Preface
Contents
Contributors
Part I: Analysis of mRNA Translation Regulation
Chapter 1: An Overview of Methods for Detecting eIF2Ξ± Phosphorylation and the Integrated Stress Response
1 Introduction
1.1 Evolutionary Perspective
1.2 Importance of eIF2Ξ± Phosphorylation and the ISR for Cell and Organismal Survival
1.3 Hypophosphorylation of eIF2Ξ± in Human Diseases Due to Mutations Impairing the ISR
1.4 Hyperphosphorylation of eIF2Ξ± as a Cause of Human Diseases
2 Measuring ISR Activation
2.1 Measuring Kinase Activity
2.2 Monitoring eIF2Ξ± Phosphorylation
2.3 In Vitro eIF2Ξ± Phosphorylation and Dephosphorylation Assays
2.4 Tracking Downstream Effectors of the ISR
2.5 UPR or ISR?
2.6 Measuring Translation
2.7 Optimal Conditions to Measure ISR
2.8 Detection of the ISR In Vivo
2.9 Roadmap to ISR Detection
3 Conclusion
References
Chapter 2: CRISPR-Based Screening for Stress Response Factors in Mammalian Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Plasmids for Generating the Reporter Cell Line
2.3 Plasmids for Generation of Lentivirus
2.4 CRISPRi Libraries
2.5 Validation of the Activity of CRISPRi
2.6 Reagents for Generation of Lentivirus
2.7 FACS
2.8 Sample Preparation
2.8.1 Genomic DNA Extraction
2.8.2 PCR Enrichment of sgRNAs
2.8.3 Home-Made SPRI Beads
2.8.4 SPRI Beads Purification of sgRNA
2.9 Next-Generation Sequencing
2.10 Individual sgRNA Cloning
3 Methods
3.1 Construction of the Reporter Cell Line
3.1.1 Establishment of a CRISPRi Cell Line
Integration of the CRISPRi into the Cells
Validation of the Activity of CRISPRi
3.1.2 Construction of the Reporter Plasmid(s)
3.1.3 Production Lentivirus of the Reporter Plasmids
3.1.4 Establishment of the Reporter Cell Line
3.2 Introduction of the sgRNA Library to the Reporter Cell Line
3.2.1 Production of Virus with sgRNA Libraries
3.2.2 Transduction of the sgRNA Library Virus to the Reporter Cell Line
Small-Scale Transduction of the sgRNA Library Virus
Large-Scale Transduction of sgRNA Library Virus
3.3 FACS-Based CRISPRi Screens
3.4 Sample Preparation
3.4.1 Genomic DNA Extraction
3.4.2 PCR Enrichment of sgRNA
Test PCR
Scale-up PCR
3.4.3 SPRI Beads Purification of the Enriched PCR Product
Home-Made SPRI Beads Solution (Skip this Step if Using Commercially Available SPRI Beads)
SPRI Bead Purification
3.5 Submission for Next-Generation Sequencing (NGS)
3.6 Data Analysis
3.7 Cloning Individual sgRNA for Post-screen Validation
4 Notes
References
Chapter 3: Multiplexed Analysis of Human uORF Regulatory Functions During the ISR Using PoLib-Seq
1 Introduction
2 Materials
3 Methods
3.1 In Vitro RNA Transcription Template Preparation
3.2 In Vitro RNA Transcription
3.3 5β² Capping Reaction
3.4 HEK 293 T Cell In Vitro RNA Transfection
3.5 Polysome Fractionation
3.6 RNA Extraction
3.7 DNase Treatment
3.8 Reverse Transcription
3.9 Sequencing Library Preparation
4 Notes
References
Chapter 4: Measuring Bulk Translation Activity in Single Mammalian Cells During the Integrated Stress Response
1 Introduction
2 Materials
2.1 SUrface SEnsing of Translation (SUnSET) Assay
2.2 Fluorescent Noncanonical Amino Acid Tagging (FUNCAT) Assay
3 Methods
3.1 SUnSET Assay
3.2 FUNCAT Assay
3.3 Imaging and Image Analysis
4 Notes
References
Chapter 5: Quantitative Translation Proteomics Using mePROD
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
2.3 SILAC Amino Acid Stock Solutions
2.4 Buffers
2.5 Standard Peptides
3 Methods
3.1 Pulse Labeling of Cells and Harvest
3.2 Sample Preparation for Mass Spectrometry
3.3 High pH Reverse Phase Fractionation
3.4 LC-MS2
3.5 Data Analysis
4 Notes
References
Chapter 6: Quantifying the Binding of Fluorescently Labeled Guanine Nucleotides and Initiator tRNA to Eukaryotic Translation I...
1 Introduction
2 Materials
2.1 Purification of eIF2
2.2 Preparation of Apo-eIF2
2.3 Fluorescence Spectroscopy Analysis
3 Methods
3.1 eIF2 Purification
3.1.1 Cell Growth, Harvest, and Lysis
3.1.2 Lysate Clarification and Nickel-Affinity Chromatography
3.1.3 Heparin HP Chromatography
3.1.4 HiTrap Q Chromatography and Final Steps
3.2 Preparation of Apo-eIF2
3.3 Fluorescence Spectroscopy Analysis of Nucleotide Binding to eIF2
3.4 Fluorescence Spectroscopy Analysis of Met-tRNAi Binding to eIF2
3.5 Fluorescence Data Analysis
4 Notes
References
Chapter 7: Mammalian In Vitro Translation Systems
1 Introduction
2 Materials
2.1 Translation with the Crude Cell Lysate
2.2 Global In Vitro Translation with Isolated Ribosomes and Non-ribosome-Containing Cytosolic Fractions
2.3 In Vitro Translation of One Specific Reporter mRNA with Isolated Ribosomes and Non-ribosome-Containing Fractions
3 Methods
3.1 In Vitro Translation with the Crude Cell Lysate
3.2 Global In Vitro Translation with Isolated Ribosomes and the Non-ribosome-Containing Cytosolic Fraction
3.2.1 Isolation of P100 from Mammalian Cell Lines, Primary Cells, or Mouse Tissues
3.2.2 Preparation of Concentrated S100
3.2.3 Setting up Translation Reaction
3.3 Translation of One Specific mRNA Reporter with Isolated Ribosomes and the Non-ribosome-Containing Cytosolic Fraction
3.3.1 Isolation of P100 from Mammalian Cell Lines, Primary Cells or Mouse Tissues
3.3.2 Prepare RR S100
3.3.3 Set up in Vitro Translation Reaction
4 Notes
References
Chapter 8: Measuring Repeat-Associated Non-AUG (RAN) Translation
1 Introduction
2 Materials
2.1 Cloning of Plasmid Constructs
2.2 Stable Cell Line Generation
2.3 Luciferase Activity Assay
2.4 Assessing the Cap-Independent RAN Translation
2.5 Investigating the RNA Template of RAN Translation
2.6 Modulation of RAN Translation by Integrated Stress Response
3 Methods
3.1 Construct Design and Cloning
3.1.1 Construct Design Strategy
3.1.2 Molecular Cloning
3.2 Stable Cell Line Generation
3.2.1 Making Stable Lines by Flp-In
3.2.2 Making Stable Lines by Retrovirus Transduction
3.2.3 Confirmation of RAN Translation Products
3.3 Luciferase Assay to Measure RAN Translation
3.4 Assessing the Cap-Independent RAN Translation
3.5 Investigating the RNA Template of RAN Translation
3.5.1 Translating Ribosome Affinity Purification
3.5.2 RNA Extraction
3.5.3 First Strand cDNA Synthesis
3.5.4 Real-Time PCR Quantification
3.6 Modulation of RAN Translation by Integrated Stress Response
3.6.1 Measuring RAN Translation Under Stress Stimuli
3.6.2 Immunofluorescence of Stress Granules
3.6.3 Inhibition of the Integrated Stress Response Pathway
4 Notes
References
Chapter 9: Analysis of Ribosome Profiling Data
1 Introduction
2 Materials
2.1 Quality Control and Codon Occupancy Estimates
2.2 Inference and Visualization of Translation Dynamics Using the TASEP
3 Methods
3.1 Quality Control
3.1.1 Installation of RiboVIEW
3.1.2 Fetching Reference Sequences and Reference Annotation
3.1.3 Setting Up Parameters
3.1.4 Preliminary Calculation and Check (Periodicity)
3.1.5 Run Calculations for Quality Control and Visualization
3.1.6 Reviewing Quality Controls
3.1.7 Codon Enrichment (Occupancy) and Further Results
3.2 Inference of Rates Under the TASEP Model
3.3 Visualization and Perturbation Analysis
4 Notes
References
Chapter 10: Analysis of Translational Control in the Integrated Stress Response by Polysome Profiling
1 Introduction
2 Materials
2.1 Cell Culture and Chemical Treatment
2.2 Making Sucrose Gradients and Other Solutions
2.3 Collecting Lysate and Ultracentrifugation
2.4 Polysome Analysis and Fractionation
2.5 Analysis of Transcript Sedimentation
2.6 Analysis of Translational Machinery Sedimentation
3 Methods
3.1 Cell Culture and Chemical Treatment
3.2 Making Sucrose Gradients and Other Solutions
3.3 Collecting Lysate and Ultracentrifugation
3.4 Polysome Analysis and Fractionation
3.5 Analysis of Transcript Sedimentation
3.6 Analysis of Translational Machinery Sedimentation
4 Notes
References
Chapter 11: High-Resolution Ribosome Profiling for Determining Ribosome Functional States During Translation Elongation
1 Introduction
2 Materials
3 Methods
3.1 Yeast Cell Harvesting by Filtration and Flash Freezing
3.2 Lyse by Freezer Mill and Polysome Isolation
3.3 Monosome Isolation by Sucrose Gradient
3.4 Construction of a Sequencing Library from Ribosome-Protected mRNA Fragments
3.4.1 Size Select Ribosome-Protected mRNA Fragments
3.4.2 Dephosphorylation
3.4.3 Linker Ligation
3.4.4 Ribosomal RNA Depletion
3.4.5 Reverse Transcription
3.4.6 Circularization
3.4.7 Preparative PCR
3.5 Bioinformatic Analysis of RPF Libraries
4 Notes
References
Chapter 12: Fluorescence Intensity-Based eIF2BΒ΄s Guanine Nucleotide-Exchange Factor Activity Assay
1 Introduction
2 Materials
2.1 Substrate eIF2 Preparation
2.2 Cell Lysate Preparation
2.3 GDP-Release Assay
3 Methods
3.1 Substrate eIF2 Preparation
3.2 Cell Extract Preparation
3.3 GDP-Exchange Assay on Purified eIF2
4 Notes
References
Part II: Analysis of Interaction Networks and RNP Granules
Chapter 13: Collective Learnings of Studies of Stress Granule Assembly and Composition
1 Introduction
2 Stress Granule Formation
3 Canonical Versus Noncanonical SGs
4 Stressors
4.1 Sodium Arsenite (SA)
4.2 Heat Stress
4.3 Osmotic Stress
4.4 ER Stress
4.5 Eukaryotic Initiation Factor (eIF) 4A Inhibitor
5 A Critical Appraisal of the Techniques to Study SG Composition
5.1 Cellular Fractionation-Based Approaches
5.2 Immunoprecipitation-Based Approaches
5.3 Proximity-Labeling Approaches
6 Insights Gained on SG Composition
6.1 Protein Components
6.2 RNA Components
7 Lessons that Can Be Learned from These Studies
7.1 Cellular Context Is Important, and Often Overlooked
7.2 There Is a Preexisting Network of SG Protein Interactions in Basal Conditions
7.3 SG Formation Is an Active Process: The Importance of ATPases
7.4 SG Formation Is a Sequential Process
7.5 SG Disassembly May Be Facilitated by a Distinct Set of Interactions
7.6 Canonical and Noncanonical SGs Are Different Entities
8 Conclusion and Future Directions
References
Chapter 14: Detecting Stress Granules in Drosophila Neurons
1 Introduction
2 Materials
2.1 Fly Lines for Expression of Fluorescent SG Proteins
2.2 Arsenite Treatment
2.3 Dissection and Fixation of Drosophila CNS Samples
2.3.1 Dissection and Fixation of Larval CNS
2.3.2 Dissection and Fixation of Adult Brains
2.4 Mounting of Drosophila CNS Samples
2.5 Image Acquisition
2.6 Image Analysis
3 Methods
3.1 Induction of Stress
3.1.1 Ectopic In Vivo Expression of Pathological Proteins
3.1.2 Ex Vivo Treatment with Arsenite
3.2 Dissection of Drosophila CNS Samples
3.2.1 Dissection of Larval CNS
3.2.2 Dissection of Adult Brains
3.3 Fixation of Drosophila CNS Samples
3.3.1 Fixation of Larval CNS
3.3.2 Fixation of Adult Brains
3.4 Mounting of Drosophila CNS Samples
3.4.1 Mounting of Larval CNS
3.4.2 Mounting of Adult Brains
3.5 Imaging of Drosophila CNS Samples
3.6 Image Analysis: Detection of Stress Granules
4 Notes
References
Chapter 15: Monitoring and Quantification of the Dynamics of Stress Granule Components in Living Cells by Fluorescence Decay A...
1 Introduction
2 Materials
2.1 Coating of Glass-Bottom Culture Dishes
2.2 PC12 Cell Culture
2.3 Stress Induction
2.4 Equipment for Image Acquisition
2.5 Software and Hardware
3 Methods
3.1 Glass Coverslip Coating
3.2 PC12 Cell Culture
3.2.1 Transfection
3.2.2 Cell Plating
3.2.3 Neuronal Differentiation
3.2.4 Stress Induction
3.3 Image Acquisition
3.4 Image Analysis
3.4.1 Intensity Measurement in Fiji
3.4.2 Background Extraction, Normalization, and Fitting of FDAP Curves
4 Notes
References
Chapter 16: Probing Protein Solubility Patterns with Proteomics for Insight into Network Dynamics
1 Introduction
2 Materials
3 Methods
3.1 Isolation of Soluble and Insoluble Proteins by Ultracentrifugation
3.2 Purification of Disease-Associated Protein Aggregates
3.3 Peptide Preparation for Mass Spectrometry
3.3.1 Trypsin Digestion
3.3.2 Peptide Desalting
3.3.3 Stable Isotope Dimethyl Labeling of Peptides
3.4 Proteomic Data Analysis
3.5 Bioinformatics Analysis and Visualization Using Cytoscape v3.8.2
3.5.1 Gene Ontology (GO) Enrichment Analysis
3.5.2 Protein-Protein Interaction Network Analysis Using Cytoscape
4 Notes
References
Chapter 17: Analyzing the Composition and Organization of Ribonucleoprotein Complexes by APEX-Seq
1 Introduction
2 Materials
2.1 RNA Proximity Labeling
2.2 Total RNA Extraction
2.3 Biotinylated RNA Enrichment
2.4 RNA Purification and Fragmentation
2.5 RNA Sequencing
3 Methods
3.1 Cell Growth and Preparation
3.2 Labeling Reaction
3.3 RNA Extraction
3.4 DNase I Digest
3.5 RNA Purification
3.6 RNA Fragmentation
3.7 Biotinylated RNA Enrichment
3.8 Fragmentation and Priming Reaction
3.9 First and Second Strand cDNA Synthesis
3.10 Purification of Double-Stranded cDNA
3.11 End Processing of cDNA Library and Adaptor Ligation
3.12 Ligation Product Purification
3.13 PCR Amplification
4 Notes
References
Chapter 18: Differential Analysis of the Nuclear and the Cytoplasmic RNA Interactomes in Living Cells
1 Introduction
2 Materials
2.1 Equipment and Disposables
2.2 Coupling of LNA Oligos to Carboxylated Beads
2.3 Cell Culture, Fractionation, and Lysate Preparation
2.4 eRIC
3 Methods
3.1 Coupling of LNA Oligos to Carboxylated Magnetic Beads
3.2 Cell Culture and In Vivo UV Cross-Linking
3.3 Cell Fractionation
3.4 Preparation of Lysates for eRIC
3.5 Capture of Polyadenylated RNAs by eRIC
3.6 Heat-Elution of the Captured RNA
3.7 RNase Elution of Captured RBPs
3.8 Second Capture Round
3.9 Washing and Storing the Beads for Reuse
4 Notes
References
Chapter 19: Identification of RNA-RBP Interactions in Subcellular Compartments by CLIP-Seq
1 Introduction
2 Materials
2.1 SNS Preparation
2.2 Cross-Linking
2.3 Immunoprecipitation and Radiolabeling
2.4 PAGE and Immunoblotting
2.5 RNA Isolation
2.6 DNA Library Preparation
3 Methods
3.1 SNS Isolation and RNA-RBP Cross-Linking
3.2 Total Homogenate Preparation and RNA-RBP Cross-Linking
3.3 Immunoprecipitation: Part 1 - Antibody Coupling to Magnetic Beads
3.4 Immunoprecipitation: Part 2 - Lysate Preparation
3.5 Immunoprecipitation: Part 3-Pull-Down
3.6 Nuclease Treatment
3.7 RNA Radiolabeling (See Note 16)
3.8 SDS-PAGE and Transfer onto a Nitrocellulose Membrane
3.9 SDS-PAGE and Immunoblot
3.10 RNA Isolation
3.11 cDNA Library Preparation and Sequencing
4 Notes
References
Chapter 20: Monitoring Virus-Induced Stress Granule Dynamics Using Long-Term Live-Cell Imaging
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Cell Culture Reagents
2.3 Production of Lentiviruses and Generation of Fluorescent SG-Reporter Cell Lines
2.4 Production of HCVTCP
2.5 Live-Cell Microscopy
3 Methods
3.1 Generation of a Fluorescent SG-Reporter Cell Line Using Lentiviral Delivery
3.1.1 Production of VSV-G Pseudotyped Lentiviral Particles (See Note 9)
3.1.2 Generation of Stable SG Reporter Cells for Live-Cell Imaging
3.2 Production and Purification of HCVTCP
3.3 Long Term Live-Cell Imaging
3.3.1 Cell Seeding and Infection with HCVTCP (See Note 18)
3.3.2 Microscope Set-up and Image Acquisition
3.3.3 Semiautomated Image Processing Pipeline Using Ilastik (See Note 25)
4 Notes
References
Chapter 21: Single-Molecule Imaging of mRNA Interactions with Stress Granules
1 Introduction
2 Materials
2.1 Sample Prep
2.2 Tissue Culture
2.3 Imaging
3 Methods
3.1 Imaging
3.2 Image Analysis
3.2.1 Preprocessing Movies
3.2.2 Global Translation Shut off Dynamics
3.2.3 Single mRNA Molecule Translation Shut off Dynamics
3.2.4 mRNA-SG Interaction Dynamics
3.2.5 Diffusion of mRNA
4 Notes
References
Chapter 22: Image-Based Screening for Stress Granule Regulators
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Stable Expression of a Fluorescently Tagged SG Marker Protein in your Cell Line of Interest
3.2 Optimization of Knockdown (kd) Efficiency
3.3 Coating of Cell Culture and Imaging Plates with an siRNA Library
3.4 High-Throughput RNAi
3.5 Cell Fixation and Staining
3.6 Image Acquisition
3.7 Image Analysis
3.8 Statistics and Data Validation
4 Notes
References
Chapter 23: APEX Proximity Labeling of Stress Granule Proteins
1 Introduction
2 Materials
2.1 APEX-Expressing Cell Line Culture
2.2 Optional Validation of Cell Lines
2.3 APEX-Mediated Biotinylation
2.4 Validation of Biotinylation by Western Blotting and/or Immunocytochemistry
2.5 Streptavidin Affinity Purification and Preparation for Mass Spectrometry
3 Methods
3.1 Experimental Design Considerations
3.2 Culture of APEX-Expressing Cell Lines
3.3 Cell Line Validation
3.3.1 Fluorescence Microscopy to Visualize SG Localization of G3BP-APEX2-GFP
3.3.2 Western Blotting to Confirm V5 Tag Expression of G3BP-GFP-APEX Fusion Protein
3.4 APEX-Mediated Biotinylation of SG Proximal Proteins
3.5 Validation of APEX Labeling: Streptavidin Western Blotting and Immunocytochemistry
3.6 Affinity Purification of Biotinylated Samples and On-Bead Peptide Digestion
4 Notes
References
Index