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

✍ Scribed by Ren-Jang Lin (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 349
• Edition: 2nd ed. 2023
• Category: Library

✦ Synopsis

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

Dedication
Preface
Contents
Contributors
Chapter 1: A Simple Method for the Detection of Wybutosine-Modified tRNAPheGAA as a Readout of Retrograde tRNA Nuclear Import ...
1 Introduction
2 Materials
2.1 HCl/Aniline Assay
2.2 Gel Electrophoresis
2.3 Transfer and UV Cross Linking
2.4 DIG-Labeled Probes
2.5 Northern Blot Hybridization and Detection of DIG-labeled Probes
3 Methods
3.1 HCl/Aniline Assay
3.2 Northern Blot
4 Notes
References
Chapter 2: Analysis of Gene Expression Patterns and RNA Localization by Fluorescence in Situ Hybridization in Whole Mount Dros...
1 Introduction
2 Materials
3 Methods
3.1 Fly Husbandry
3.2 RNA FISH Probe Design
3.3 RNA FISH
3.4 Combining RNA FISH with Protein Localization
4 Notes
References
Chapter 3: Electrophoretic Mobility Shift Assay (EMSA) and Microscale Thermophoresis (MST) Methods to Measure Interactions Bet...
1 Introduction
2 Materials
2.1 Materials for Preparing DNA Templates
2.2 In Vitro Transcription (IVT)
2.3 RNA Purification Using Fast Performance Liquid Chromatography (FPLC)
2.4 Denaturing Urea PAGE Analysis and Visualization
2.5 RNA Annealing
2.6 Nucleic Acid Precipitation
2.7 Enzyme-RNA Interaction Assay
2.8 EMSA
2.9 MST
3 Methods
3.1 DNA Template Preparation
3.1.1 Primer Design for Single Construct Assembly (60-300 nt)
3.1.2 PCR Assembly of DNA Template
3.1.3 Perform DNA Precipitation
3.2 Run-Off In Vitro Transcription
3.2.1 In Vitro Transcription Reaction
3.2.2 Remove DNA Template
3.2.3 Denaturing Urea PAGE Analysis and Visualization
3.3 RNA Purification Using an Ion Exchange Column
3.3.1 Purification Using a DEAE Ion Exchange Column
3.3.2 Refolding/Annealing of tRNA
3.4 Purification of the Homogenous Form of RNA of Interest
3.5 EMSA
3.6 MST
3.6.1 Screening of Capillaries and Optimization of the Buffer Conditions
3.6.2 Determination of the Concentration of the Fluorescently Labeled Molecule
3.6.3 Sample Preparation for MST Measurements
4 Notes
References
&spi1;Chapter 4: Mapping of RNase P Ribozyme Regions in Proximity with a Human RNase P Subunit Protein Using Fe(II)-EDTA Cleavage and Nuclease Footprint Analyses
1 Introduction
2 Materials
2.1 Chemicals and Solutions
2.2 Solutions and Buffers
2.3 Enzymes and Reagents
2.4 RPP29 Protein, RNAs, and Plasmids
2.5 Equipment Items
3 Methods
3.1 Generation of M1GS Ribozyme
3.1.1 DNA Template
3.1.2 Synthesis of M1GS Ribozyme
3.2 Ribozyme Activity Assay—In Vitro Cleavage of an mRNA Substrate by M1GS RNA With and Without RPP29 Protein
3.2.1 M1GS Cleavage of Substrate csp38 In Vitro Without Rpp29
3.2.2 M1GS Ribozyme Cleavage of Substrate csp38 In Vitro with RPP29
3.3 Nuclease Cleavage Footprint Analysis to Determine the Regions of M1GS RNA Potentially in Close Contact with RPP29 Protein
3.3.1 Dephosphorylation of M1GS Ribozyme
3.3.2 5′ End Labeling with γ-[32P] ATP
3.3.3 3′ End Labeling with [32P]-pCp
3.3.4 RNase Mapping
3.3.5 T1 Control
3.3.6 Alkaline Hydrolysis Control
3.4 Fe(II)-EDTA-Mediated Mapping of the M1GS RNA Regions Potentially Interacting with RPP29 Protein
4 Notes
References
Chapter 5: SHAPE to Probe RNA Structure and RNA-Protein Interactions In Vitro
1 Introduction
2 Materials
2.1 RNA Folding
2.2 SHAPE Assay
2.3 Reverse Transcription
2.4 Sequencing Gel and Imaging
3 Methods
3.1 SHAPE Assay
3.2 5′-End Labeling of Reverse Transcription Primers
3.3 Reverse Transcription
3.4 Polyacrylamide Gel-Pouring, Running, and Exposure
3.5 Data Collection and Data Analysis
4 Notes
References
Chapter 6: Chemical Probing of RNA Structure In Vivo Using SHAPE-MaP and DMS-MaP
1 Introduction
2 Materials
2.1 In Vivo Probing with NAI (for SHAPE-MaP)
2.2 Preparation of Denatured Control (for SHAPE-MaP)
2.3 In Vivo Probing with DMS (for DMS-MaP)
2.4 RNA Extraction (for SHAPE-MaP and DMS-MaP)
2.5 Reverse Transcription (for SHAPE-MaP)
2.6 Reverse Transcription (for DMS-MaP)
2.7 Library Preparation (for SHAPE-MaP and DMS-MaP)
2.8 Data Analysis (for SHAPE-MaP and DMS-MaP)
3 Methods
3.1 In Vivo SHAPE Treatment (for SHAPE-MaP)
3.2 In Vivo DMS Treatment (for DMS-MaP)
3.3 RNA Extraction (for SHAPE-MaP and DMS-MaP)
3.4 Preparation of Denatured Control (for SHAPE-MaP)
3.5 Reverse Transcription (for SHAPE-MaP)
3.6 Reverse Transcription (for DMS-MaP)
3.7 Library Preparation (for SHAPE-MaP and DMS-MaP)
3.8 Data Analysis (for SHAPE-MaP)
3.9 Data Analysis (for DMS-MaP)
4 Notes
References
Chapter 7: Analysis of RNA-Protein Interaction Networks Using RNP-MaP
1 Introduction
2 Materials
2.1 In Vivo Cross-Linking
2.2 Proteinase K Digestion and RNA Extraction
2.3 MaP Reverse Transcription
2.4 Sequencing Library Preparation for Mutational Profiling
2.5 Data Analysis
3 Methods
3.1 In Vivo Cross-Linking
3.2 Protease Digestion and RNA Extraction
3.3 MaP Reverse Transcription
3.4 Two-Step PCR for Small RNA MaP Libraries and Deep Sequencing
3.5 Mutation Counting and RNP-MaP Analysis
3.6 RNP-MaP Correlation
4 Notes
References
Chapter 8: Native RNA Immunoprecipitation (RIP) for Precise Detection and Quantification of Protein-Interacting RNA
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cell Lysis and RIP
2.3 RNA Extraction, Purification, and Quantification
3 Methods
3.1 Cell Harvesting
3.2 Cell Lysis
3.3 Immunoprecipitation of Protein-RNA Complex
3.4 RNA Purification
3.5 cDNA Synthesis
3.6 Real-Time PCR
4 Notes
References
Chapter 9: In Vivo Cross-Linking and Co-Immunoprecipitation Procedure to Analyze Nuclear tRNA Export Complexes in Yeast Cells
1 Introduction
2 Materials
2.1 Formaldehyde Cross-Linking and Cryolysis of Yeast Cells
2.2 Co-Immunoprecipitation
2.3 Gel Electrophoresis of Proteins
2.4 Protein Staining
2.5 Western Blot
2.6 RT-PCR and RT-qPCR
2.7 Agarose Gel Electrophoresis
3 Methods
3.1 In Vivo Cross-Linking
3.2 Immunoprecipitation of tRNA-Tagged Export Complexes
3.3 SYPRO RUBY Staining
3.4 Western Blot of Immunoprecipitated Proteins
3.4.1 RT-PCR Analysis of Co-Immunoprecipitated RNA
3.4.2 RT-qPCR Analysis of Co-Immunoprecipitated RNA
4 Notes
References
Chapter 10: Identify MicroRNA Targets Using AGO2-CLASH (Cross-linking, Ligation, and Sequencing of Hybrids) and AGO2-CLIP (Cro...
1 Introduction
2 Materials
3 Methods
3.1 AGO2-CLASH (See Fig. 1, Left Side)
3.1.1 Cross-Linking and RNA Isolation
3.1.2 RNA Ligation and Sequencing (See Note 8)
3.1.3 CLASH Data Analysis
3.2 AGO2-IP (AGO2-CLIP) (Fig. 1 Right Side)
3.2.1 Cross-Linking and RNA Isolation
3.2.2 RNA Sequencing
3.2.3 AGO2-CLIP Data Analysis
4 Notes
References
&spi1;Chapter 11: Ribosomal Profiling by Gradient Fractionation of Cell Lysates
1 Introduction
2 Materials
2.1 Stock Solutions
2.2 Other Reagents and Equipment
2.3 Buffers and Solutions
3 Methods
3.1 Gradient Preparation
3.2 Harvesting Cells/Biological Material
3.3 Centrifugation
3.4 Fraction Collection
4 Notes
References
Chapter 12: Global Assessment of Protein Translation in Mammalian Cells Using Polysome Fractionation
1 Introduction
2 Materials
2.1 Sucrose Gradient Components
2.2 Cell Lysis Components
2.3 Ultracentrifugation Components
2.4 Fractionation Components
3 Methods
3.1 Sucrose Gradients Preparation
3.2 Cell Preparation
3.3 Cell Lysis
3.4 Sucrose Gradient Centrifugation
3.5 Fractionation and Collection
4 Notes
References
Chapter 13: Fluorescent In Situ Detection of RNA-Protein Interactions in Intact Cells by RNA-PLA
1 Introduction
2 Materials
2.1 Cell Culture
2.2 RNA-PLA Probes and Oligonucleotides
2.3 Cell Fixation and Permeabilization (see Note 2)
2.4 Antibody and RCA Assay
2.5 Equipment
3 Methods
3.1 Cell Fixation, Permeabilization, and Blocking for PLA
3.2 PLUS Probe and Primary Antibody Incubation
3.3 Secondary Antibody (MINUS Probe) Incubation
3.4 Ligation and Amplification
3.5 Image Capture and Analysis (see Note 14)
4 Notes
References
Chapter 14: In Vitro Reconstitution of Pseudouridylation Catalyzed by Human Box H/ACA Ribonucleoprotein Particles
1 Introduction
2 Materials
2.1 Human Cell Culture and HeLa Cell Extract Preparation
2.2 Box H/ACA Guide RNA Preparation
2.3 Radiolabeled Substrate RNA Preparation
2.4 In Vitro Reconstitution Assay
3 Methods
3.1 Human Cell Culture and HeLa Cell Extract Preparation
3.2 Box H/ACA Guide RNA Preparation
3.3 Radiolabeled Substrate RNA Preparation
3.4 In Vitro Reconstitution Assay
4 Notes
References
Chapter 15: Arresting Spliceosome Intermediates at Various Stages of the Splicing Pathway
1 Introduction
2 Materials
2.1 Preparation of 32P-Labled Actin Pre-mRNA
2.2 Gel-Purification of RNA Substrate
2.3 Splicing Extract Preparation
2.4 In Vitro Pre-mRNA Splicing
2.5 Immunodepletion and Immunoprecipitation
3 Methods
3.1 Preparation of Substrate Pre-mRNA
3.2 Preparation of Splicing Extracts
3.3 In Vitro Pre-mRNA Splicing Reaction
3.4 Immunodepletion
3.5 Immunoprecipitation
3.6 Use of Dominant-Negative Mutants of DExD/H-Box Proteins
3.7 Stalling the Spliceosome at Various Stages
3.7.1 Arresting the Pre-Activated Spliceosome by Limiting ATP Concentration
3.7.2 Arresting the Pre-Catalytic Spliceosome at the Post-Activation Stage
3.7.3 Arresting the Spliceosome in the Second Step
3.7.4 Arresting the Spliceosome After Exon Ligation
3.7.5 Arresting the Spliceosome Prior to Disassembly
4 Notes
References
Chapter 16: Streamlined Purification of RNA-Protein Complexes Using UV Cross-Linking and RNA Antisense Purification
1 Introduction
2 Materials
2.1 Specialized Equipment and Materials
2.2 Materials for Tissue Culture and Cell Harvesting
2.3 Buffers for Cell Lysis and RNA-Protein Complex Purification
3 Methods
3.1 Growth of Large-Scale Cultures of Cancer Cells
3.2 Cross-Linking and Harvesting Cells
3.3 Designing Antisense Capture Probes
3.4 Preparation of Whole Cell Lysates
3.5 Capture and Purification of UV Cross-Linked RNA-Protein Complexes
3.6 Elution of Captured RNA
3.7 Elution of Captured Protein
3.8 Quantitation of Captured Proteins by Mass Spectrometry
3.9 Mass Spectrometry Data Analysis
4 Notes
References
Chapter 17: MS2-MBP-Based Affinity Purification of Nucleus- or Cytoplasm-Localized lncRNA-Protein Complexes Formed In Vivo
1 Introduction
2 Materials
2.1 Establishment of Inducible Stable Cell Line with Flp-In T-Rex System
2.2 Determination of the Time Course for lncRNA Export
2.3 Expression and Purification of MS2-MBP Fusion Protein
2.4 Preparation of NE and CE
2.5 LncRNP Purification for Mass Spectrometry Analysis
3 Methods
3.1 Establishment of Inducible Stable Cell Line with Flp-In T-Rex System
3.2 Determination of the Time Course for lncRNA Expression and Export
3.3 Expression and Purification of MS2-MBP Fusion Protein
3.4 Preparation of Nuclear Extract (NE) and Cytoplasmic Extract (CE)
3.5 LncRNP Purification for Mass Spectrometry Analysis
4 Notes
References
Chapter 18: RNA and Protein Interactomes of an RNA-Binding Protein Tagged with FLAG Epitopes Using Combinatory Approaches of G...
1 Introduction
2 Materials
2.1 HDR-Mediated Flag Knock-In
2.2 Plasmid Construction for Flag-Tagged RBP
2.3 Transfection and Selection for Stable Cell Line
2.4 CRISPR-Cas9 gRNA Design and Transfection
2.5 Cross-Linking and Immunoprecipitation
3 Methods
3.1 HDR-Mediated Flag Insertion to the Endogenous Gene Locus
3.1.1 To Design and Clone the Cas9-sgRNA Plasmid (Adapted from the Method)
3.1.2 To Design and Construct the Donor Template Plasmid
3.1.3 Transfection
3.2 Plasmid Construction for Stable Flag-Tagged RBP Expression
3.3 Generation of Stable Cell Line Containing Flag-Tagged RBP
3.4 Knockout of Endogenous RBP Using CRISPR-Cas9
3.5 Immunoprecipitation Using Anti-Flag Antibody for RNA-Protein Interactome Studies
3.5.1 Cross-Linking of RNA-Protein Interaction Using Ultraviolet Light (UV) or Formaldehyde
3.5.2 Immunoprecipitation
3.5.3 Library Construction for RBP Footprinting
3.6 Immunoprecipitation Using Anti-Flag Antibody for Protein-Protein Interactome Studies
3.6.1 Preparation of Cell Extracts and Immunoprecipitation
3.6.2 Preparative Gel Electrophoresis of Protein Complexes for In-Gel Digestion
4 Notes
References
Chapter 19: Detecting R-Loop Formation Using a Plasmid-Based In Vitro Transcription Assay
1 Introduction
2 Materials
2.1 In Vitro Transcription
2.2 RNase A and RNase H Treatment
2.3 Agarose Gel Electrophoresis
2.4 Phenol/Chloroform Extraction and Ethanol Precipitation
2.5 Dot-Blot Analysis
2.6 DRIP and Quantitative PCR Analysis
3 Methods
3.1 In Vitro Transcription
3.2 Agarose Gel Electrophoresis
3.3 Phenol/Chloroform Extraction and Ethanol Precipitation
3.4 Dot-Blot Analysis
3.5 DRIP and Quantitative PCR (DRIP-qPCR) Analysis
4 Notes
References
Chapter 20: Methods to Study RNA-Chromatin Interactions
1 Introduction
2 One RNA Versus Whole-Genome Profiling Techniques
2.1 Chromatin Isolation by RNA Purification (ChIRP)
2.2 RNA Antisense Purification (RAP)
2.3 Capture Hybridization Analysis of RNA Targets (CHART)
3 All RNAs Versus Whole-Genome Profiling Methods
3.1 Mapping of RNA-Genome Interactome (MARGI)
3.2 Global RNA Interaction with DNA Sequencing (GRID-seq)
3.3 Chromatin-Associated RNA Sequencing (ChAR-seq)
3.4 RNA and DNA Interacting Complexes Ligated and Sequenced (RADICL-seq)
4 Other Methods for Detecting RNA-DNA Interactions
5 Conclusion
References
Chapter 21: Challenges for Studying and Isolating Extracellular Vesicles from Cell-Conditioned Media
1 Introduction
1.1 Considerations Before Working with EV
1.2 Methods for Enriching EV
1.2.1 Differential Ultracentrifugation
1.2.2 Size Exclusion Chromatography
1.2.3 Precipitation-Based Methods
1.2.4 Density Gradients
1.2.5 Functional Assays with EV
2 Materials
3 Methods
3.1 Generating Cell-Conditioned Media from Adherent Cells
3.2 Differential Ultracentrifugation
3.3 Extracting RNA from EV with Trizol
4 Notes
References
Chapter 22: Evolution of Cell-Type-Specific RNA Aptamers via Live Cell-Based SELEX
1 Introduction
2 Materials
2.1 Cell Culture and Cell-Surface Target Protein Detection by Flow Cytometry
2.2 Generation of Initial DNA Template by PCR Amplification
2.3 Preparation of RNA Library by T7 RNA Polymerase
2.4 8% Denaturing Polyacrylamide Gel (7 M Urea)
2.5 Live Cell-Based SELEX
2.6 Monitor the Progress of SELEX or Detect Binding Affinity by qRT-PCR
2.7 Illumina High-Throughput Sequencing (HTS) and Data Analysis
2.8 Cell-Surface Binding and Internalization Studies by Flow Cytometry
2.9 Cell-Surface Binding and Internalization Studies by Live Cell Confocal Microscopy
2.10 Validate the Target Protein via Gel Shift Assay
2.11 Validate the Target Protein via siRNA Knockdown
3 Methods
3.1 Detection of the Cell-Surface Target Protein
3.2 Generation of Initial Library DNA Template by PCR Amplification
3.3 Preparation of RNA Library by T7 RNA Polymerase
3.4 8% Denaturing Polyacrylamide Gel (7 M Urea)
3.5 Live Cell-Based SELEX
3.6 Monitor the Progress of SELEX or Detect Binding Affinity by qRT-PCR
3.7 Illumina High-Throughput Sequencing (HTS) and Data Analysis
3.8 Cell-Surface Binding and Internalization Studies by Flow Cytometry
3.9 Cell-Surface Binding and Internalization Studies by Live Cell Confocal Microscopy
3.10 Validate Aptamer Binding to Target Protein via Gel Shift Assay
3.11 Validate the Target Protein via siRNA Knockdown
4 Notes
References
Index