Analysis of Nonsense‐Mediated mRNA Decay in Mammalian Cells

Pamela Nicholson1, Raphael Joncourt1, Oliver Mühlemann1

1 Department for Chemistry and Biochemistry, University of Bern, Bern, Switzerland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 27.4
DOI:  10.1002/0471143030.cb2704s55
Online Posting Date:  June, 2012
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Abstract

The nonsense‐mediated mRNA decay (NMD) pathway acts to selectively identify and degrade mRNAs that contain a premature translation termination codon (PTC), and hence reduce the accumulation of potentially toxic truncated proteins. NMD is one of the best studied mRNA quality‐control mechanisms in eukaryotes, and it has become clear during recent years that many physiological mRNAs are also NMD substrates, signifying a role for NMD beyond mRNA quality control as a translation‐dependent post‐transcriptional regulator of gene expression. Despite a great deal of scientific research for over twenty years, the process of NMD is far from being fully understood with regard to its physiological relevance to the cell, the molecular mechanisms that underpin this pathway, all of the factors that are involved, and the exact cellular locations of NMD. This unit details some of the fundamental RNA based approaches taken to examine aspects of NMD, such as creating PTC+ reporter genes, knocking down key NMD factors via RNAi, elucidating the important functions of NMD factors by complementation assays or Tethered Function Assays, and measuring RNA levels by reverse‐transcription quantitative PCR. Curr. Protoc. Cell Biol. 55:27.4.1‐27.4.61. © 2012 by John Wiley & Sons, Inc.

Keywords: NMD; site‐directed mutagenesis; RNAi; complementation assay; Tethered Function Assay; RNA; RT‐qPCR

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Creation of PTC+ mRNAs and Mutant NMD Proteins by Site‐Directed Mutagenesis
  • Basic Protocol 2: pSUPuro‐Based RNAi to Knock Down NMD Factors
  • Basic Protocol 3: NMD Complementation Assays
  • Extraction of RNA and Analysis of RNA Levels by Reverse Transcription–Quantitative PCR
  • Basic Protocol 4: Guanidium Thiocyanate–Phenol–Chloroform Extraction of RNA
  • Basic Protocol 5: Synthesis of Mammalian cDNA Using Total Cellular RNA
  • Basic Protocol 6: Quantitative PCR
  • Basic Protocol 7: The Use of Tethered Function Assays to Study NMD
  • Support Protocol 1: Annealing the Oligonucleotides
  • Support Protocol 2: Harvesting Cells for Eventual Protein Analysis
  • Support Protocol 3: Spectrophotometric Quantification of RNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Creation of PTC+ mRNAs and Mutant NMD Proteins by Site‐Directed Mutagenesis

  Materials
  • Mutagenic primers (see Strategic Planning)
  • Template dsDNA of interest (see Strategic Planning)
  • Kits containing PCR reaction components (Table 27.4.2)
  • Necessary controls (see Table 27.4.2)
  • Restriction endonuclease DpnI
  • Ultracompetent cells (e.g., from XL10‐Gold Ultracompetent cells from Agilent Technologies)
  • 2‐mercaptoethanol (optional)
  • 0.1 ng/µl pUC18 transformation control plasmid
  • Luria‐Bertani (LB) liquid medium ( appendix 2A) with and without appropriate antibiotic (usually 100 µg/ml ampicillin or 50 µg/ml kanamycin; see recipe for antibiotic stocks) for the plasmid vector
  • LB agar plates ( appendix 2A) containing 100 µg/ml ampicillin (see recipe for antibiotic stocks), 80 µg/ml Xgal (prepare from 50 mg/ml stock; see recipe), and 0.5 mM IPTG (prepare from 0.1 M stock; see recipe)
  • Plasmid DNA alkaline lysis miniprep kit
  • 0.2‐ml thin‐walled PCR tubes
  • Thermal cycler
  • 42°C water bath
  • 14‐ml polypropylene round‐bottom tubes (e.g., BD Falcon)
  • 37°C shaking incubator
NOTE: In theory, for simple alterations, it is possible to follow the protocol described below (excluding the control reactions) for the QuikChange II kit and provide the following reagents from elsewhere: dNTP mix, high‐fidelity DNA polymerase, and reaction buffer containing Mg2+, DpnI restriction endonuclease, and competent bacteria.

Basic Protocol 2: pSUPuro‐Based RNAi to Knock Down NMD Factors

  Materials
  • Human cervix epithelioid carcinoma cell line (HeLa cells) or human cell line of choice
  • pSUPuro plasmid with inserted oligonucleotides that contain the shRNA‐expressing sequences targeting gene of choice (see Strategic Planning and Fig. )
  • pSUPuro plasmid with no inserted oligonucleotides (pSUPuro‐empty)
  • DMEM+/+ medium (see recipe)
  • DMEM–/– medium (see recipe)
  • DreamFect transfection reagent (OZ Biosciences, http://www.ozbiosciences.com/)
  • DMEM+/– medium (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.05% trypsin/EDTA (e.g., Invitrogen, cat. no. 25300‐062)
  • 0.5 mg/ml puromycin stock solution (see recipe)
  • Hemacytometer
  • 6‐well culture plates
  • 25‐cm2 culture flasks
  • Tissue culture inverted microscope
  • Additional reagents and equipment for basic cell culture techniques including counting cells (unit 1.1), harvesting cells ( protocol 9), protein blotting (unit 6.2), and RT‐qPCR (Basic Protocols protocol 55 and protocol 66)

Basic Protocol 3: NMD Complementation Assays

  Materials
  • pSUPuro empty (pSUPuro‐E)
  • pSUPuro Vector that expresses a short interfering RNA, to knockdown SMG6 gene (pSUPuro‐SMG6, see Fig. C)
  • Empty vector is from Invitrogen (pcDNA3‐E) or pcDNA3 plasmid expressing another NMD factor
  • Plasmid exogenously expressing SMG6 (pcDNA3‐HA‐SMG6)
  • Plasmid exogenously expressing RNAi resistant version of SMG6 gene (pcDNA3‐HA‐SMG6‐RNAiR, see Fig. D)
  • Plasmid exogenously expressing RNAi resistant version of SMG6 gene that has mutations to make it endonucleolytically inactive, as shown in Figure B (pcDNA3‐HA‐SMG6 PIN mut‐RNAiR)
  • Plasmid expressing green fluorescence protein (GFP) to act as a transfection control (p‐EGFP‐C1 BD Biosciences, Clontech)
  • pBeta‐actin‐Ig mini µ wild‐type (Ig mu wt)
  • pBeta‐actin‐Ig mini µ Ter 310 (Ig mu ter 310; see Fig. A)

Basic Protocol 4: Guanidium Thiocyanate–Phenol–Chloroform Extraction of RNA

  Materials
  • Cell samples for extraction of RNA (see protocols above)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • TRI‐mix (see recipe; very toxic)
  • Chloroform (potential narcotic)
  • 20 mg/ml glycogen, molecular biology grade (e.g., Sigma, cat. no. G1767)
  • Isopropanol (2‐propanol)
  • 75% ethanol in DEPC‐treated water
  • RNase‐free water: filtered or DEPC treated as described in appendix 2A
  • Ambion TURBO DNA‐free DNase Treatment and Removal Reagents (Invitrogen Applied Biosystems:
    • 10 × TURBO DNase Buffer
    • TURBO DNase, 2 U/µl
    • DNase inactivation solution and nuclease‐free water
  • Aerosol‐barrier pipet tips
  • Nuclease‐free 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • Thermomixer heat block (e.g., Eppendorf Thermomixer Compact)
  • Additional reagents and equipment for spectrophotometric quantitation of RNA ( protocol 10)

Basic Protocol 5: Synthesis of Mammalian cDNA Using Total Cellular RNA

  Materials
  • 1 µg of total RNA ( protocol 4)
  • RNase‐free water: filtered or DEPC treated as described in appendix 2A
  • 150 ng/µl random hexamers (e.g., Applied Biosystems, cat. no. N8080127)
  • Reagents for RT master mix (Table 27.4.5)
    • 10 × AffinityScript RT buffer (Agilent Technologies)
    • 100 mM DTT
    • 10 mM each dNTP
    • RNasin
    • AffinityScript Multiple Temperature Reverse Transcriptase (Agilent Technologies)
  • Aerosol‐barrier pipet tips
  • Nuclease‐free 1.5‐ml microcentrifuge tubes
  • Thermomixer heat block (e.g., Eppendorf Thermomixer Compact)
    Table 7.4.5   Materials   Components and Amounts for the RT Reaction e   Components and Amounts for the RT Reaction

    Components Amount per reaction Volume per reaction
    AffinityScript buffer (10×) 2.0 µl
    DTT (100 mM) 10 mM 2.0 µl
    dNTPs (10 mM each) 0.4 mM each 0.8 µl
    RNasin 40 U 0.5 µl
    AffinityScript Multi‐Temp Reverse Transcriptase 50 U 1.0 µl
    Total/reaction 6.3 µl

     eDue to the small volumes that are required for each component, it is highly recommended to make a master mix of the above components tailored to the number of RNA samples that need to be reverse transcribed. This will minimize variations between the samples due to pipetting errors.

Basic Protocol 6: Quantitative PCR

  Materials
  • 8 ng/µl cDNA samples from protocol 5
  • TaqMan or SYBR Green assay components (see Strategic Planning and Table 27.4.6):
    • SYBR Green Assay: forward and reverse primers and 2× KAPA SYBR FAST Universal qPCR Master Mix (KAPA Biosystems; http://www.kapabiosystems.com)
    • TaqMan Assay: forward and reverse primers, TaqMan Probe and 2× Brillant II Fast chemistry qPCR master mix (Agilent Technologies)
    • DNase‐free and RNase‐free highly pure deionized water
  • Aerosol‐barrier pipet tips
  • Real‐time PCR machine, accessory machines, and analysis software (we use the Corbett RotorGene 6200 system, robot, and heat sealer; http://corbettlifescience.com/)
    Table 7.4.6   Materials   The Required Components and Volumes for Both TaqMan and SYBR Green Assays f   The Required Components and Volumes for Both TaqMan and SYBR Green Assays

    TaqMan assay Volume per reaction SYBR Green assay Volume per reaction
    qPCR standard water 3.75 µl qPCR standard water 6.20 µl
    2× Brillant II Fast chemistry qPCR master mix (Agilent Technologies) 7.50 µl 2× KAPA SYBR FAST Universal qPCR Master Mix (KAPA Biosystems) 10.00 µl
    20× TaqMan probe/primer mix (made by mixing 16 µM forward primer, 16 µM reverse primer, and 4 µM TaqMan probe. 0.75 µl forward primer 10 µM reverse primer 10 µM 0.40 µl 0.40 µl
    8 ng/µl cDNA sample 3.00 µl 8 ng/µl cDNA sample 3.00 µl
    Total 15.00 µl 20.00 µl

     fImportantly, these PCR master mixes contain nucleoside triphosphates, dye, buffer, magnesium, and a DNA polymerase. Importantly, the DNA polymerase must be heat‐stable, robust, and efficient, and in the case of TaqMan assays, it must also contain 5′‐3′ exonuclease activity. Such master mixes are recommended for qPCR assays, as they are optimized and consistent in composition. Avoid multiple freeze‐thaw cycles with these master mixes. The 2× master mixes, primers, and probes and water volumes should be assembled as a master mix tailored to the number of reactions that are to be carried out in total; this reduces inaccuracies due to pipetting very small volumes. Quantitative PCR standard water means highly pure deionized water that is nuclease‐free.

Basic Protocol 7: The Use of Tethered Function Assays to Study NMD

  Materials
  • Forward and reverse oligonucleotides
  • Annealing solution (see recipe)
  • Thermomixer heat block (e.g., Eppendorf Thermomixer Compact)

Support Protocol 1: Annealing the Oligonucleotides

  Materials
  • Cells (any particular kind)
  • Modified radio immunoprecipitation assay (RIPA) buffer (see recipe), ice cold
  • 2× SDS‐PAGE sample buffer ( appendix 2A)
  • Refrigerated shaker
  • Refrigerated centrifuge
  • Additional reagents and equipment for counting cells

Support Protocol 2: Harvesting Cells for Eventual Protein Analysis

  Materials
  • RNA samples to be quantified
  • Solution used to dissolve RNA pellets in protocol 4
  • NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific)
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Figures

Videos

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Internet Resources
  http://www.geneinfinity.org/sp/sp_oligo.html
  For designing all types of primers:
  http://www.bioinformatics.org/primerx/
  For creating mutagenic primers:
  http://www.my‐whiteboard.com/the‐human‐codon‐usage‐table/
  Codon usage table:
  http://www.dharmacon.com/designcenter/designcenterpage.aspx
  To design shRNA oligonucleotides:
  http://www.eu.idtdna.com/Scitools/Applications/shRNA/default.aspx
  For designing qPCR primers/probes:
  http://www.oligoengine.com
  To BLAST designed primers/probes against databases to check their specificity:
  http://www.eu.idtdna.com/scitools/Applications/RealTimePCR/
  A Web site that offers software products and services that are desirable for aiding with qPCR primer/probe design (as well as for many other techniques such as mutagenesis, cloning, PCR, etc.):
  http://www.primer3.sourceforge.net
  Software's to help find the best normalizer for your experiment:
  http://www.ncbi.nlm.nih.gov/tools/primer‐blast/
  http://www.PremierBiosoft.com
  geNorm software (Vandesompele et al., )
  BestKeeper software (Pfaffl et al., )
  Norm‐Finder software (Andersen et al., )
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