Analysis of Nonsense‐Mediated mRNA Decay in Saccharomyces cerevisiae

Bessie W. Kebaara1, Kristian E. Baker2, Krista D. Patefield3, Audrey L. Atkin3

1 Department of Biology, Baylor University, Waco, Texas, 2 Center for RNA Molecular Biology, Case Western Reserve University, School of Medicine, Cleveland, Ohio, 3 School of Biological Sciences, University of Nebraska‐Lincoln, Lincoln, Nebraska
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 27.3
DOI:  10.1002/0471143030.cb2703s54
Online Posting Date:  March, 2012
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Abstract

Nonsense‐mediated mRNA decay is a highly conserved pathway that degrades mRNAs with premature termination codons. These mRNAs include mRNAs transcribed from nonsense or frameshift alleles as well as wild‐type mRNA with signals that direct ribosomes to terminate prematurely. This unit describes techniques to monitor steady‐state mRNA levels, decay rates, and structural features of mRNAs targeted by this pathway, as well as in vivo analysis of nonsense suppression and allosuppression in the yeast Saccharomyces cerevisiae. Protocols for the structural features of mRNA include analysis of cap status, 5′ and 3′ untranslated region (UTR) lengths, and poly(A) tail length. Curr. Protoc. Cell Biol. 54:27.3.1‐27.3.39. © 2012 by John Wiley & Sons, Inc.

Keywords: steady‐state mRNA level; mRNA decay rate; untranslated region length; 5′‐cap; poly(A) tail length; nonsense suppression; allosuppression

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Total Yeast RNA
  • Basic Protocol 2: Northern Blotting of RNA
  • Alternate Protocol 1: Riboprobe Synthesis and Hybridization
  • Support Protocol 1: Stripping Radiolabeled Probe from a Blot
  • Support Protocol 2: Removal of Radiolabeled DNA Probes from Northern Blots by Boiling
  • Basic Protocol 3: Measurement of mRNA Half‐Life Using rpb1‐1
  • Alternate Protocol 2: Measurement of mRNA Half‐Lives Using Thiolutin
  • Basic Protocol 4: Rapid Amplification of cDNA Ends (RACE)
  • Basic Protocol 5: Splinted Ligation‐Mediated RT‐PCR to Detect mRNA Lacking a 5′‐m7Gpp Cap
  • Basic Protocol 6: Analysis of mRNA Poly(A) Tail Length
  • Basic Protocol 7: Suppression Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Total Yeast RNA

  Materials
  • Isogenic nmd and NMD+ yeast strains
  • Yeast growth medium (selective or rich medium)
  • DEPC‐treated water
  • Dry ice/ethanol bath
  • TES solution (see recipe)
  • 5:1 (v/v) acid phenol/chloroform, pH 4.5 (Applied Biosystems/Ambion), 65°C
  • 1:1 UltraPure TE‐buffered saturated phenol (Invitrogen)/chloroform, 37°C
  • Chloroform
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • Absolute and 70% ethanol, ice cold
  • 18 × 150‐mm culture tubes
  • Spectrophotometer
  • 45‐ml screw‐capped centrifuge tubes
  • High‐speed centrifuge
  • RNase‐free 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • RNase‐free pipet tips
  • Vortex
  • 65°C water bath
  • 30° and 37°C incubators

Basic Protocol 2: Northern Blotting of RNA

  Materials
  • Agarose
  • DEPC‐treated water
  • 10× MOPS gel running buffer (see recipe)
  • Formaldehyde
  • RNA samples of known concentration (see protocol 1)
  • RNA ladder (Invitrogen Life Technologies)
  • Formaldehyde loading dye (Ambion, cat. no. 8552)
  • GeneScreen Plus membrane (PerkinElmer)
  • NorthernMax transfer buffer (Ambion, cat. no. 8672)
  • 10 mg/ml ethidium bromide
  • 2× SSC (see recipe)
  • 25 ng probe DNA (in water or TE)
  • TE, pH 8.0 ( appendix 2A)
  • RadPrime DNA labeling system (Invitrogen, cat. no. 18428‐011) containing:
    • 500 mM dATP
    • 500 mM dGTP
    • 500 mM dTTP
    • 2.5× random primers solution
    • Klenow enzyme
    • Stop buffer
  • [α‐32 P]dCTP (3000 Ci/mmol, 10 mCi/ml)
  • Sephadex G‐50 (see recipe)
  • Scintillation cocktail
  • NorthernMax prehybridization/hybridization buffer (Ambion, cat. no. 8677)
  • 2× SSPE/2% SDS wash solution, pre‐warmed to 65°C
  • 2× SSPE wash solution (see recipe for 20×)
  • 500‐ml Erlenmeyer flasks
  • Microwave oven
  • 37°, 42°, and 60°C water baths
  • Horizontal gel electrophoresis apparatus
  • Recirculating pump
  • Razor blades
  • Whatman 3 MM filter paper
  • Paper towels
  • Glass or plastic flat‐bottomed trays or containers with low side walls
  • RNase‐free glass plate
  • Gel photographic equipment and fluorescent ruler
  • Forceps
  • 80°C vacuum oven
  • Plastic wrap
  • 37°C heating block
  • 1‐ml disposable syringes, without needle
  • Glass wool
  • 17 × 100–mm culture test tubes with snap caps (or equivalent 15‐ml plastic test tubes)
  • 2‐ml screw‐capped microcentrifuge tubes
  • 1.5‐ml snap‐cap microcentrifuge tubes
  • Scintillation vials
  • Scintillation counter
  • Hybridization bottles
  • 42°C hybridization oven
  • Phosphorscreen and cassette
  • Phosphorimager and quantification software
  • Phosphorscreen eraser
  • Autoradiography film and cassette
  • Film developer

Alternate Protocol 1: Riboprobe Synthesis and Hybridization

  • Linear DNA template from linearized plasmid DNA or PCR products that contain a T7 RNA polymerase promoter upstream of DNA fragment cloned in the antisense direction with respect to the promoter (plasmid templates should be linearized using a restriction site on the amino‐terminal (5′) side of the coding region of the protein)
  • T7 RNA polymerase
  • 10× T7 RNA polymerase transcription buffer (see recipe)
  • 10 mM ATP
  • 10 mM CTP
  • 10 mM GTP
  • 800 Ci/mmol [α‐32P]UTP (10 mCi/ml)
  • RNase‐free DNase I
  • 0.5 M EDTA ( appendix 2A)
  • 0.1× SSC/0.1% SDS
  • 1.5‐ml RNase‐free microcentrifuge tubes

Support Protocol 1: Stripping Radiolabeled Probe from a Blot

  Materials
  • 0.01% SDS/0.01× SSC stripping solution
  • Northern hybridization membrane with bound probe (see protocol 2)
  • 2‐liter beaker containing stir bar
  • Magnetic hot plate
  • Plastic wrap
  • Clean glass tray
  • Shaking platform
  • Hand‐held radiation monitor

Support Protocol 2: Removal of Radiolabeled DNA Probes from Northern Blots by Boiling

  Materials
  • Northern hybridization membrane with bound probe (see protocol 2)
  • 0.1× SSC/1.0% SDS
  • 2‐liter beaker
  • Hot plate
  • Plastic wrap

Basic Protocol 3: Measurement of mRNA Half‐Life Using rpb1‐1

  Materials
  • Isogenic nmd and NMD+ yeast strains in rpb1‐1 background (grown at 28°C)
  • Selective or rich culture medium, 60°C
  • Dry ice/ethanol bath
  • Spectrophotometer
  • Sterile 250‐ml screw‐cap centrifuge bottles
  • High‐speed centrifuge
  • Sterile 250‐ml flask
  • 39° and 60°C water baths
  • RNase‐free 10‐ml pipets
  • RNase‐free 1.5‐ml microcentrifuge tubes (pre‐labeled with time points of yeast cell harvest)
  • Microcentrifuge (with rapid deceleration, e.g., Tomy Capsule microcentrifuge)
  • Timer
  • −70°C freezer

Alternate Protocol 2: Measurement of mRNA Half‐Lives Using Thiolutin

  • Isogenic nmd and NMD+ yeast strains
  • Culture medium
  • 2 mg/ml thiolutin (Pfizer or Sigma‐Aldrich) in DMSO

Basic Protocol 4: Rapid Amplification of cDNA Ends (RACE)

  Materials
  • 1 to 5 µg total yeast RNA (see protocol 1)
  • Sterile DEPC‐treated distilled water
  • Gene‐specific primer (GSP)
  • 5′ RACE system for rapid amplification of cDNA ends (Invitrogen) containing:
    • 10× PCR buffer
    • 25 mM MgCl 2
    • 10 mM dNTPs
    • 0.1 M DTT
    • Superscript II RT
    • RNase mix
    • SNAP column DNA purification system
    • 5× tailing buffer
    • 2 mM dCTP
    • TdT
    • Abridged anchor primer
    • Abridged universal amplification primer (AUAP)
    • Universal amplification primer
  • Gene‐specific primer 2 (GSP2)
  • Taq DNA polymerase (Invitrogen)
  • Agarose gels
  • DNA markers
  • Nested gene‐specific primer
  • 3′ RACE system for rapid amplification of cDNA ends (Invitrogen) containing:
    • Adapter primer (AP)
    • 10× PCR buffer
    • 25 mM MgCl 2
    • 10 mM dNTPs
    • 0.1 M DTT
    • Superscript II RT
    • RNase H
    • Abridged universal amplification primer (AUAP)
    • Universal amplification primer (AUP)
  • RNase‐free, sterile 0.5‐ or 0.2‐ml PCR tubes
  • 37°, 42°, 70°, and 94°C water baths
  • Microcentrifuge
  • Timer
  • PCR tubes
  • Thermal cycler
  • Gel apparatus

Basic Protocol 5: Splinted Ligation‐Mediated RT‐PCR to Detect mRNA Lacking a 5′‐m7Gpp Cap

  Materials
  • 15 µg total yeast RNA sample resuspended in DEPC‐treated water (see protocol 1)
  • DNA oligonucleotide splint (DNA oligonucleotide complementary to both the 5′ end of an mRNA lacking its m7Gpp cap and the RNA oligonucleotide adaptor; see Fig. ) resuspended in DEPC‐treated water
  • RNA oligonucleotide adaptor (RNA oligonucleotide complementary to the 3′ end of the DNA splint; oligonucleotide should terminate in hydroxyl groups on both the 5′ and 3′ ends; see Fig. ) resuspended in DEPC‐treated water
  • 500 mM KCl ( appendix 2A)
  • 1 mM EDTA ( appendix 2A)
  • DEPC‐treated water
  • 10× T4 DNA ligase buffer (Roche)
  • RNase inhibitor (RNasin; Promega)
  • T4 DNA ligase (Roche)
  • 50 mM CaCl 2 ( appendix 2A)
  • DNase I (Roche)
  • LET buffer (see recipe)
  • LET‐buffered phenol/chloroform (1:1)
  • 7.5 M ammonium acetate ( appendix 2A)
  • Glycogen coprecipitant (e.g., glycoblue; Ambion)
  • 70% and 95% ethanol in sterile DEPC‐treated water
  • 5× first strand synthesis buffer (Invitrogen)
  • Gene‐specific DNA oligonucleotide primer (see Fig. )
  • 100 mM DTT ( appendix 2A)
  • 10 mM deoxynucleotide mix (10 mM each of dATP, dCTP, dGTP, and dTTP in DEPC‐treated water)
  • Superscript II reverse transcriptase (Invitrogen)
  • Forward PCR primer (complementary to RNA oligonucleotide adaptor; see Fig. ) resuspended in DEPC‐treated water
  • 8% polyacrylamide gel
  • Ethidium bromide
  • 0.2‐ and 1.5‐ml sterile, RNase‐free microcentrifuge tubes
  • RNase‐free pipet tips and micropipettors
  • 30°, 37°, 42°, 56°, and 65°C heating blocks
  • Microcentrifuge
  • Vortex
  • Thermal cycler
  • Vertical electrophoresis unit
  • Additional reagents and equipment for PCR ( appendix 3F)

Basic Protocol 6: Analysis of mRNA Poly(A) Tail Length

  Materials
  • 20 to 40 µg total yeast RNA sample resuspended in DEPC‐treated water (see protocol 2)
  • Deoxythymidylic acid oligonucleotide 20 residues in length [oligo(dT) 20] (Life Technologies)
  • RNA‐oligo hybridization buffer (see recipe)
  • RNAse H cleavage buffer (see recipe)
  • RNase H (Promega)
  • RNase H reaction stop mix (see recipe)
  • 70% and 95% ethanol in sterile DEPC‐treated water
  • Formamide loading dye (see recipe)
  • 6% to 8% denaturing polyacrylamide gel
  • Sterile, RNase‐free pipet tips and 1.5‐ml microcentrifuge tubes
  • SpeedVac sample concentrator
  • 30°, 48°, 65°, and 100°C heating blocks
  • Microcentrifuge
  • Micropipettors
  • Gel apparatus

Basic Protocol 7: Suppression Assay

  Materials
  • Isogenic nmd and NMD+ yeast strains (any yeast strain with a mutant upf1, upf2, or upf3 allele and a suppressible allele; Table 27.3.1; and the isogenic UPF1, UPF2, and UPF3 control strain)
  • Liquid yeast growth medium (see Commentary)
  • Sterile water
  • Control plates: YPD or synthetic complete plates, prewarmed to room temperature
  • Suppression phenotype tester plates (see Table 27.3.1), prewarmed to room temperature
  • Sterile microtiter plates with lids, optional
  • Multichannel pipettor with sterile pipet tips
  • Spectrophotometer (that can read culture in test tubes)
  • Incubators
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Figures

Videos

Literature Cited

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