A Screening Protocol for Identification of Functional Mutants of RNA Editing Adenosine Deaminases

Tristan Eifler1, Dalen Chan1, Peter A. Beal1

1 Department of Chemistry, University of California, Davis, Davis, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch120139
Online Posting Date:  December, 2012
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Abstract

Genetic screens can be used to evaluate a spectrum of mutations and thereby infer the function of particular residues within a protein. The Adenosine Deaminase Acting on RNA (ADAR) family of RNA‐editing enzymes selectively deaminate adenosines (A) in double‐helical RNA, generating inosine (I). The protocol described here exploits the editing activity of ADAR2 in a yeast‐based screen by inserting an editing substrate sequence with a stop codon incorporated at the editing site upstream from the sequence encoding the reporter α‐galactosidase. A‐to‐I editing changes the stop codon to a tryptophan codon, allowing normal expression of the reporter. This technique is particularly well‐suited for screening ADAR and ADAR substrate mutant libraries for editing activity. Curr. Protoc. Chem. Biol. 4:357‐369 © 2012 by John Wiley & Sons, Inc.

Keywords: RNA editing; yeast; screen; A‐to‐I editing; ADAR substrate

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Screen for Functional Adenosine‐to‐Inosine Editing in Yeast by α‐Galactosidase Expression
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Screen for Functional Adenosine‐to‐Inosine Editing in Yeast by α‐Galactosidase Expression

  Materials
  • Wild‐type ADAR plasmid, 50 ng/µl
  • Reporter plasmid, 50 ng/µl
  • Milli‐Q water
  • 5× Phusion HF reaction buffer (New England Biolabs, cat. no. M0530S)
  • 10 mM dNTP mix
  • Forward and reverse primers containing randomized nucleotides
  • Phusion high‐fidelity DNA polymerase, 2.0 U/µl (New England Biolabs, cat. no. M0530S)
  • Restriction enzyme DpnI, 20 U/µl
  • Chemically competent E. coli
  • Luria‐Bertani + ampicillin (LB + amp) medium, liquid and agar plate (see recipe)
  • QIAprep Spin Miniprep kit (QIAGEN, cat. no. 27104) containing:
    • P1 buffer
    • P2 buffer
    • N3 buffer
  • INVSc1 S. cerevisiae, or another strain with the appropriate selection marker genotype
  • YPD liquid medium and agar plate (see recipe)
  • YPAD liquid medium (see recipe)
  • Lithium acetate solution (see recipe)
  • Salmon sperm ssDNA, 10 mg/ml
  • 40% PEG solution (see recipe)
  • Complete medium (CM) –ura +glu, liquid and agar plate (see recipe)
  • Complete medium (CM) –ura –W +glu, agar plate (see recipe)
  • Complete medium (CM) –ura –W +gal, agar plate (see recipe)
  • 5‐bromo‐4‐chloro‐3‐indolyl α‐D‐galactopyranoside (X‐α‐gal; see recipe)
  • Acid washed silica or zirconium beads (0.5‐mm diameter)
  • 0.2‐ml PCR reaction tubes
  • Thermal cycler suitable for PCR
  • 1.5‐ml microcentrifuge tubes
  • Cell incubator with shaker
  • Heating block
  • Cell spreader
  • 15‐ml centrifuge tubes
  • Centrifuge
  • Sterile velvet replica plating pad
  • Replica plating apparatus
  • Vortex mixer
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Figures

Videos

Literature Cited

   Bass, B.L. 1997. RNA editing and hypermutation by adenosine deamination. Trends Biochem. Sci. 22:157‐162.
   Eggington, J.M., Greene, T., and Bass, B.L. 2011. Predicting sites of ADAR editing in double‐stranded RNA. Nat. Commun. 2:319.
   Elbing, K. and Brent, R. 2002. Growth on solid media. Curr. Protoc. Mol. Biol. 59:1.3.1‐1.3.6.
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   Gommans, W.M., McCane, J., Nacarelli, G.S., and Maas, S. 2010. A mammalian reporter system for fast and quantitative detection of intracellular A‐to‐I RNA editing levels. Anal. Biochem. 399:230‐236.
   Hundley, H.A. and Bass, B.L. 2010. ADAR editing in double‐stranded UTRs and other noncoding RNA sequences. Trends Biochem. Sci. 35:377‐383.
   Iwamoto, K. and Kato, T. 2003. RNA editing of serotonin 2C receptor in human postmortem brains of major mental disorders. Neurosci. Lett. 346:169‐172.
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   Macbeth, M.R. and Bass, B.L. 2007. Large‐scale overexpression and purification of ADARs from Saccharomyces cerevisiae for biophysical and biochemical studies. Methods Enzymol. 424:319‐331.
   Macbeth, M.R., Schubert, H.L., Vandemark, A.P., Lingam, A.T., Hill, C.P., and Bass, B.L. 2005. Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 309:1534‐1539.
   Maydanovych, O. and Beal, P.A. 2006. Breaking the central dogma by RNA editing. Chem. Rev. 106:3397‐3411.
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   Pokharel, S. and Beal, P.A. 2006. High‐throughput screening for functional adenosine to inosine RNA editing systems. ACS Chem. Biol. 1:761‐765.
   Pokharel, S., Jayalath, P., Maydanovych, O., Goodman, R.A., Wang, S.C., Tantillo, D.J., and Beal, P.A. 2009. Matching active site and substrate structures for an RNA editing reaction. J. Am. Chem. Soc. 131:11882‐11891.
   Roberson, L.M. and Rosenthal, J.J. 2006. An accurate fluorescent assay for quantifying the extent of RNA editing. RNA 12:1907‐1912.
   Rupp, S. 2002. LacZ assays in yeast. Methods Enzymol. 350:112‐131.
   Sanchis, J., Fernandez, L., Carballeira, J.D., Drone, J., Gumulya, Y., Hobenreich, H., Kahakeaw, D., Kille, S., Lohmer, R., Peyralans, J.J., Podtetenieff, J., Prasad, S., Soni, P., Taglieber, A., Wu, S., Zilly, F.E., and Reetz, M.T. 2008. Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: Application to difficult‐to‐amplify templates. Appl. Microbiol. Biotechnol. 81:387‐397.
   Schirle, N.T., Goodman, R.A., Krishnamurthy, M., and Beal, P.A. 2010. Selective inhibition of ADAR2‐catalyzed editing of the serotonin 2c receptor pre‐mRNA by a helix‐threading peptide. Org. Biomol. Chem. 8:4898‐4904.
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   Vissel, B., Royle, G.A., Christie, B.R., Schiffer, H.H., Ghetti, A., Tritto, T., Perez‐Otano, I., Radcliffe, R.A., Seamans, J., Sejnowski, T., Wehner, J.M., Collins, A.C., O'Gorman, S., and Heinemann, S.F. 2001. The role of RNA editing of kainate receptors in synaptic plasticity and seizures. Neuron 29:217‐227.
   Yeo, J., Goodman, R.A., Schirle, N.T., David, S.S., and Beal, P.A. 2010. RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1. Proc. Natl. Acad. Sci. U.S.A. 107:20715‐20719.
Internet Resources
   http://www.microbelibrary.org/component/resource/laboratory‐test/3152‐transformation‐of‐escherichia‐coli‐made‐competent‐by‐calcium‐chloride‐protocol
  American Society for Microbiology Microbe Library. This contains a detailed protocol for the creation and transformation of chemically competent Escherichia coli.
  http://www.yeastgenome.org/
  Saccharomyces Genome Database (SGD). The SGD is a large database of biological information on S. cerevisiae, including a genome browser, lists of yeast strains, additional protocols, and more.
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