Post-Transcriptional Gene Regulation

Preface
Part I: Bioinformatics
Part II: Expression Studies
Part III: Interactomics
Part IV: The RNA Lifecycle
Part V: RNA Modifications
Contents
Contributors
Part I: Bioinformatics
Chapter 1: Introduction to Bioinformatics Resources for Post-transcriptional Regulation of Gene Expression
1 Introduction
2 Tools for Omics Datasets Analysis
2.1 Expression Profiling
2.2 Small RNA Profiling
2.3 Binding Sites Identification
3 PTR Databases
3.1 RBPs Binding Sites
3.2 miRNAs Binding Sites and ncRNAs
3.3 Cis-Elements
3.4 Integrative
4 Prediction Tools
4.1 RBP Targets
4.2 miRNA Targets
4.3 SNPs Impact on RNA Secondary Structure
5 Tools for RNA Sequence and Structure Motif Search
6 Pipelines for PTR: Two Case Studies
6.1 Case Study 1: Impact of a Treatment on Translation
6.2 Case Study 2: Identification of Targets and Binding Preference for an RBP
References
Chapter 2: Predicting RNA Secondary Structure Using In Vitro and In Vivo Data
1 Introduction
2 Materials
2.1 Artificial Neural Network
2.2 Protein-RNA Interactions Using the catRAPID Algorithm
3 Methods
3.1 Predicting In Vitro Data
3.1.1 Selecting Higher-Propensity Nucleotides from Different Experimental Techniques
3.1.2 Encoding Sequence Information into the Predictive Approach
3.1.3 In Vitro ANN Architecture: Training and Testing
3.2 Predicting In Vivo Data
3.2.1 Filtering and Selecting In Vivo Data
3.2.2 Integrating Protein Data into the In Vivo Predictive Approach
3.2.3 In Vivo ANN Architecture Including Protein Data: Training and Testing
4 Notes
References
Chapter 3: RBPmap: A Tool for Mapping and Predicting the Binding Sites of RNA-Binding Proteins Considering the Motif Environme...
1 Introduction
2 The RBPmap Tool
2.1 RBPmap Algorithm
2.1.1 The Match Score
2.1.2 The Weighted Rank (WR) Score
2.2 RBPmap Features
2.2.1 RBPmap Motif Database
2.2.2 Output Features
3 RBPmap Applications
3.1 Predicting RBP-RNA Interactions Involved in Post-transcriptional Regulation
3.2 Predicting RBP-RNA Interactions Associated with Viral Infections
3.3 Evaluating RBPmap Predictions Based on High-Throughput Binding Data
4 Notes
References
Part II: Expression Studies
Chapter 4: Analysis of mRNA Translation by Polysome Profiling
1 Introduction
2 Materials
2.1 Cell Lysis
2.2 Sucrose Gradient
2.3 Fractionation
2.4 RNA Analysis
2.5 Protein Analysis
3 Methods
3.1 Cell Lysis
3.2 Sucrose Gradient Formation, Loading, and Ultracentrifugation (Fig. 1b)
3.3 Fractionation
3.4 RNA Analysis
3.5 Protein Analysis
4 Notes
References
Chapter 5: Exploring Ribosome-Positioning on Translating Transcripts with Ribosome Profiling
1 Introduction
2 Materials
2.1 Library Preparation Components
3 Methods
3.1 Culture and Pre-treatments
3.1.1 Culture: E. coli
3.1.2 Culture: S. cerevisiae
3.1.3 Culture: Mammalian Cells
3.2 Cell Harvest by Filtration
3.2.1 E. coli and S. cerevisiae
3.2.2 Mammalian Cells
3.3 Cell Lysis
3.3.1 Cell Lysis Using Freezer Mill: E. coli and S. cerevisiae (See Note 7)
3.3.2 Cell Lysis Using Mortar and Pestle: E. coli and S. cerevisiae
3.3.3 Cell Lysis: Mammalian Cells
3.4 Pellet Ribosome-Associated mRNAs
3.5 Quantification for Nuclease Digestion
3.5.1 Nuclease Digestion: E. coli (See Note 9)
3.5.2 Nuclease Digestion: S. cerevisiae and Mammalian Cells
3.6 Sucrose Gradient and Monosome Isolation
3.6.1 Monosome Isolation
3.6.2 Extract RNA from Monosome Fraction
3.7 Footprint Fragment Purification
3.7.1 Gel Size Selection
3.7.2 Gel Extraction
3.8 Ribosomal RNA Depletion Using RiboZero Plus (Illumina, 20036696) [Optional: See Note 14]
3.8.1 Clean-up RNA (Using Zymo Oligo Clean and Concentrator)
3.9 3′ Linker Ligation
3.9.1 Preparation of 20 μM Preadenylated 3′ Linker
3.9.2 3′ Dephosphorylation
3.9.3 3′ Ligation with Pre-adenylated Linker
3.9.4 Selective Degradation of Pre-adenylated Linkers
3.9.5 Pool, Clean, and Concentrate Samples
3.9.6 Evaluate Samples and Controls
3.10 5′ Linker Ligation
3.10.1 5′ Phosphorylation
3.10.2 5′ Ligation
3.11 Hybridization of RT Primer
3.12 Reverse Transcription
3.12.1 Reaction Mix
3.12.2 RNA Hydrolyzation and Neutralization
3.12.3 RT Size-Selection Gel
3.12.4 RT Gel Extraction
3.13 PCR
3.13.1 Pilot PCR
3.13.2 Pilot PCR Gel
3.13.3 Preparative PCR Amplification
3.13.4 Preparative PCR Gel
3.13.5 Preparative Gel Extraction
3.14 Data Analysis
4 Notes
References
Chapter 6: Identification of RNA Binding Partners of CRISPR-Cas Proteins in Prokaryotes Using RIP-Seq
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Bacterial Culture and Collection
2.3 RNA Co-immunoprecipitation
2.4 Quality Control and DNase I Treatment
3 Methods
3.1 Bacterial Culture and Pellet Collection
3.2 Cell Lysis and Incubation with Antibody/Protein A-Sepharose Beads
3.3 Quality Control for Verification of Successful CjeCas9-3xFLAG Immunoprecipitation
3.3.1 Western Blot
3.3.2 Northern Blot
3.4 DNase I Treatment
3.5 cDNA Library Preparation, Sequencing, and Analysis
3.6 Outlook
4 Notes
References
Chapter 7: Rapidly Characterizing CRISPR-Cas13 Nucleases Using Cell-Free Transcription-Translation Systems
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Design of the Expression Constructs
3.1.1 Nuclease
3.1.2 gRNA
3.1.3 Acrs
3.2 Purification of DNA
3.2.1 Plasmid DNA
3.2.2 Linear DNA
3.3 TXTL Pre-expression and RNA Cleavage Assay to Observe On-target and Collateral RNA Cleavage Using Fluorescence Reporters
3.4 TXTL Pre-expression and RNA Cleavage Assay to Assess Inhibition by Acrs
3.4.1 Pre-expression of Nuclease and gRNA
3.4.2 Pre-expression of Acrs
3.4.3 RNA Cleavage Assay
3.5 Data Processing
4 Notes
References
Part III: Interactomics
Chapter 8: Studying RNP Composition with RIP
1 Introduction
2 Materials
2.1 Tissue Cell Culture Components
2.2 Sample Collection Components
2.3 RNP Immunoprecipitation Components
3 Methods
3.1 Tissue Culture and RNP Lysate Collection
3.2 Antibody Coating of Protein A/G Beads
3.3 Immunoprecipitation Reaction and RNA Extraction
3.4 RNP Immunoprecipitation Controls
4 Notes
References
Chapter 9: PAR-CLIP: A Method for Transcriptome-Wide Identification of RNA Binding Protein Interaction Sites
1 Introduction
2 Materials
3 Methods
3.1 Preparation of UV-Crosslinked RNPs
3.1.1 Expanding Cells
3.1.2 UV-Crosslinking for Adherent Cells
3.1.3 UV-Crosslinking for Cells Grown in Suspension
3.1.4 Cell Lysis and RNase T1 Digest
3.2 Immunoprecipitation and Recovery of Crosslinked Target RNA Fragments
3.2.1 Preparation of Magnetic Beads
3.2.2 Immunoprecipitation (IP), Second RNase T1 Digestion, and Dephosphorylation
3.2.3 Radiolabeling of RNA Segments Crosslinked to Immunoprecipitated Proteins
3.2.4 SDS Polyacrylamide Gel Electrophoresis, Transfer, and Recovery of RNA from Nitrocellulose Membrane
3.2.5 Proteinase K Digestion
3.3 cDNA Library Preparation and Deep Sequencing
3.3.1 3′ Adapter Ligation
3.3.2 5′ Adapter Ligation
3.3.3 Reverse Transcription
3.3.4 PCR Amplification
Optional: Determination of Incorporation Levels of 4-Thiouridine into Total RNA
3.4 PAR-CLIP Analysis
4 Notes
References
Chapter 10: A Pipeline for Analyzing eCLIP and iCLIP Data with Htseq-clip and DEWSeq
1 Introduction
1.1 Properties of e/iCLIP Data
1.2 Controls
1.3 From Raw to Aligned Reads
2 Data Analysis
2.1 System Requirements
2.2 Data Requirements
2.3 From Aligned Reads to Counts with Htseq-Clip
2.3.1 Installation Requirements
2.3.2 Quick Installation
2.3.3 Conda Environment
2.3.4 Version Used in this Chapter
2.3.5 Help
2.3.6 Overview
2.4 A Minimal Reproducible Example
2.4.1 First Steps
2.4.2 Download BAM Files
2.4.3 Download Annotation
2.4.4 Prepare Annotation
2.4.5 Flatten Annotation
2.4.6 Create Sliding Windows
2.4.7 Create Mapping
2.5 Extracting and Counting Truncation Sites
2.5.1 Extract Crosslink Sites
2.5.2 Count and Aggregate Sites
2.5.3 Create Count Matrix
2.6 Additional Help
2.7 Differential Analysis with DEWSeq
2.7.1 Installation of DEWSeq and Required Packages
2.7.2 Loading Libraries
2.7.3 Help
2.7.4 Data Import
2.7.5 Estimate Size Factors
2.7.6 Prefiltering
2.7.7 Estimate Dispersion and Model Fit
2.7.8 Differential Expressed Windows Analysis
2.7.9 Multiple Hypothesis Correction
2.7.10 Combining Windows to Regions
2.7.11 Exporting Results
2.7.12 Visualization: Volcano Plot
2.7.13 Session Info
2.7.14 Additional Help
3 Further Improvements and Variations of the Analysis
3.1 Advanced Models
3.2 Quality Control
3.3 Additional Variations
4 Other Tools
References
Chapter 11: Identification of miRNAs Bound to an RNA of Interest by MicroRNA Capture Affinity Technology (miR-CATCH)
1 Introduction
2 Materials
2.1 Formaldehyde Cross-Linking and Cell Lysis
2.2 Streptavidin Bead Preparation and mRNAs Pull-Down
2.3 miR-CATCH: Total RNA and Capture Validations
2.4 Analysis of Pulled-Down miRNAs
2.5 Laboratory Equipment
2.6 Other Labware and Consumables
3 Methods
3.1 Design of Biotinylated Capture and Control DNA Oligonucleotides
3.2 Formaldehyde Cross-Linking
3.3 Cell Lysis
3.4 Streptavidin Beads Preparation, Oligonucleotide Immobilization, and mRNAs Pull-Down
3.5 Elution and Cross-Link Reversal
3.6 miR-CATCH: Total RNA Validation
3.7 miR-CATCH: Capture Validation
3.8 Analysis of Captured miRNAs
4 Notes
References
Chapter 12: Identifying Protein Interactomes of Target RNAs Using HyPR-MS
1 Introduction
2 Materials
2.1 Formaldehyde Cross-Linking
2.2 Cell Lysis, Target RNA Isolation, and RT-qPCR
2.3 Protein Preparation and Mass Spectrometric Analysis
3 Methods
3.1 Experimental Design
3.1.1 Design of Control Experiment(s)
3.1.2 Design of Capture Oligonucleotides
3.1.3 Design of qPCR Assay(s)
3.2 Formaldehyde Cross-Linking
3.3 Cell Lysis and RNA Solubilization
3.4 Hybridization Capture and Elution
3.5 RNA Purification and RT-qPCR Analysis
3.5.1 RNA Purification
3.5.2 Reverse Transcription
3.5.3 qPCR
3.6 Protein Preparation and Mass Spectrometric Analysis
3.6.1 eFASP
3.6.2 C18 Solid-Phase Extraction
3.6.3 Mass Spectrometry
3.7 Mass Spectrometric Data Analysis
4 Notes
References
Part IV: The RNA Lifecycle
Chapter 13: Visualization and Quantification of Subcellular RNA Localization Using Single-Molecule RNA Fluorescence In Situ Hy...
1 Introduction
2 Materials
2.1 Stable or Transiently Transfected Cell Lines
2.2 Probe
2.2.1 Commercially Available Probes
2.2.2 smiFISH
2.2.3 Enzymatically Labeled Probes
2.3 smFISH
2.4 Microscopy and Image Analysis
3 Methods
3.1 Stellaris
3.2 smiFISH
3.2.1 smiFISH Probe Design
3.2.2 smiFISH Probe Synthesis
3.3 Enzymatically Labeled Probes
3.3.1 Probe Design
3.3.2 Probe Synthesis
3.3.3 PAGE Analysis of the Labeled Oligonucleotides
3.4 Hybridization of Probes
3.5 Imaging
3.6 Data Analysis: FISH-Quant
4 Notes
References
Chapter 14: Single-Molecule RNA Imaging Using Mango II Arrays
1 Introduction
2 Materials
2.1 Cell Lines and Media
2.2 Transfection and Fixation Reagents
2.3 Fluorescent Dyes
2.4 Microscopy
3 Methods
3.1 Cell Culture
3.2 Transfecting Cells
3.3 Fixation and Immunostaining
3.4 Microscopy
3.4.1 Microscopy for Fixed Samples
3.4.2 Microscopy for Live Samples
3.5 Image Analysis
3.5.1 Image Processing
3.5.2 Polarization Index Calculation
4 Notes
References
Chapter 15: Genome-Wide Identification of Polyadenylation Dynamics with TED-Seq
1 Introduction
2 Materials
2.1 Poly(A) RNA Isolation
2.2 3′ RNA Adaptor Ligation
2.3 RNA Cleanup
2.4 RNA Fragmentation
2.5 5′ RNA Phosphorylation
2.6 5′ RNA Adaptor Ligation
2.7 Reverse Transcription
2.8 First Round Amplification of the Library
2.9 PCR Cleanup Using SPRI Beads
2.10 Size Selection of the Library
2.11 Second Round Full Amplification of the Library
2.12 Second Size Selection and PCR Cleanup
2.13 TED-seq Data Analysis
3 Methods
3.1 Poly-A RNA Isolation
3.2 3′ RNA Adaptor Ligation
3.3 RNA Cleanup
3.4 RNA Fragmentation
3.5 5′ RNA Phosphorylation
3.6 5′ RNA Adaptor Ligation
3.7 Reverse Transcription (RT)
3.8 First Round Amplification of the Library
3.9 PCR Cleanup Using SPRI Beads
3.10 Size Selection of the Library
3.11 Second Round Full Amplification of the Library
3.12 Second Size Selection and Cleanup
3.13 TED-seq Data Visualization
4 Notes
References
Chapter 16: In Vivo RNA Structure Probing with DMS-MaPseq
1 Introduction
2 Materials
2.1 Buffers
2.2 Oligos
3 Methods
3.1 In Vivo Dimethyl Sulfate (DMS) Modification in Drosophila Ovary
3.2 In Vivo Dimethyl Sulfate (DMS) Modification in HEK293T Cells (See Note 4)
3.3 TRIzol RNA Extraction
3.4 RNase H-Based Ribosomal RNA Extraction
3.5 DNase Treatment (See Note 16)
3.6 Reverse Transcription Polymerase Chain Reaction (RT-PCR)
3.6.1 Reverse Transcription
3.6.2 Polymerase Chain Reaction (See Note 19)-For Advantage HF Kit
3.6.3 Polymerase Chain Reaction (See Note 19)-For Phusion Kit
3.7 Library Preparation Strategy
3.7.1 Fragmentation (See Note 20)
3.7.2 ZYMO Column Cleanup (see Note 23)
3.7.3 Dephosphorylation
3.7.4 Linker Ligation (See Note 24)
3.7.5 Linker Degradation (See Note 25)
3.7.6 Reverse Transcription (See Note 26)
3.7.7 Size Selection on Gel (See Note 27)
3.7.8 Gel Extraction (Using 300 mM NaCl to Extract)
3.7.9 Circular Ligation
3.7.10 PCR to Add Sequencing Handles
3.7.11 PCR Gel
3.8 Sequencing
4 Notes
References
Chapter 17: Transcriptome-Wide Profiling of RNA Stability
1 Introduction
2 Materials
2.1 General Equipment
2.2 Reagents and Buffers
2.3 Cell Lines
3 Methods
3.1 HeLa Cell Culture Procedures
3.2 Assessment of s4U-Induced Cytotoxicity
3.3 General Considerations When Measuring Transcriptome Stability in HeLa Cells
3.4 Pulse-Chase Metabolic RNA Labeling
3.5 RNA Extraction
3.6 s4U-Alkylation (Iodoacetamide Treatment)
3.7 DNase Treatment
3.8 Depletion of Ribosomal RNA
3.8.1 Hybridization
3.8.2 Bead Washing
3.8.3 Depletion
3.8.4 Purification
3.9 RNA Library Preparation
3.9.1 RNA Fragmentation and Priming
3.9.2 First Strand cDNA Synthesis
3.9.3 Second Strand cDNA Synthesis
3.9.4 Purification of Double-Stranded cDNA
3.9.5 End Prep of cDNA Library
3.9.6 Adaptor Ligation
3.9.7 Purification of the Ligation Reaction
3.9.8 PCR Enrichment of Adaptor-Ligated DNA
3.9.9 Purification of the PCR Reaction
3.9.10 Assess Library Quantity and Quality
3.10 High-Throughput Sequencing
3.11 Data Analysis
4 Notes
References
Chapter 18: High-Throughput Quantitation of Yeast uORF Regulatory Impacts Using FACS-uORF
1 Introduction
2 Materials
3 Methods
3.1 Oligo Library PCR Amplification
3.2 Restriction Digest-Digest the Oligo PCR Products and Vector
3.3 Vector Phosphatase Treatment
3.4 Ligation
3.5 E. coli Transformation
3.6 Harvesting Colonies and Extracting Massively Parallel Reporter (MPR) Plasmid DNA
3.7 Making Competent Yeast
3.8 Transforming Saccharomyces yeast with the pGM-YFP, and pGM-mCherry Controls
3.9 Transforming Saccharomyces yeast with the MPR Library
3.10 FACS
3.11 Prepare Plasmid DNA Libraries
4 Notes
References
Part V: RNA Modifications
Chapter 19: m6A RNA Immunoprecipitation Followed by High-Throughput Sequencing to Map N6-Methyladenosine
1 Introduction
2 Materials
2.1 Buffers
2.2 m6A MeRIP
3 Methods
3.1 Prepare RNA
3.2 RNA Fragmentation and Purification
3.3 Preparation of Anti-m6A Antibody Conjugated to Dynabeads Protein A for Immunoprecipitation
3.4 Immunoprecipitation
3.5 Elution of m6A-Containing mRNA
4 Notes
References
Chapter 20: Detecting m6A with In Vitro DART-Seq
1 Introduction
2 Materials
2.1 Induction of DART Protein Expression in E. coli
2.2 Purification of DART Protein by Affinity Chromatography
2.3 Dialysis of Purified DART Protein
2.4 Assessing Protein Quality and Purity by Coomassie and Immunoblot
2.5 Long-Term Storage of APOBEC1-YTH Purified Proteins
2.6 In Vitro DART Assay
3 Methods
3.1 Induction of DART Protein Expression in E. coli
3.2 Purification of DART Protein by Affinity Chromatography
3.3 Dialysis of Purified DART Protein
3.4 Assessing Protein Quality and Purity by Coomassie Stain and Immunoblot
3.5 Long-Term Storage of APOBEC1-YTH Purified Proteins
3.6 In Vitro DART Assay
4 Notes
References
Chapter 21: Target-Specific Profiling of RNA m5C Methylation Level Using Amplicon Sequencing
1 Introduction
2 Materials
2.1 In Vitro Transcription Components
2.2 Sodium Bisulfite Conversion Components
2.3 cDNA Synthesis Components
2.4 PCR Amplification Components
2.5 Agarose Gel Electrophoresis and PCR Purification Components
2.6 Library Preparation Components
3 Methods
3.1 RNA Extraction and DNase Treatment
3.2 Generation of the Renilla Luciferase (R-Luc) In Vitro Transcript Spike-in Control (See Note 7)
3.3 Bisulfite Conversion of RNA
3.4 cDNA Synthesis
3.5 Bisulfite PCR Primer Design
3.6 PCR Amplification and Pooling of Amplicons
3.7 MiSeq Amplicon Sequencing Library Preparation
3.8 Validation and Quantification of the Libraries
3.9 Preparation of the Sample Sheet
3.10 Dilution of the Libraries and Loading of the Cartridge (See Note 30)
3.11 Alignment of the MiSeq Data
4 Notes
References
Chapter 22: Transcriptome-Wide Identification of 2′-O-Methylation Sites with RibOxi-Seq
1 Introduction
2 Materials
2.1 RNA Fragmentation
2.2 RNA Oxidation, β-Elimination, and Dephosphorylation
2.3 3′-DNA Linker Ligation
2.4 PAGE Gel Purification
2.5 5′-RNA Linker Ligation
2.6 cDNA Synthesis
2.7 Library Amplification
2.8 Library Quantification and Visualization
3 Methods
3.1 RNA Preparation and Fragmentation
3.2 RNA Oxidation, β-Elimination, and Dephosphorylation
3.3 3′-DNA Linker Ligation
3.4 5′-RNA Linker Ligation
3.5 cDNA Synthesis
3.6 Library Amplification and Library QC
3.7 Data Processing and Analysis
4 Notes
References
Index