RNA-Protein Complexes and Interactions: Methods and Protocols(2666 Methods in Molecular Biology)

This second edition updates, complements, and expands upon the first edition by providing a collection of cutting-edge techniques developed or refined in the past few years along with tried-and-true methods. Chapters explore the isolation and characterization of RNA-protein complexes, the analysis and measurement of RNA-protein interaction, and related novel techniques and strategies. Written in the highly successful Methods in Molecular Biology series format, the chapters include brief introductions to the material, lists of necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and a Notes section which highlights tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, RNA-Protein Complexes and Interactions: Methods and Protocols, Second Edition aims to be comprehensive guide for researchers in the field.

Table of Contents:
In vivo cross-linking and co-immunoprecipitation procedure to analyze nuclear tRNA export complexes in yeast cells.- Analysis of Gene Expression Patterns and RNA Localization by Fluorescence in situ Hybridization in Whole Mount Drosophila Testes.- Electrophoretic mobility shift assay (EMSA) and microscale thermophoresis (MST) methods to measure interactions between tRNAs and their modifying enzymes.- Mapping of RNase P ribozyme regions in proximity with a human RNase P subunit protein using Fe(II)-EDTA cleavage and nuclease footprint analyses.- SHAPE to probe RNA structure and RNA-protein interactions in vitro.- Chemical probing of RNA structure in vivo using SHAPE-MaP and DMS-MaP.- Analysis of RNA-protein interaction networks using RNP-MaP.- Native RNA immunoprecipitation (RIP) for precise detection and quantification of protein-interacting RNA.- In vivo cross-linking and co-immunoprecipitation procedure to analyze nuclear tRNA export complexes in yeast cells.- Identify microRNA targets using AGO2-CLASH (Cross-linking, Ligation, And Sequencing of Hybrids) and AGO2-CLIP (Cross-Linking and Immuno-Precipitation) in cells with or without the microRNA of interest depleted.- Ribosomal Profiling by Gradient Fractionation of Cell Lysates.- Global assessment of protein translation in mammalian cells using polysome fractionation.- Fluorescent in situ detection of RNA-Protein interactions in intact cells by RNA-PLA.- Arresting Spliceosome Intermediates at Various Stages of the Splicing Pathway.- Streamlined purification of RNA-protein complexes using UV crosslinking and RNA antisense purification.- MS2-MBP based affinity purification of nucleus- or cytoplasm-localized lncRNA-protein complexes formed in vivo.- RNA and protein interactomes of an RNA-binding protein tagged with FLAG epitopes using combinatory approaches of genome engineering and stable transfection.- Detecting R-loop formation using a plasmid-based in vitro transcription assayMethods to Study RNA-Chromatin Interactions.- Challenges for Studying and Isolating Extracellular Vesicles from Cell-conditioned Media.- Evolution of Cell-Type-Specific RNA Aptamers Via Live Cell-Based SELEX.