Mitochondrial Ribosome (Mitoribosome) Profiling for Monitoring Mitochondrial Translation In Vivo

Mary T. Couvillion1, L. Stirling Churchman1

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.28
DOI:  10.1002/cpmb.41
Online Posting Date:  July, 2017
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Abstract

Translation in the mitochondria is regulated by mechanisms distinct from those acting in the cytosol and in bacteria, yet precise methods for investigating it have lagged behind. This unit describes an approach, mitochondrial ribosome (mitoribosome) profiling, to quantitatively monitor mitochondrial translation with high temporal and spatial resolution in Saccharomyces cerevisiae. Mitoribosomes are immunoprecipitated from whole‐cell lysate and the protected mRNA fragments are isolated. These fragments are then converted to sequencing libraries or analyzed by northern blot hybridization to reveal the distribution of mitoribosomes across the mitochondrial transcriptome. As information about RNA abundance is required to resolve translational from RNA effects, we also present an RNA sequencing approach that can be performed in parallel. Accurately capturing the biologically relevant distribution of mitoribosome positions depends on several critical parameters that are discussed. Application of mitoribosome profiling can reveal mechanisms of mitochondrial translational control that were not previously possible to uncover. © 2017 by John Wiley & Sons, Inc.

Keywords: mitochondria; ribosome profiling; ribosome immunoprecipitation; RNA‐seq; northern blot; translation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolate Mitoribosome‐Protected Fragments by Immunoprecipitation
  • Basic Protocol 2: Construct DNA Sequencing Libraries from Mitoribosome Footprints and/or Fragmented Total mRNA
  • Alternate Protocol 1: Quantify Mitoribosome‐Protected Fragments by Northern Blotting
  • Support Protocol 1: Isolate mRNA for RNA Sequencing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolate Mitoribosome‐Protected Fragments by Immunoprecipitation

  Materials
  • Yeast strain expressing a C‐terminal epitope tagged (3X‐FLAG) MrpS17 grown in medium of choice
  • RNase‐free water
  • Lysis buffer (including protease inhibitors and lauryl maltoside; see recipe)
  • 50 U/μl RNase I (RNase I f; New England Biolabs, cat. no. M0243)
  • 5× SDS‐PAGE sample buffer (see recipe)
  • 20 U/μl SUPERase‐In
  • Agarose slurry conjugated with FLAG antibody (Sigma‐Aldrich)
  • Wash buffer (including Triton X‐100; see recipe)
  • 5 mg/ml 3X‐FLAG peptide in tris‐buffered saline (TBS; aliquot and store at −80°C, avoid freeze‐thaw; Sigma‐Aldrich, cat. no. F4799)
  • 125:24:1 (v/v/v) acid‐phenol/chloroform/isoamyl alcohol, pH 4.5
  • 3 M sodium acetate, pH 5.5 (RNase‐free; )
  • 25 mg/ml linear polyacrylamide (LPA)
  • Isopropanol
  • 70% (v/v) ethanol
  • 10 mM Tris·Cl, pH 7.0 (RNase‐free; )
  • Anti‐FLAG M2 antibody (Sigma‐Aldrich, cat. no. F3165)
  • Liquid nitrogen
  • Microfiltration assembly (90‐mm, ULTRA‐WARE)
    • 4‐liter side‐arm flask with a fritted glass support base
    • Glass funnel
    • Anodized aluminum clamp
    • No. 8 silicone stopper
  • Nitrocellulose membrane (0.45‐μm, 90‐mm diameter membranes; Whatman)
  • Mixer mill, 50‐ml chambers and 25‐mm stainless steel ball (Retsch)
  • 100°C heat block
  • Floor centrifuge with rotor able to reach 20,000 × g (e.g., Sorvall with SS‐34 rotor and Oak Ridge tubes)
  • End‐over‐end rotator
  • Costar Spin‐X centrifuge tube filter (0.45‐μm cellulose acetate in 2‐ml tube; Corning)
  • Spectrophotometer
  • Vacuum source
  • Styrofoam container
  • Forceps
  • 15‐ and 50‐ml conical tubes
  • 20‐G needles
  • Plastic spatula (Cell Lifter, 18 cm; GeneMate, cat. no. T‐2443‐4)
  • Metal spatulas with curved ends
  • Tongs
  • Cryo‐gloves
  • Kimwipes
  • −80°C freezer
  • Refrigerated centrifuge
  • Vortex
CAUTION: Phenol/chloroform is harmful if swallowed or comes in contact with skin, causes severe skin burns and eye damage, is fatal if inhaled, and is potentially carcinogenic. It should be used with appropriate safety measures such as protective gloves, glasses, clothing, and sufficient ventilation. All waste should be handled according to hazardous waste regulations.

Basic Protocol 2: Construct DNA Sequencing Libraries from Mitoribosome Footprints and/or Fragmented Total mRNA

  Materials
  • Mitoribosome‐associated RNA (from protocol 1) and/or fragmented and size‐selected mRNA (from protocol 4Support Protocol)
  • 2× urea RNA loading buffer (see recipe)
  • 1× TBE buffer (diluted from UltraPure 10× stock; Invitrogen, cat. no. 15581044)
  • 10‐bp DNA ladder
  • 15% and 10%TBE‐urea, 8% TBE polyacrylamide gels
  • SYBR gold (10,000× concentrate)
  • RNase‐free water
  • 3 M sodium acetate, pH 5.5 (RNase‐free)
  • 25 mg/ml linear polyacrylamide (LPA)
  • Isopropanol
  • 70% (v/v) ethanol
  • 10× T4 polynucleotide kinase (PNK) buffer
  • 10 U/μl T4 polynucleotide kinase (PNK)
  • Oligonucleotide linker and primers (see Table 4.28.1)
  • 50% (w/v) PEG 8000 (RNase‐free)
  • 10× T4 RNA ligase reaction buffer (New England Biolabs, cat. no. B0216L)
  • 200 U/μl T4 RNA ligase 2 (RNL2), truncated (New England Biolabs)
  • 10 mM Tris⋅Cl, pH 8.0 (RNase‐free; )
  • 5× First‐strand (FS) RT reaction buffer (Invitrogen, cat. no. 18080‐993)
  • 10 mM dNTPs
  • 0.1 M DTT
  • 20 U/μl SUPERase‐In
  • 200 U/μl Superscript III
  • 1 N sodium hydroxide
  • 3 M sodium chloride
  • 10× CircLigase reaction buffer (Epicentre, cat. no. CL4111K)
  • 1 mM ATP
  • 50 mM manganese chloride
  • 100 U/μl CircLigase ssDNA ligase
  • 5× Phusion HF reaction buffer (New England Biolabs, cat. no. M0530S)
  • 2 U/μl Phusion HF DNA polymerase
  • 10× DNA loading buffer (see recipe)
  • 100‐bp DNA ladder
  • DNA gel extraction buffer (see recipe)
  • 10 mM Tris⋅Cl, pH 8.5 ( )
  • Qubit dsDNA HS assay kit
  • Heat blocks: 80°C, 70°C, 37°C, 25°C
  • Typhoon fluorescent/phosphorescent image scanner, or UV transilluminator with camera
  • Blue light transilluminator (or UV transilluminator) box
  • End‐over‐end rotator
  • Costar Spin‐X centrifuge tube filter (0.45‐μm cellulose acetate in 2‐ml tube; Corning)
  • Qubit Fluorometer
  • Bioanalyzer DNA high sensitivity chip and reagents
  • Polyacrylamide electrophoresis apparatus and power source
  • 0.5‐ and 1.5‐ml RNase‐free non‐stick microcentrifuge tubes
  • Plastic sheet protector
  • 20‐G needle
  • Razor blades or scalpels
  • Thermocycler and tubes (PCR tubes)

Alternate Protocol 1: Quantify Mitoribosome‐Protected Fragments by Northern Blotting

  Additional Materials (also see protocol 2)
  • 20% (w/v) SDS (RNase‐free)
  • 20 mg/ml proteinase K
  • 24:1 chloroform/isoamyl alcohol
  • 100% ice‐cold ethanol
  • 10× RQ1 RNase‐free DNase reaction buffer (Promega, cat. no. M6101)
  • 1 U/μl RQ1 RNase‐free DNase (Promega, cat. no. M6101)
  • Ribo‐Zero Gold rRNA Removal Kit for yeast (Illumina, cat. no. MRZY13)
  • 2× alkaline fragmentation buffer (see recipe)
  • Heat blocks: 42°C, 37°C, 70°C, 80°C
  • NanoDrop UV/Vis Spectrophotometer
CAUTION: Chloroform/isoamyl alcohol is harmful if swallowed, causes skin and eye irritation, and is potentially carcinogenic. It should be used with appropriate safety measures such as protective gloves, glasses, clothing, and sufficient ventilation. All waste should be handled according to hazardous waste regulations.
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Figures

Videos

Literature Cited

Literature Cited
   Couvillion, M. T. , Soto, I. C. , Shipkovenska, G. , & Churchman, L. S. (2016). Synchronized mitochondrial and cytosolic translation programs. Nature, 533, 499–503. doi: 10.1038/nature18015.
   Deshpande, A. P. , & Patel, S. S. (2012). Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase. Biochimica et Biophysica Acta, 1819, 930–938. doi: 10.1016/j.bbagrm.2012.02.003.
   Fox, T. D. , Folley, L. S. , Mulero, J. J. , McMullin, T. W. , Thorsness, P. E. , Hedin, L. O. , & Costanzo, M. C. (1991). Analysis and manipulation of yeast mitochondrial genes. In C. V. Kumar (Ed.), Methods in enzymology, Vol. 194, (pp. 149–165). doi: 10.1016/0076‐6879(91)94013‐3. New York: Springer Science+Business Media.
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   Ingolia, N. T. , Brar, G. A. , Rouskin, S. , McGeachy, A. M. , & Weissman, J. S. (2012). The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome‐protected mRNA fragments. Nature Protocols, 7, 1534–1550. doi: 10.1038/nprot.2012.086.
   Ingolia, N. T. , Ghaemmaghami, S. , Newman, J. R. , & Weissman, J. S. (2009). Genome‐wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science, 324, 218–223. doi: 10.1126/science.1168978.
   Kehrein, K. , Bonnefoy, N. , & Ott, M. (2013). Mitochondrial protein synthesis: Efficiency and accuracy. Antioxidants & Redox Signaling, 19, 1928–1939. doi: 10.1089/ars.2012.4896.
   Ni, D. , Xu, P. , Sabanayagam, D. , & Gallagher, S. R. (2016). Protein blotting: Immunoblotting. Current Protocols Essential Laboratory Techniques, 12, 8.3.1–8.3.40. doi: 10.1002/9780470089941.et0803s12.
   Nilsen, T. W. (2012). Selective precipitation of large RNAs. Cold Spring Harbor Protocols, 2012. doi: 10.1101/pdb.prot072322.
   Ott, M. , Amunts, A. , & Brown, A. (2016). Organization and regulation of mitochondrial protein synthesis. Annual Review of Biochemistry, 85, 77–101. doi: 10.1146/annurev‐biochem‐060815‐014334.
   Sherman, F. (2002). Getting started with yeast. In C. Guthrie & G. R. Fink (Eds.), Methods in enzymology, Vol. 350, (pp. 3–41). doi: 10.1016/S0076‐6879(02)50954‐X. New York: Springer Science+Business Media.
   Treco, D. A. , & Winston, F. (2008). Growth and manipulation of yeast. Current Protocols in Molecular Biology, 82, 13.2.1–13.2.12. doi: 10.1002/0471142727.mb1302s82.
   Vignais, P. V. , Stevens, B. J. , Huet, J. , & Andre, J. (1972). Mitoribosomes from Candida utilis. Morphological, physical, and chemical characterization of the monomer form and of its subunits. The Journal of Cell Biology, 54, 468–492. doi: 10.1083/jcb.54.3.468.
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