The Mitoribosome: Methods and Protocols

Preface
Contents
Contributors
Part I: General Concepts
Chapter 1: Discovery of Mitochondrial Ribosomes
References
Chapter 2: Evolution: Mitochondrial Ribosomes Across Species
1 Introduction
2 Evolution of the Mitochondrial Ribosome
2.1 Mitoribosomal RNAs
2.2 Mitoribosomal Proteins
2.3 Species-Specific Structural Differences
3 Structural and Functional Complementation Between the Mitoribosome and Its Binding Ligands
4 Concluding Remarks
References
Chapter 3: Mitoribosome Biogenesis
1 Introduction
2 Mitoribosome Structural Features
3 Mitoribosome Assembly Pathway and Factors Involved
3.1 General Concepts
3.2 Methods to Study Mitoribosome Biogenesis
3.2.1 Pulse-Chase SILAC
3.2.2 Screens for Mitochondrial RNA-Binding and Mitoribosome-Interacting Proteins
3.2.3 Screens for Mutations in Ribosomal Components and Assembly Factors in Patients Suffering from Mitochondrial Disorders As...
3.2.4 Determination of Cryo-EM Structures of Stable Assembly Intermediates
3.3 Cotranscriptional Mitoribosome Assembly
3.4 mtSSU Assembly
3.4.1 Early and Intermediate Stages of mtSSU Assembly
3.4.2 Late Stages of mtSSU Assembly
3.5 mtLSU Assembly
3.5.1 Early and Intermediate Stages of mtLSU Assembly
3.5.2 Late Stages of mtLSU Assembly
4 Conclusions and Perspectives
References
Chapter 4: Translation in Mitochondrial Ribosomes
1 Introduction
2 How Are Mitochondrial Transcripts Prepared for Translation?
3 Critical Factors and Mechanisms of Translation Initiation
4 Translational Elongation
5 How Is Mitochondrial Translation Termination Effected?
6 How Are Mitoribosomes Recycled?
7 Final Comments
References
Part II: Methods to Study Mitoribosome Structure, Function, and Biogenesis
Chapter 5: Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation
1 Introduction
2 Materials
2.1 Isolation of Mitochondria from HEK Suspension Cells
2.2 Isolation of Mitochondria from Yeast Cells
2.3 Grid Preparation
2.4 Image Analysis and Visualization Software
3 Methods
3.1 Mitochondria Isolation Human
3.2 Mitochondria Isolation Yeast
3.3 Grid Preparation
3.4 Tomogram Segmentation
3.4.1 Tomogram Segmentation
3.4.2 Mapping Particles in the Segmentation
4 Notes
References
Chapter 6: Cryo-EM for Structure Determination of Mitochondrial Ribosome Samples
1 Introduction
2 Materials
2.1 For Sample Preparation and Cryo-EM Imaging
2.2 For Image Processing and 3D Reconstruction
2.2.1 Computer Hardware Requirements
2.2.2 Computer Software Requirements
2.3 Example Data
3 Methods
3.1 Cryo-EM Sample Preparation and Data Collection
3.2 Image Preprocessing and Particle Extraction
3.3 Image Processing, Classification, and Reconstruction
3.4 Model Building and Refinement
4 Notes
References
Chapter 7: Sucrose Gradient Analysis of Human Mitochondrial Ribosomes and RNA
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cell Lysis Buffer for Mitochondrial Ribosome Isolation
2.3 Preparation of 10-30% Linear Sucrose Gradient
2.4 RNA Isolation from Sucrose Gradients
2.5 Bioanalyzer Analysis of Purified RNA to Assess Quality
2.6 Northern Blotting
2.6.1 Preparation of RNA Sample
2.6.2 Preparation of Denaturing Gel
2.6.3 Membrane Transfer
2.6.4 5′ End Labeling of Oligonucleotide with 32P
2.6.5 Oligonucleotide Hybridization
2.7 Next-Generation RNA Sequencing
2.7.1 RNA Library Preparation with InGex TGIRT-III Reverse Transcriptase
2.8 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
3 Methods
3.1 Cell Culture
3.2 Preparation of 10-30% Linear Sucrose Gradient
3.3 Cell Lysis
3.4 Sucrose Gradient Sedimentation
3.5 RNA Isolation
3.6 Bioanalyzer Analysis of Purified RNA
3.7 Northern Blotting
3.7.1 Preparation of Denaturing Gel
3.7.2 Preparation and Separation of RNA
3.7.3 RNA Transfer onto a Hybridization Membrane
3.7.4 5′ End Labeling of Oligonucleotide Probes
3.7.5 Hybridization
3.8 Library Preparation for RNA Sequencing of the Entire Mitochondrial Transcriptome on the Illumina Platform
3.9 Protein Analysis of Sucrose Gradient Fractions
3.9.1 TCA Precipitation of Proteins from Collected Fractions
3.9.2 SDS-PAGE and Immunoblotting
4 Notes
References
Chapter 8: Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation
1 Introduction
2 Materials
2.1 FLAG Affinity Purification of Mitoribosomes
2.2 Sucrose Cushions for Purification and/or Analysis of Mitoribosomes
2.3 Linear Sucrose Gradients for the Biochemical Analysis of Mitoribosomes
3 Methods
3.1 FLAG Affinity Purification of Mitoribosomes
3.1.1 Yeast Cell Culturing and Freezing of ``Popcorn´´
3.1.2 Cell Lysis
3.1.3 Immunoprecipitation of FLAG-Tagged Mitoribosomes
3.1.4 TCA Precipitation, SDS-PAGE, and Immunoblot Analysis
3.2 Sucrose Cushions for Purification and/or Analysis of Mitoribosomes
3.3 Linear Sucrose Gradients for the Biochemical Analysis of Mitoribosomes
3.3.1 Lysis of Mitochondria and Sucrose Gradient
3.3.2 Downstream Analysis: Preparation for Immunoblotting
3.3.3 Downstream Analysis: Preparation for Blue Native PAGE
4 Notes
References
Chapter 9: Rapid Cryopurification of the Yeast Mitochondrial Ribosome
1 Introduction
2 Materials
2.1 Preparing Frozen Lysis Buffer
2.2 Yeast Cell Culture and Collection
2.3 Cryogenic Cell Lysis Using Mixer Mill (Cryomilling)
2.4 Immunoprecipitate Mitoribosome
3 Methods
3.1 Preparing Frozen Lysis Buffer
3.2 Yeast Cell Culture and Collection
3.3 Cryogenic Cell Lysis Using Mixer Mill (Cryomilling)
3.4 Immunoprecipitate Mitoribosome
4 Notes
References
Chapter 10: Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast
1 Introduction
1.1 Protein Import Routes into Mitochondria
1.2 Import of Mitoribosomal Proteins
2 Materials
2.1 Isolation of Mitochondria from Yeast Cells
2.2 Import of Mitoribosomal Proteins into Isolated Mitochondria
2.3 Immunoprecipitation of MRPs and Sample Preparation for Mass Spectrometry
3 Methods
3.1 Isolation of Mitochondria from S. cerevisiae
3.2 Import of Mitoribosomal Proteins into Isolated Mitochondria
3.3 Immunoprecipitation of MRPs and Sample Preparation for Mass Spectrometry
4 Notes
References
Chapter 11: Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway
1 Introduction
2 Materials
2.1 Generation of a Collection of Yeast Strains KO for Genes Coding for Mitoribosome Proteins and Assembly Factors
2.1.1 Amplification of Gene-Targeted KanMX4 Deletion Cassettes
2.1.2 Yeast Transformation
2.1.3 Genomic DNA Extraction and Validation of KanMX4 Cassette Integration
2.2 Sucrose Gradient Analysis to Establish the Mitoribosome Profile and Detect Assembly Intermediates
2.2.1 Growth of Yeast Cells
2.2.2 Isolation of Mitochondria
2.2.3 Preparation of a 10-30% Linear Sucrose Gradient
2.2.4 Mitoribosome Extraction and Sedimentation Analysis
2.2.5 SDS-PAGE and Immunoblot Analysis of Sedimented Mitoribosomal Particles
2.3 Mass Spectrometry Analysis of Assembly Intermediates Compositions and Clustering Analysis
2.3.1 Methanol/Chloroform Protein Precipitation of Sucrose Gradient Fractions Containing Assembly Intermediates and Mitoriboso...
2.3.2 Clustering Analysis of Mass Spectrometry Data to Assign Hierarchical Assembly Clusters
3 Methods
3.1 Generation of a Collection of Yeast Strains KO for Genes Coding for Mitoribosome Proteins and Assembly Factors
3.1.1 Amplification of Gene-Targeted KanMX4 Deletion Cassettes
3.1.2 Yeast Transformation
3.1.3 Genomic DNA Extraction and Validation of KanMX4 Cassette Integration
3.2 Sucrose Gradient Analysis to Establish the Mitoribosome Profile and Detect Assembly Intermediates
3.2.1 Culture of Yeast Cells
3.2.2 Isolation of Mitochondria
3.2.3 Preparation of a 10-30% Linear Sucrose Gradient
3.2.4 Mitoribosome Extraction and Sedimentation Analysis
3.2.5 SDS-PAGE and Immunoblot Analysis of Sedimented Mitoribosomal Particles
3.3 Mass Spectrometry Analysis of Assembly Intermediates Compositions and Clustering Analysis
3.3.1 Methanol/Chloroform Protein Precipitation of Sucrose Gradient Fractions Containing Assembly Intermediates and Mitoriboso...
3.3.2 Quantitative Mass Spectrometry Analysis of Assembly Intermediates and Data Clustering Analysis to Determine Hierarchical...
4 Notes
References
Chapter 12: Metabolic Labeling of Mitochondrial Translation Products in Whole Cells and Isolated Organelles
1 Introduction
2 Materials
2.1 Labeling of Mitochondrion-Encoded Proteins in Yeast Cells
2.2 Labeling of Newly Synthesized Proteins in Isolated Yeast Mitochondria
2.2.1 Mitochondria Isolation
2.2.2 In Organello Translation
2.3 Labeling of Mitochondrion-Encoded Proteins in Mammalian Cell Cultures
2.4 Labeling of Newly Synthesized Proteins in Isolated Mammalian Mitochondria
2.4.1 Mitochondria Isolation
2.4.2 In Organello Translation
2.5 Protein Separation and Detection by Electrophoresis, Electrotransfer, and Autoradiography
3 Methods
3.1 Labeling of Mitochondrion-Encoded Proteins in Yeast Cells
3.2 Labeling of Newly Synthesized Proteins in Isolated Yeast Mitochondria
3.2.1 Mitochondria Isolation
3.2.2 In Organello Translation
3.3 Labeling of Mitochondrion-Encoded Proteins in Mammalian Cell Cultures
3.4 Labeling of Newly Synthesized Proteins in Isolated Mammalian Mitochondria
3.4.1 Mitochondria Isolation
3.4.2 In Organello Translation
3.5 Protein Separation and Detection by Electrophoresis, Electrotransfer, and Autoradiography
4 Notes
References
Chapter 13: Assembly of the Mitochondrial Translation Initiation Complex
1 Introduction
2 Materials
2.1 Purification of 55S Ribosomes
2.1.1 Purification of Mitochondria from HEK Cells
2.1.2 Purification of Crude Mitoribosomes
2.2 Double Labelling of Mitoribosomes
2.2.1 Fluorescent Labelling of Mitoribosomes
2.2.2 Dissociation of Labelled Mitoribosomes
2.2.3 Reassociation of Ribosomal Subunits Labelled with Different Fluorophores
2.3 Preparation of E. coli fMet-tRNAfMet
2.3.1 Preparation of Immobilized 3′-Biotinylated Oligonucleotide Matrix
2.3.2 Preparation of Immobilized 3′-Amine Oligonucleotide Matrix
2.3.3 Isolation of E. coli fMet-tRNAfMet from Bulk E. coli tRNA
2.3.4 Isolation of Bulk E. coli Synthetase
2.3.5 Charging and Formylation of E. coli tRNAfMet
2.4 Overexpression and Purification of Mitochondrial Translation Initiation Factors
2.5 Assembly of the Mitochondrial Translation Initiation Complex
3 Methods
3.1 Purification of Mitoribosomes from HEK Cells
3.1.1 Purification of Mitochondria from HEK Cells
3.1.2 Sucrose Gradient Purification of 55S Monosomes
3.2 Double Labelling of Mitoribosomes
3.2.1 Labelling of the Full Monosome
3.2.2 Dissociation of the Labelled Monosome into Subunits
3.2.3 Reassociation of Small and Large Subunits Labelled with Different Fluorophores
3.3 Preparation of E. coli fMet-tRNAfMet
3.3.1 Preparation of Immobilized 3′-Biotinylated Oligonucleotide Matrix
3.3.2 Preparation of Immobilized 3´-Amine Oligonucleotide Matrix (See Note 12)
3.3.3 Isolation of E. coli tRNAfMet from Bulk E. coli tRNA
3.3.4 Isolation of Bulk E. coli Synthetase
3.3.5 Charging and Formylation of E. coli tRNAfMet
3.4 Overexpression and Purification of Mitochondrial Translation Initiation Factors
3.4.1 Plasmid Transformation
3.4.2 Large-Scale Protein Expression
3.4.3 Protein Purification
3.5 Assembly of the Mitochondrial Translation Initiation Complex
4 Notes
References
Chapter 14: Reconstitution of Mammalian Mitochondrial Translation System Capable of Long Polypeptide Synthesis
1 Introduction
2 Materials
2.1 IF-2mt Preparation
2.2 IF-3mt Preparation
2.3 EF-G1mt Preparation
2.4 EF-G2mt Preparation
2.5 EF-Tumt Preparation
2.6 EF-Tsmt Preparation
2.7 RF-1Lmt Preparation
2.8 RRFmt Preparation
2.9 55S Ribosome Preparation
2.10 In Vitro Translation
2.11 Analysis of Translation Products
3 Methods
3.1 Preparation of Translation Factors (IF-2mt, IF-3mt, EF-G1mt, EF-G2mt, EF-Tumt, EF-Tsmt, RF-1Lmt and RRFmt)
3.1.1 Cell Culture
3.1.2 First-Step Purification by Ni-NTA Agarose (Table 1, Row 5)
3.1.3 Histidine-Tag Cleavage (Table 1, Row 6)
3.1.4 Second-Step Purification by Ion-exchange Column (Table 1, Row 7)
3.2 55S Ribosome Preparation
3.2.1 Crude Ribosome Preparation (from 30 g of Mitoplasts)
3.2.2 55S Ribosome Preparation (from Crude Ribosomes)
3.3 In Vitro Translation
3.4 Analysis of Translation Products
3.4.1 Measurement of Nanoluciferase Activity
3.4.2 Detection of 35S-Met-Labeled Proteins by SDS-PAGE or Tricine-PAGE
4 Notes
References
Chapter 15: Human Mitoribosome Profiling: A Re-engineered Approach Tailored to Study Mitochondrial Translation
1 Introduction
2 Materials
2.1 Cell Growth
2.2 Cell Lysis and Sample Collection
2.3 RNase Digestion
2.4 Mitochondrial Ribosome Isolation
2.4.1 Sucrose Gradient Sedimentation
2.4.2 Mitochondrial Ribosome Visualization in Sucrose Gradients
2.5 Mitochondrial Footprint Isolation
2.6 Mitochondrial Footprint Size Purification
2.7 Library Preparation
2.7.1 RNA Dephosphorylation
2.7.2 3′ End Linker Ligation
2.7.3 Purification of 3′ End Ligation Product
2.7.4 Reverse Transcription
2.7.5 Purification of Reverse Transcription Product
2.7.6 Circularization
2.7.7 PCR Amplification
2.7.8 PCR Product Purification
2.8 Library Quality Controls and Quantitation
2.9 Library Sequencing
2.10 Data Analysis
3 Methods
3.1 Cell Growth
3.2 Cell Lysis and Sample Collection
3.3 RNase Digestion
3.4 Mitochondrial Ribosome Isolation
3.4.1 Sucrose Gradient Sedimentation
3.4.2 Mitochondrial Ribosome Visualization
3.5 Mitochondrial RPF Isolation
3.6 MitoRPF Gel Purification
3.7 Library Preparation
3.7.1 RNA Dephosphorylation
3.7.2 3′ End Linker Ligation
3.7.3 Purification of 3′ End Ligation Product
3.7.4 Reverse Transcription
3.7.5 Purification of Reverse Transcription Product
3.7.6 Circularization
3.7.7 PCR Amplification of Libraries
PCR Cycle Number Determination
PCR Amplification with Determined Cycle Number
3.7.8 PCR Product Purification
3.8 PCR Product Quality Controls and Quantitation
3.9 Sequencing
3.10 Data Analysis
3.10.1 Raw FASTQ Files Initial Processing
3.10.2 Transform the Read Signal to Ribosome A Site Positions
4 Notes
References
Chapter 16: The ARG8m Reporter for the Study of Yeast Mitochondrial Translation
1 Introduction
2 Materials
2.1 Media for Yeast Cell Growth
2.2 Mitopreps and Western blot
2.3 Tetrad Dissection
2.4 Mitochondrial Transformation
2.5 Serial Dilutions
3 Methods
3.1 Mitochondrial Transformation to Insert the ARG8m Reporter
3.1.1 Yeast Strain Preparation for Six Transformation Reactions by Microprojectile Bombardment
3.1.2 Tungsten Particle Preparation
3.1.3 Precipitation of Plasmid DNA on Tungsten Particles
3.1.4 Bombardment
3.1.5 Genetic Recombination to Integrate the ARG8m Reporter in the Mitochondrial Genome
3.2 Growth of Yeast Cells on Media Lacking Arginine to Assess ARG8m mRNA Translation
3.3 Mitochondrial Translation in Whole Cells or in Isolated Organelles
3.4 Rapid Mitochondrial Preparations and Western Blot Assays to Detect Arg8 Production
3.5 Search for Mitochondrial Translation Defect Suppressor Genes
3.5.1 Method to Find and Isolate Suppressors
3.5.2 Method to Determine Whether the Suppressor Is Nuclear or Mitochondrial
3.5.3 A Second Method to Analyze if the Suppressor has a Mitochondrial or Nuclear Origin
4 Notes
References
Chapter 17: Four-Color STED Super-Resolution RNA Fluorescent In Situ Hybridization and Immunocytochemistry to Visualize Mitoch...
1 Introduction
1.1 Background
2 Materials
2.1 General Supplies, Solutions, and Equipment
2.2 Buffers
2.3 RNA FISH Probes and Antibodies
2.3.1 RNA FISH Probes
2.3.2 Mitochondrial Antibodies
2.3.3 Microscopy
3 Methods
3.1 Cell Culture and Fixation
3.2 Immunofluorescence (IF) Staining
3.3 RNA Fluorescence In Situ Hybridization
3.4 Imaging
4 Notes
References
Chapter 18: Digital RNase Footprinting of RNA-Protein Complexes and Ribosomes in Mitochondria
1 Introduction
2 Materials
2.1 Isolation of Mitochondria from Tissues
2.2 Isolation of Mitochondria from Cells
2.3 RNase Treatment and Library Construction
2.4 Software Packages Required for Analyses
3 Methods
3.1 Isolation of Mitochondria from Mouse Tissues
3.2 Isolation of Mitochondria from Cells
3.3 RNase Treatment of Mitochondria from Mouse Tissues
3.4 RNase Treatment of Mitochondria from Cells
3.5 Library Construction
3.6 Sequence Alignment
3.7 Footprint Analysis
4 Notes
References
Chapter 19: Dead-Seq: Discovering Synthetic Lethal Interactions from Dead Cells Genomics
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Drugs
2.3 Annexin V Labeling
2.4 Genetic Perturbation Library
2.5 Annexin V-Based MACS Purification
3 Methods
3.1 Cell Line Maintenance
3.2 Prescreen: Validating a Suitable Synthetic Lethal Interaction
3.3 Screen
3.3.1 Library Titration for the Cell Line Used
3.3.2 Infection
3.3.3 Selection with Puromycin and Confirmation of the MOI
3.3.4 Cell Expansion, Early Time Point, and Preparation of Cell Stocks
3.3.5 Glucose/Galactose Treatment
3.3.6 Select the Annexin V Positive (Dead) Cells by MACS for DNA Isolation
3.4 DNA Isolation and Data Analysis
4 Notes
References
Index