberglass
  • Column‐based RNA isolation kit (Qiagen RNeasy Mini Kit or similar)
  • Nanodrop or other spectrophotometer
NOTE: Until elution from FLAG‐beads, all solutions, glassware, centrifuge tubes, and equipment should be pre‐cooled to 4°C. Samples should be kept on ice and incubations performed at 4°C. All solutions and reagents should be maintained free of RNases.

Alternate Protocol 1: Immunopurification of FLAG‐Tagged Ribosomes Using Anti‐FLAG Magnetic Beads

  • 8 M guanidine hydrochloride
  • Ethanol, molecular biology grade
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Literature Cited

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Key Reference
  Zanetti et al., 2005. See above.
  This manuscript makes a thorough comparison between polysome purification by density gradients and tag‐based immunoprecipitation protocols in Arabidopsis. In addition, use of different FLAG‐tagged ribosomal subunits demonstrates that RPL18 is the ideal subunit for use in TRAP techniques. Any researcher interested in TRAP should read this manuscript to understand the rationale and biochemistry behind the development of this technique.
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