Methods in enzymology. Volume four hundred and thirty-one, Translation initiation. Cell biology, high-throughput methods, and chemical-based approaches

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. II......Page 1 V-IX......Page 2 XI-XV......Page 7 XVII......Page 12 XIX-XLIV......Page 13 Purification of FLAG-Tagged Eukaryotic Initiation Factor 2B Complexes, Subcomplexes, and Fragments from Saccharomyces......Page 39 Introduction......Page 40 Plasmid Vectors and Yeast Strains Used......Page 42 Buffers for protein purification......Page 44 Cell lysis......Page 46 Dialysis to remove 3XFLAG peptide......Page 47 Acknowledgments......Page 48 References......Page 49 Synthesis of Anti-Reverse Cap Analogs (ARCAs) and their Applications in mRNA Translation and Stability......Page 52 Introduction......Page 53 Ni2+ Affinity Purification of eIF3 Using a Polyhistidine Tag......Page 58 A Reverse Chemical Genetic Assay Probing eIF4E:eIF4G and eIF4A: eIF4G Interactions......Page 239 Preparation of mRNAs......Page 263 Analysis of the purified proteins......Page 60 Deletion/Mutational Analysis of eIF3 Subunits and Ni2+ Affinity Purification of Their Subcomplexes......Page 61 Typical example......Page 62 Immunoaffinity purification of eIF3 containing hemagglutinin-tagged eIF3i/TIF34 (HA pull-down)......Page 64 Immunoaffinity purification of eIF3 containing FLAG-tagged eIF3g/TIF35 (FLAG pull-down)......Page 65 CAM (Clustered-10-Alanine Mutagenesis)......Page 66 Acknowledgments......Page 67 References......Page 68 Analysis of mRNA Translation in Cultured Hippocampal Neurons......Page 70 Introduction......Page 71 Translation initiation......Page 300 Amplification of epitope tagging cassette......Page 72 Yeast transformation......Page 74 Downstream targets......Page 157 PCR analysis to verify tagged genes......Page 75 FRAP analysis......Page 76 Preparation of cells and slides for FRAP analysis......Page 77 Analysis of FRAP experiments......Page 78 Fixed cell imaging of RNA......Page 79 Additional In Vivo Techniques......Page 80 References......Page 81 4......Page 83 Methods for Identifying Compounds that Specifically Target Translation......Page 260 Background......Page 84 Preparation of rough microsomes and ribosome-free reticulocyte lysate......Page 85 In vitro translation......Page 87 Ribosomal assembly status-ER membrane-restricted initiation......Page 88 Background......Page 89 Modulating polyribosome loading......Page 90 Sequential detergent extraction......Page 92 Immunoprecipitation for kinase assay against a protein substrate......Page 166 Translational efficiency of ARCA-capped transcripts......Page 94 References......Page 95 Methods to Analyze MicroRNA-Mediated Control of mRNA Translation......Page 97 Introduction......Page 98 Detection and Identification of SGs and PBs......Page 99 Buffers and solutions......Page 106 Staining procedure......Page 107 Stimuli for the Induction of SGs and PBs......Page 112 Transfection-Induced SGs......Page 113 Induction of PBs......Page 114 References......Page 115 6......Page 118 Introduction......Page 236 Systems to Measure miR-Mediated Repression......Page 120 The PI 3-kinase pathway......Page 150 mRNA transfection into HeLa cells......Page 122 Normalization of data......Page 123 Systematic differences between plasmid and mRNA transfections......Page 125 Preparation of the amplicon templates......Page 265 Use of phosphospecific antisera......Page 162 RNase protection assay......Page 128 Measurement of mRNA functional half-life......Page 131 Synthesis of a Bioactive Biotin-Pateamine A (B-PatA) Conjugate......Page 343 Measuring the Distribution of miRNP Components in Polysome Profiles......Page 134 Preparation of polysome gradients......Page 135 Extraction of RNA from sucrose gradients......Page 137 mRNA reverse transcription and qPCR......Page 138 miR reverse transcription and qPCR......Page 139 Localization of miRNP Components to P-Bodies......Page 140 References......Page 143 Methods for Studying Signal-Dependent Regulation of Translation Factor Activity......Page 147 Introduction to Signaling Pathways and the Control of Translation Factors......Page 148 Two fractions......Page 212 Assays for pathway activation......Page 152 Signaling linked to MAP kinases......Page 153 Inhibitors of MAP kinase signaling......Page 155 mTOR......Page 156 Inhibitors......Page 158 Assays for pathway activation......Page 159 Total Synthesis of Pateamine A......Page 160 Lysis buffer......Page 161 Isoelectric focusing (IEF) analysis......Page 163 Isoelectric focusing (IEF) analysis......Page 164 General immunoprecipitation method......Page 165 Immunoprecipitation method for eIF2B assay (Immunoprecipitation of endogenous eIF2B complexes)......Page 169 Analysis of eIF4F complex formation......Page 170 Protein synthesis assays......Page 171 References......Page 172 8......Page 177 Materials and Reagents for Primary Neuron Culture......Page 178 Hippocampal cell culture......Page 179 RNA Transfection and Reporter Assays......Page 183 Synthesis of Derivatives of Pateamine A and Structure Activity Relationship Studies......Page 187 Characterization of Inhibitors of Translation Identified in Chemical Genetic Screens......Page 191 Synaptoneurosome Isolation and 35S-met/cys Labeling......Page 192 UV-Crosslinking, Immunoprecipitation of an RNA-Binding Protein, CPEB3......Page 193 References......Page 195 9......Page 197 General Concept......Page 198 Reagents......Page 200 Cell lysis and separation of polysomes......Page 202 Fraction collection and RNase H cleavage......Page 203 Separation of cleavage product......Page 204 Determination of sedimentation position......Page 206 References......Page 257 References......Page 208 Genome-Wide Analysis of mRNA Polysomal Profiles with Spotted DNA Microarrays......Page 210 Introduction......Page 211 Direct comparison......Page 214 Indirect comparison......Page 216 Three or more fractions......Page 217 Inclusion of exogenous RNA (spike-in controls)......Page 221 Structure and conformation of ARCAs by NMR......Page 222 Cell lysis......Page 223 Fractions collection......Page 224 Preparation of bacterial S30......Page 269 RNA extraction from fractionated gradients......Page 226 Preparation of a reference sample......Page 227 Reverse transcription and amino-allyl coupling......Page 228 cDNA synthesis......Page 229 Microarray slides preparation......Page 230 Washing......Page 231 Creating a reliable dataset......Page 232 Data verification......Page 233 References......Page 295 References......Page 234 11......Page 235 Synthesis of methylene ARCAs......Page 242 In Vitro and In Vivo Assays......Page 245 Incorporation of ARCAs into RNA by In Vitro Transcription......Page 246 Efficiency of cap incorporation during in vitro transcription......Page 248 Analysis of cap orientation......Page 250 Properties of ARCAs and ARCA-Capped mRNAs in Cell-Free Translation Systems......Page 251 Inhibition of cap-dependent translation by ARCAs......Page 252 Properties of ARCA-Capped mRNAs in Mammalian Cells......Page 253 Translational efficiency of ARCA-capped mRNAs......Page 255 Principle of the approach......Page 256 Introduction......Page 261 Chemicals and fine chemicals......Page 262 Anion exchange chromatography......Page 266 Affinity chromatography on oligo d(T) cellulose-for small-scale preparations......Page 267 Preparation of Thr-tRNA, Ile-tRNA, and [14C] Phe-tRNA......Page 268 Preparation of human HeLa cells S30......Page 270 Assessment of the in vivo target of an inhibitor......Page 271 Macromolecular syntheses in gram-positive bacteria......Page 272 Translation of mRNAs (standard) in E. coli......Page 273 Translation of Renilla luciferase mRNAs (luciferase synthesis)......Page 274 Translation of mRNAs (standard) in yeast......Page 277 Cap-independent translation......Page 279 Cap-dependent vs. cap-independent translation in yeast......Page 280 Translational test for the identification of inhibitors of tRNA aminoacylation......Page 281 Methods to quantify the translation level......Page 282 Detection by immunoblotting......Page 283 Enzymatic (luminescent) determination of the level of translation......Page 284 fMet-tRNA binding to 30S or 70S ribosomes......Page 285 Kinetics of fMet-tRNA binding to 30S ribosomal subunit......Page 286 fMet-puromycin formation......Page 288 Initiation dipeptide and tripeptide formation......Page 289 Screening for IF2 inhibitors......Page 290 IF2-dependent GTPase......Page 291 IF2-fMet-tRNA interaction......Page 293 Electrophoretic band-shift......Page 294 Identifying Small Molecule Inhibitors of Eukaryotic Translation Initiation......Page 299 Translation initiation inhibitors......Page 303 Principle of the approach......Page 305 Material and equipment......Page 307 Preparation of recombinant protein......Page 310 Optimizing protein concentrations......Page 311 Use of selective inhibitors as assay development tools......Page 312 Counterscreens and secondary confirmatory assays......Page 313 Screening for inhibitors of eIF4E:eIF4G517-606 interaction......Page 314 Material and equipment......Page 315 In vitro synthesis of reporter mRNA......Page 318 Screening for inhibitors of translation......Page 319 Monitoring initiation complex formation by sedimentation velocity centrifugation......Page 321 Monitoring cap-dependent RNA binding of eIF4F, eIF4A, and eIF4B......Page 323 Monitoring 35S-methionine/cysteine incorporation......Page 324 Polysome profile analysis......Page 325 Discussion and Concluding Remarks......Page 326 References......Page 327 Isolation and Identification of Eukaryotic Initiation Factor 4A as a Molecular Target for the Marine Natural Product Pateamine A......Page 333 Synthesis of PatA derivatives......Page 340 Preparation of cell lysate......Page 345 Procedures......Page 346 Identification of PatA-Binding Proteins......Page 349 Acknowledgments......Page 351 References......Page 352 Author Index......Page 355 Subject Index......Page 372 Purification of FLAG-tagged eukaryotic initation factor 2B complexes, subcomplexes, and fragments from Saccharomyces cerevisiae / Sarah S. Mohammad-Qureshi ... [et al.] In vivo deletion analysis of the architecture of a multiprotein complex of translation initiation factors / Klaus H. Nielsen and Leos̆ Valás̆ek An approach to studying the localization and dynamics of eukaryotic translation factors in live yeast cells / Susan G. Campbell and Mark P. Ashe In vitro and tissue culture methods for analysis of translation initiation on the endoplasmic reticulum / Samuel B. Stephens and Christopher V. Nicchitta Mammalian stress granules and processing bodies / Nancy Kedersha and Paul Anderson Methods to analyze micro-RNA-mediated control of mRNA translation / Jennifer L. Clancy ... [et al.] Methods for studying signal-dependent regulation of translation factor activity / Xuemin Wang and Christopher G. Proud Analysis of mRNA translation in cultured hippocampal neurons / Yi-Shuian Huang and Joel D. Richter Detecting ribosomal association with the 5' leader of mRNAs by ribosome density mapping (RDM) / Naama Eldad and Yoav Arava Genome-wide analysis of mRNA polysomal profiles with spotted DNA microarrays / Danial Melamed and Yoad Arava Synthesis of anti-reverse cap analogs (ARCAs) and their applications in mRNA translation and stability / Ewa Grudzien-Nogalska ... [et al.] Methods for identifying compounds that specifically target translation / Letizia Brandi ... [et al.] Identifying small molecule inhibitors of eukaryotic translation initiation / Regina Cencic, Francis Robert, and Jerry Pelletier Isolation and identification of eukaryotic initiation factor 4A as a molecular target for the marine natural product pateamine A / Woon-Kai Low ... [et al.]. For over fifty years the Methods in Enzymology series has been the critically acclaimed 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 Part of the "Methods in Enzymology" series, which is a publication in the field of biochemistry, this volume, the third of three on the topic of "Translation Initiation" includes articles written by leaders in the field.