Elsevier S & T

Translation Initiation: Cell Biology, High-throughput and Chemical-based Approaches: Cell Biology, High-throughput and Chemical-based Approaches

Lorsch, Jon

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Table of contents
  • Contentsv
  • Contributorsxi
  • Prefacexvii
  • Volumes in Seriesxix
  • Chapter 1: Purification of FLAG-Tagged Eukaryotic Initiation Factor 2B Complexes, Subcomplexes, and1
  • 1. Introduction2
  • 2. Plasmid Vectors and Yeast Strains Used4
  • 3. Expression and Purification of eIF2B6
  • 3.1. Buffers for protein purification6
  • 3.2. Cell growth and harvest8
  • 3.3. Cell lysis8
  • 3.4. Purification using anti-FLAG M2 affinity resin9
  • 3.5. Dialysis to remove 3XFLAG peptide9
  • 4. Functional Analysis of Purified Proteins10
  • 5. Conclusions10
  • Acknowledgments10
  • References11
  • Chapter 2: In Vivo Deletion Analysis of the Architecture of a Multiprotein Complex of Translation In15
  • 1. Introduction16
  • 2. Ni2+ Affinity Purification of eIF3 Using a Polyhistidine Tag21
  • 2.1. Affinity-tagging21
  • 2.2. Whole-cell extract (WCE) preparation22
  • 2.3. Batch Ni2+ affinity purification of eIF3 (Ni pull-down [Ni PD])23
  • 2.4. Analysis of the purified proteins23
  • 2.5. Troubleshooting24
  • 3. Deletion/Mutational Analysis of eIF3 Subunits and Ni2+ Affinity Purification of Their Subcomplexe24
  • 3.1. Typical example25
  • 4. eIF3 Purification Using Other Epitope Tags27
  • 4.1. Immunoaffinity purification of eIF3 containing hemagglutinin-tagged eIF3i/TIF34 (HA pull-down)27
  • 4.2. Immunoaffinity purification of eIF3 containing FLAG-tagged eIF3g/TIF35 (FLAG pull-down)28
  • 5. CAM (Clustered-10-Alanine Mutagenesis)29
  • Acknowledgments30
  • References31
  • Chapter 3: An Approach to Studying the Localization and Dynamics of Eukaryotic Translation Factors i33
  • 1. Introduction34
  • 1.1. Epitope tagging34
  • 1.2. Amplification of epitope tagging cassette35
  • 2. Yeast Strains and Growth Conditions37
  • 2.1. Yeast transformation37
  • 2.2. Confirmation of epitope-tagged proteins38
  • 2.3. PCR analysis to verify tagged genes38
  • 2.4. Live cell imaging39
  • 3. Application of Live Cell Imaging39
  • 3.1. FRAP analysis39
  • 3.2. Microscopy and FRAP analysis40
  • 3.3. Analysis of FRAP experiments41
  • 3.4. RNA localization42
  • 4. Additional In Vivo Techniques43
  • 5. Conclusions44
  • Acknowledgments44
  • References44
  • Chapter 4: In Vitro and Tissue Culture Methods for Analysis of Translation Initiation on the Endopla47
  • 1. Background48
  • 2. Methods49
  • 2.1. In vitro analysis of protein synthesis initiation on membrane-bound ribosomes49
  • 2.2. In vitro translation51
  • 2.3. Ribosomal assembly status„ER membrane-restricted initiation52
  • 3. Analysis of Protein Synthesis Initiation on ER Membrane-Bound Ribosomes of Tissue Culture Cells53
  • 3.1. Background53
  • 3.2. Modulating polyribosome loading54
  • 3.3. Sequential detergent extraction56
  • 4. Velocity Sedimentation57
  • 4.1. Reagents58
  • 4.2. Velocity sedimentation59
  • Acknowledgments59
  • References59
  • Chapter 5: Mammalian Stress Granules and Processing Bodies61
  • 1. Introduction62
  • 2. Detection and Identification of SGs and PBs63
  • 3. Immunostaining Protocol70
  • 3.1. Buffers and solutions70
  • 3.2. Staining procedure71
  • 4. Stimuli for the Induction of SGs and PBs76
  • 5. Transfection-Induced SGs77
  • 6. Induction of PBs78
  • 7. Conclusions79
  • References79
  • Chapter 6: Methods to Analyze MicroRNA-Mediated Control of mRNA Translation83
  • 1. Introduction84
  • 2. Systems to Measure miR-Mediated Repression85
  • 2.1. Co-transfection of synthetic miRs and target plasmids into HeLa cells86
  • 2.2. mRNA transfection into HeLa cells87
  • 2.3. Systematic differences between plasmid and mRNA transfections90
  • 2.4. Internal ribosome entry sites90
  • 3. Detection of miR-Mediated Changes in mRNA Stability93
  • 3.1. RNA extraction93
  • 3.2. RNase protection assay93
  • 3.3. Measurement of mRNA functional half-life96
  • 4. Measurement of miR Target mRNA Deadenylation97
  • 5. Measuring the Distribution of miRNP Components in Polysome Profiles99
  • 5.1. Preparation of polysome gradients100
  • 5.2. Extraction of RNA from sucrose gradients102
  • 5.3. Detection of miRs by reverse transcription (RT) and quantitative PCR (qPCR)103
  • 6. Localization of miRNP Components to P-Bodies105
  • 7. Concluding Remarks108
  • Acknowledgments108
  • References108
  • Chapter 7: Methods for Studying Signal-Dependent Regulation of Translation Factor Activity113
  • 1. Introduction to Signaling Pathways and the Control of Translation Factors114
  • 1.1. The PI 3-kinase pathway116
  • 1.2. Signaling linked to MAP kinases119
  • 1.3. mTOR122
  • 1.4. Downstream targets123
  • 2. Experimental Protocols126
  • 2.1. General procedures126
  • 3. Analysis of the Phosphorylation States of Translation Factors and Signaling Components„Overview128
  • 3.1. Use of phosphospecific antisera128
  • 3.2. Isoelectric focusing (IEF) analysis130
  • 3.3. General immunoprecipitation method131
  • 3.4. Immunoprecipitation for western blot analysis132
  • 3.5. Immunoprecipitation for protein kinase assays using peptide substrates132
  • 4. Assays for Specific Protein Kinases132
  • 4.1. Immunoprecipitation for kinase assay against a protein substrate132
  • 5. Assays for Initiation Factor Function135
  • 5.1. eIF2B135
  • 5.2. Analysis of eIF4F complex formation136
  • 5.3. Protein synthesis assays137
  • References138
  • Chapter 8: Analysis of mRNA Translation in Cultured Hippocampal Neurons143
  • 1. Materials and Reagents for Primary Neuron Culture144
  • 1.1. Hippocampal cell culture145
  • 2. RNA Transfection and Reporter Assays149
  • 3. shRNA Design and Lenti-shRNA Virus Production153
  • 4. shRNA Knockdown of CPEB3 in Cultured Neurons157
  • 5. Synaptoneurosome Isolation and 35S-met/cys Labeling158
  • 6. UV-Crosslinking, Immunoprecipitation of an RNA-Binding Protein, CPEB3159
  • References161
  • Chapter 9: Detecting Ribosomal Association with the 5' Leader of mRNAs by Ribosome Density Mapping (163
  • 1. Introduction164
  • 2. General Concept164
  • 2.1. Reagents166
  • 3. Methods168
  • 3.1. Cell lysis and separation of polysomes168
  • 3.2. Fraction collection and RNase H cleavage169
  • 3.3. Separation of cleavage product170
  • 3.4. Determination of sedimentation position172
  • Acknowledgments174
  • References174
  • Chapter 10: Genome-Wide Analysis of mRNA Polysomal Profiles with Spotted DNA Microarrays177
  • 1. Introduction178
  • 2. Experimental Designs179
  • 2.1. Number of fractions into which the gradient is separated179
  • 2.2. Inclusion of exogenous RNA (spike-in controls)188
  • 2.3. Analysis of changes at the transcriptome level189
  • 3. Methods189
  • 3.1. Gradient preparation190
  • 3.2. Cell lysis190
  • 3.3. Fractions collection191
  • 3.4. Adding spike-in controls to fractions192
  • 3.5. RNA extraction from fractionated gradients193
  • 3.6. Preparation of a reference sample194
  • 3.7. Reverse transcription and amino-allyl coupling195
  • 3.8. Microarray slides preparation197
  • 3.9. Hybridization198
  • 3.10. Washing198
  • 4. Data Acquisition and Analysis199
  • 4.1. Microarray scanning199
  • 4.2. Creating a reliable dataset199
  • 4.3. Data verification200
  • Acknowledgments200
  • References201
  • Chapter 11: Synthesis of Anti-Reverse Cap Analogs (ARCAs) and their Applications in mRNA Translation203
  • 1. Introduction204
  • 2. Chemical Synthesis207
  • 2.1. Synthesis of ARCAs containing conventional phosphate chains207
  • 2.2. Synthesis of methylene ARCAs210
  • 2.3. Structure and conformation of ARCAs by NMR213
  • 3. In Vitro and In Vivo Assays213
  • 4. Binding Affinity of ARCAs for eIF4E214
  • 5. Incorporation of ARCAs into RNA by In Vitro Transcription214
  • 5.1. Efficiency of cap incorporation during in vitro transcription216
  • 5.2. Analysis of cap orientation218
  • 6. Properties of ARCAs and ARCA-Capped mRNAs in Cell-Free Translation Systems219
  • 6.1. Inhibition of cap-dependent translation by ARCAs220
  • 6.2. Translational efficiency of ARCA-capped transcripts220
  • 7. Properties of ARCA-Capped mRNAs in Mammalian Cells221
  • 7.1. Translational efficiency of ARCA-capped mRNAs223
  • 7.2. Stability of ARCA-capped mRNAs in cultured mammalian cells224
  • Acknowledgments225
  • References225
  • Chapter 12: Methods for Identifying Compounds that Specifically Target Translation229
  • 1. Introduction230
  • 2. Materials Required231
  • 2.1. Chemicals and fine chemicals231
  • 2.2. Biologicals232
  • 2.3. Preparation of mRNAs232
  • 2.4. Preparation and linearization of the plasmid templates234
  • 2.5. Preparation of the amplicon templates234
  • 2.6. In vitro transcription and purification of mRNAs235
  • 2.7. Preparation of cellfree extracts238
  • 3. Methods and Tests240
  • 3.1. Assessment of the in vivo target of an inhibitor240
  • 3.2. Tests to detect translational inhibitors242
  • 3.3. Product detection251
  • 3.4. Partial reactions254
  • 3.5. Screening for IF2 inhibitors259
  • Acknowledgments264
  • References264
  • Chapter 13: Identifying Small Molecule Inhibitors of Eukaryotic Translation Initiation269
  • 1. Introduction270
  • 1.1. Translation initiation270
  • 1.2. Translation initiation and cancer273
  • 1.3. Translation initiation inhibitors273
  • 2. A Reverse Chemical Genetic Assay Probing eIF4E:eIF4G and eIF4A: eIF4G Interactions275
  • 2.1. Principle of the approach275
  • 2.2. Material and equipment277
  • 2.3. Preparation of recombinant protein280
  • 2.4. Assay development281
  • 2.5. Screening for inhibitors of eIF4E:eIF4G517-606 interaction284
  • 3. A Forward Chemical Genetic Screen to Identify Inhibitors of Eukaryotic Translation285
  • 3.1. Principle of the approach285
  • 3.2. Material and equipment285
  • 3.3. Assay development288
  • 3.4. In vitro synthesis of reporter mRNA288
  • 3.5. Screening for inhibitors of translation289
  • 4. Characterization of Inhibitors of Translation Identified in Chemical Genetic Screens291
  • 4.1. Compound sensitivity toward different IRESes291
  • 4.2. Testing for nucleic acid binding291
  • 4.3. Monitoring initiation complex formation by sedimentation velocity centrifugation291
  • 4.4. Monitoring cap-dependent RNA binding of eIF4F, eIF4A, and eIF4B293
  • 4.5. Monitoring compound inhibition in vivo294
  • 5. Discussion and Concluding Remarks296
  • Acknowledgments297
  • References297
  • Chapter 14: Isolation and Identification of Eukaryotic Initiation Factor 4A as a Molecular Target fo303
  • 1. Introduction304
  • 2. Total Synthesis of Pateamine A307
  • 3. Synthesis of Derivatives of Pateamine A and Structure Activity Relationship Studies309
  • 3.1. Synthesis of PatA derivatives310
  • 4. Structural Analysis of PatA Leading to DMDA-PatA and Viable Positions for Derivatization313
  • 5. Synthesis of a Bioactive Biotin-Pateamine A (B-PatA) Conjugate313
  • 6. Affinity Pull-Down of PatA-Binding Proteins315
  • 6.1. Preparation of cell lysate315
  • 6.2. Affinity capture of PatA-binding proteins using biotin-PatA and streptavidin-agarose316
  • 7. Identification of PatA-Binding Proteins319
  • 8. Concluding Remarks321
  • Acknowledgments321
  • References322
  • Author Index325
  • Subject Index343
Book details
  • Vendor Elsevier S & T
  • SKU 9780123739643
  • ISBN-13 9780080553481
  • Author Lorsch, Jon
  • Category Science
  • Subject Microbiology

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For over fifty years the Methods in Enzymology series has been the critically aclaimed laboratory standard and one of the most respected publications in the field of biochemistry. The highly relevant material makes it an essential publication for researchers in all fields of life and related sciences. This volume, the third of three on the topic of Translation Initiation includes articles written by leaders in the field.