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This volume provides an up-to-date collection of protocols describing some of the key methods to investigate the integrated stress response (ISR), a vital evolutionarily conserved mechanism that enables eukaryotic cells to adapt to stress conditions and alter their gene expression programs. The content of the book is split between techniques to analyze mRNA translation regulation and methods to analyze interaction networks and ribonucleoprotein (RNP) granules. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, The Integrated Stress Response: Methods and Protocols serves as an ideal guide to help accelerate research into the complex and fascinating biology of the ISR.
Sommario
An Overview of Methods for Detecting eIF2 Phosphorylation and the Integrated Stress Response.- CRISPR-Based Screening for Stress Response Factors in Mammalian Cells.- Multiplexed Analysis of Human uORF Regulatory Functions during the ISR Using PoLib-Seq.- Measuring Bulk Translation Activity in Single Mammalian Cells during the Integrated Stress Response.- Quantitative Translation Proteomics Using mePROD.- Quantifying the Binding of Fluorescently Labelled Guanine Nucleotides and Initiator tRNA to Eukaryotic Translation Initiation Factor 2.- Mammalian In Vitro Translation Systems.- Measuring Repeat-Associated Non-AUG (RAN) Translation.- Analysis of Ribosome Profiling Data.- Analysis of Translational Control in the Integrated Stress Response by Polysome Profiling.- High-Resolution Ribosome Profiling for Determining Ribosome Functional States during Translation Elongation.- Fluorescence Intensity-Based eIF2B's Guanine Nucleotide-Exchange Factor Activity Assay.- Collective Learnings of Studies of Stress Granule Assembly and Composition.- Detecting Stress Granules in Drosophila Neurons.- Monitoring and Quantification of the Dynamics of Stress Granule Components in Living Cells by Fluorescence Decay after Photoactivation.- Probing Protein Solubility Patterns with Proteomics for Insight into Network Dynamics.- Analyzing the Composition and Organization of Ribonucleoprotein Complexes by APEX-Seq.- Differential Analysis of the Nuclear and the Cytoplasmic RNA Interactomes in Living Cells.- Identification of RNA-RBP Interactions in Subcellular Compartments by CLIP-Seq.- Monitoring Virus-Induced Stress Granule Dynamics Using Long-Term Live-Cell Imaging.- Single-Molecule Imaging of mRNA Interactions with Stress Granules.- Image-Based Screening for Stress Granule Regulators.- APEX Proximity Labeling of Stress Granule Proteins.
