Alternative Splicing: Methods and Protocols (Methods in Molecular Biology, 2537)

Description

This detailed volume collects commonly used and cutting-edge methods to analyze alternative splicing, a key step in gene regulation. After an introduction of the alternative splicing mechanism and its targeting for therapeutic strategies, the book continues with techniques for analyzing alternative splicing profiles in complex biological systems, visualizing and localizing alternative spliced transcripts with cellular and sub-cellular resolution, probing regulators of alternative splicing, as well as assessing the functional consequences of alternative splicing. Written for the highly successful Methods in Molecular Biology series, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, reproducible protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and practical, Alternative Splicing: Methods and Protocols serves as an ideal guide for both RNA aficionados that want to implement novel approaches in their labs and novices undertaking alternative splicing projects. Preface Contents Contributors Chapter 1: Alternative Splicing in Human Biology and Disease 1 Introduction 2 Mechanism of Pre-mRNA Splicing 3 Regulation of Alternative Splicing 4 Alternative Splicing in Human Disease 5 Myotonic Dystrophy Type I (DM1) 6 Spinal Muscular Atrophy (SMA) 7 Amyotrophic Lateral Sclerosis Linked to TDP-43 and FUS 8 Frontotemporal Dementia with Parkinsonism Linked to Chromosome 17 (FTDP-17) 9 Conclusion and Outlook References Chapter 2: Targeting Alternative Splicing for Therapeutic Interventions 1 Introduction 2 Pre-mRNA Splicing 2.1 Constitutive Splicing 2.2 Alternative Splicing 3 Strategies for Therapeutically Manipulating Splicing 3.1 Antisense Oligonucleotides 3.1.1 Exon Skipping 3.1.2 Blocking Cryptic Splice Sites 3.1.3 Modulating Alternative Splicing 3.2 Small Molecules 3.2.1 Activators 3.2.2 Inhibitors 3.3 Emerging Technologies 4 Conclusion and Future Perspectives References Chapter 3: An Optimized Protocol for the Mapping of Cell Type-Specific Ribosome-Associated Transcript Isoforms from Small Mous... 1 Introduction 2 Materials 2.1 Preparation of Work Environment 2.2 Preparation for Immunoprecipitation of Tagged Ribosome and Associated RNAs 2.3 Reagents and Equipment for RNA Purification and Clean-up 2.4 RNA Quantification and Library Preparation 2.5 Transgenic Mouse Lines 2.6 Time Considerations 3 Methods 3.1 Preparation of Reagents and Sterile Equipment 3.2 Preparation of Tissue Homogenate 3.3 Immunoprecipitation of Epitope-Tagged Ribosomes 3.4 RNA Extraction and Purification 3.5 Testing RNA Concentration and Quality 4 Notes References Chapter 4: FACS-Based Neuronal Cell Type-Specific RNA Isolation and Alternative Splicing Analysis 1 Introduction 2 Materials 2.1 Brain Tissue Harvest 2.2 Tissue Digestion and Dissociation 2.3 Cell Suspension Purification 2.4 Cell Sorting 2.5 RNA Extraction 3 Methods 3.1 Brain Tissue Harvest 3.2 Tissue Digestion and Dissociation 3.3 Cell Suspension Purification 3.4 Cell Sorting 3.5 RNA Extraction 3.6 Analysis of Alternative Splicing 4 Notes References Chapter 5: Quantitative Measurement of Alternatively Spliced RNA Isoform Levels 1 Introduction 2 Materials 2.1 RNA Extraction and cDNA Synthesis 2.2 PCR and Agarose Gel Electrophoresis 2.3 RT-qPCR 2.4 Computational and Bioinformatic Resources 3 Methods 3.1 Design of RT-qPCR Junction Primers for an Exclusion Isoform 3.2 Design of RT-qPCR Primers for an Inclusion Isoform 3.3 RNA Extraction and cDNA Synthesis 3.4 Validation and Quality Control of Isoform-Specific Primers 3.5 Quantitative Analysis of Alternative Isoforms Using RT-qPCR 4 Notes References Chapter 6: Analysis of Splicing Regulation by Third-Generation Sequencing 1 Introduction 2 Materials 2.1 RNA Extraction 2.2 Reverse Transcription and PCR 2.3 Barcode Tagging and Library Preparation 2.4 Sequencing 2.5 Bioinformatic Program Repositories 3 Methods 3.1 Total RNA Isolation 3.2 Amplification of Specific Transcripts 3.3 Barcoding and Preparation of a Multiplexed TGS Library 3.4 PacBio and ONT Sequencing Principles 3.4.1 PacBio (Left Panel Fig. 3) 3.4.2 ONT (Right Panel Fig. 3) 3.5 Demultiplexing and Clustering of Alternative Isoforms 3.5.1 Demultiplexing 3.5.2 Clustering of Identical Sequences 3.5.3 Other Pipelines Which Can Be Used for the Analysis 4 Notes References Chapter 7: Computational Analysis of Alternative Splicing Using VAST-TOOLS and the VastDB Framework 1 Introduction to Alternative Splicing and the VastDB Framework 2 vast-tools: Quantification and Differential Alternative Splicing Analyses 2.1 Introduction 2.2 vast-tools align 2.3 vast-tools merge 2.4 vast-tools combine and vast-tools tidy 2.5 vast-tools compare, vast-tools diff, and vast-tools plot 2.6 Quantification of Gene Expression and vast-tools compare_expr 3 Matt: Differential Splicing Feature Analyses 3.1 Introduction 3.2 matt cmpr_exons and matt cmpr_introns 3.3 matt cmpr_features 3.4 Motif Enrichment Analyses and matt rna_maps 4 ExOrthist: Evolutionary Conservation of Exons 4.1 Introduction 4.2 main.nf 4.3 compare_exon_sets and exint_plotter 5 VastDB: Looking at the Big Picture 5.1 Introduction 5.2 Gene View Pages 5.3 Event View Pages 5.4 Functions of Events 6 A Case Example: Identification and Characterization of Conserved Srrm4 Targets in Mammals References Chapter 8: Identification and Quantification of Microexons Using Bulk and Single-Cell RNA-Seq Data 1 Introduction 2 Materials 2.1 Hardware 2.2 Installation 2.3 Setup 2.3.1 Cluster Configuration 2.3.2 Listing Input Datasets 2.3.3 Create a Configuration File 3 Discovery and Quantification of Microexons 3.1 Minimal Configuration and Optional Parameters 3.2 Execution 3.2.1 Snakemake Additional Parameters 3.2.2 Using Target Rules for Workflow Control 3.3 Output 4 Detecting Differentially Included Microexons in Bulk RNA-Seq 4.1 Using Whippet to Assess Alternative Splicing of Microexons 4.1.1 Installing Whippet 4.1.2 Configure MicroExonator to Use Whippet 4.1.3 Running Pairwise Comparison between Experiments 4.1.4 Description of Output 5 Using scRNA-Seq Data to Quantify Microexons 5.1 Quantifying Microexons at the Single-Cell Level 5.2 Microexon Quantification Across Cell Types 5.2.1 Quantifying Microexons as Pseudobulk 5.2.2 Performing Pairwise Comparisons Between Cell Types 6 Troubleshooting References Chapter 9: Functional Annotation of Custom Transcriptomes 1 Introduction 2 Materials 2.1 Computer 2.2 R, RStudio and factR 2.3 Preassembled Transcriptome Files 2.4 Shell Scripts 2.5 Additional Programs 3 Methods 3.1 Custom Transcriptome Assembly Using Bulk RNA-Seq Data 3.2 Custom Transcriptome Assembly Using Single-Cell RNA-Seq Data 3.3 Custom Transcriptome Assembly Using Long-Read RNA-Seq Data 3.4 Functional Annotation of Custom Transcriptomes Using factR 3.5 Biological Significance and Concluding Remarks 4 Notes References Chapter 10: Design, Labeling, and Application of Probes for RNA smFISH 1 Introduction 2 Materials 2.1 Designing Custom Probes 2.2 Probe Labeling 2.3 RNA FISH Fixation and Hybridization (for 4 Coverslips) 3 Methods (See Fig. 1) 3.1 Designing Custom Probes 3.1.1 Download and Configure the Workflow 3.1.2 Prepare Genome Indexes for Specificity Filtering 3.1.3 Run the Workflow to Generate RNA FISH Probes 3.2 Probe Labeling 3.2.1 Conjugating ddUTP and Dye 3.2.2 Conjugating Oligonucleotides and ddUTP-Dye 3.2.3 Assessing the Labeling Efficiency 3.3 RNA smFISH 3.3.1 Fixation of Samples 3.3.2 Probe Hybridization 3.4 Image Acquisition 4 Notes References Chapter 11: BaseScope Approach to Visualize Alternative Splice Variants in Tissue 1 Introduction 2 Materials 3 Methods 3.1 Tissue Preparation 3.2 Target Retrieval 3.3 Protease Reaction 3.4 BaseScope 4 Notes References Chapter 12: In Situ Imaging of mRNA Splicing Variants by SpliceRCA 1 Introduction 2 Materials 2.1 Oligonucleotide Probes 2.2 Cell Culture 2.3 In Situ Amplification 2.4 General Equipment 3 Methods 3.1 Probe Design 3.2 Cell Culture and Padlock Probe Phosphorylation 3.3 Cell Fixation and Permeabilization 3.4 Padlock Probe Hybridization 3.5 T4 DNA Ligation 3.6 Primer Hybridization 3.7 In Situ Rolling Circle Amplification 3.8 Fluo-Probe Hybridization 3.9 Mounting and Imaging 3.10 Typical/Anticipated Results 4 Notes References Chapter 13: Two-Color Fluorescent Reporters for Analysis of Alternative Splicing 1 Introduction 2 Materials 2.1 Available Vectors 2.2 Molecular Cloning 2.3 Microinjection of C. elegans for CRISPR/Cas9 Genome Editing and Screening Animals 2.4 Fluorescence Microscopy 2.5 RNA Purification and Reverse Transcription Polymerase Chain Reaction (RT-PCR) 3 Methods 3.1 Two-Color Reporter Design Considerations for Minigene Sequence 3.2 Synthesis of Minigene Sequence or Amplification by PCR 3.2.1 Synthesis of Minigene Sequence by Company 3.2.2 Amplification of Minigene Sequence by PCR 3.3 Obtaining Promoters of Interest for Expression in Specific Cell Types and Digestion of Two-Color Vector for Cloning Promot... 3.4 Gibson Assembly Reaction to Clone Promoter into Two-Color Vector 3.5 Digestion of Promoter-Containing Vector to Prepare for Insertion of Minigene Fragment 3.6 Insertion of Minigene Fragment into Two-Color Reporter Vector by Gibson Assembly 3.7 Preparing Two-Color Reporter Injection Solutions and Microinjection 3.8 Screening for Animals with Two-Color Reporter Expression and Establishing Stable Extrachromosomal Array Lines 3.9 Guidelines for Imaging of Two-Color Reporters via Fluorescence Microscopy 3.10 Preparing Animals for Imaging of Two-Color Reporters via Fluorescence Microscopy 3.11 Total RNA Extraction from Reporter Expressing Animals 3.12 Semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) 4 Notes References Chapter 14: Quantitative Detection of Protein Splice Variants by Selected Reaction Monitoring (SRM) Mass Spectrometry 1 Introduction 2 Materials 2.1 Selection of Peptide Sequences for SRM Assays 2.2 SRM Assay Generation 2.3 Preparation of Synaptic Proteins Enriched Triton-Resistant Membranes (TRM) from Mouse Brain 2.4 Protein Digestion and Preparation for SID-SRM Analysis 2.5 HPLC Buffers for SID-SRM Analysis 3 Methods 3.1 Selection of Peptide Sequences for SRM Assays 3.2 SRM Assay Generation 3.3 Preparation of Synaptic Proteins Enriched Triton-Resistant Membranes (TRM) from Mouse Brain 3.4 Tryptic Digest and Column Purification of Peptide 3.5 SRM LC-MS Analysis 4 Notes References Chapter 15: Probing the Interactions of Splicing Regulatory Small Molecules and Proteins with U1 snRNP Using NMR Spectroscopy 1 Introduction 2 Materials 2.1 Expression and Purification of the U1 snRNP Protein Components 2.2 Production of the U1 snRNA and Variants Using In Vitro Transcription 2.3 Assembly and Purification of U1 snRNP 2.4 Methyl Labelling of Splicing Factors 2.5 NMR Spectroscopy Analysis of U1 snRNP Ligand Binding 2.6 Time Considerations 3 Methods 3.1 Expression and Purification of the Sm Proteins 3.1.1 Sm B-D3 3.1.2 Sm D1-D2 3.1.3 Sm E-F-G 3.2 Expression and Purification of U1 Specific Proteins 3.2.1 U1-70K 3.2.2 U1-A 3.2.3 U1-C 3.3 Preparation of the U1 snRNA 3.4 Reconstitution and Purification of U1 snRNP for NMR Spectroscopy 3.5 Preparation of the U1 snRNP Ligand 3.5.1 Preparation of ILV Labelled Splicing Factors 3.5.2 Preparation of Small Molecule Ligands 3.6 NMR Titrations 4 Notes References Chapter 16: Genome-Wide CRISPR Screening to Identify Mammalian Factors that Regulate Intron Retention 1 Introduction 1.1 General Strategy 1.2 Reporter Design 1.3 Cell Type Selection, Reporter Integration, and Validation 1.4 Lentiviral Library Selection 1.5 CRISPR Screen Timing 2 Materials 2.1 Determination of CRISPR Lentiviral Library Titer 2.2 CRISPR Screen 2.3 DNA Preparation 2.4 Library Amplification 2.5 PCR Cleanup and Sequencing 3 Methods 3.1 Determination of CRISPR Lentiviral Library Titer 3.2 CRISPR Screen 3.3 DNA Preparation 3.4 Library Amplification 3.5 PCR Cleanup and Sequencing 3.6 Timeline of CRISPR Screening 4 Notes References Chapter 17: Tethered Function Assays to Elucidate the Role of RNA-Binding Proteins 1 Introduction 1.1 Designing and Cloning of Reporter Genes Containing MS2 Binding Sites 1.2 Cloning the MS2 Fusion Protein 1.3 Expression of Both Constructs in Cells 1.4 Functional Analysis of the Protein of Interest 2 Materials 2.1 Cloning of Plasmids 2.2 Expression of Constructs in Cells 2.3 Functional Analysis of the Protein of Interest 3 Methods 3.1 Designing and Cloning of Reporter Genes Containing MS2 Binding Sites 3.2 Cloning the MS2 Fusion Protein 3.2.1 Cloning of pCMV-PABPC1-MS2-HA 3.2.2 Cloning of Controls: MS2CP Alone (pCMV-MS2-HA) 3.2.3 Cloning of Controls: PABPC1 Without MS2CP (pCMV-PABPC1-HA) 3.2.4 Cloning of Controls: Unrelated Protein with MS2CP (pCMV-lacZ-MS2-HA) 3.3 Expression of Constructs in Cells 3.4 Functional Analysis of the Protein of Interest 4 Notes References Chapter 18: Probing Liquid-Liquid Phase Separation of RNA-Binding Proteins In Vitro and In Vivo 1 Introduction LLPS and LLPS in Splicing 1.1 Liquid-Liquid Phase Separation Is a Major Organizing Principle of Cellular Biochemistry 1.2 Molecular Basis and Cellular Importance of LLPS 1.3 LLPS in Splicing 1.4 Characterization of Condensation Phenomena 2 Materials 2.1 Materials Required for Purification of Dhh1-mCherry 2.2 Materials Required for an In Vitro Reconstitution Experiment with Recombinant Proteins 2.3 Materials for Yeast Cell Growth and Hexanediol Treatment 2.4 General Imaging Requirements 2.5 Time Considerations 3 Methods 3.1 Protein Purification under Condensate-Preventing Conditions (Fig. 1) 3.2 In Vitro Reconstitution of Biomolecular Condensates 3.3 Yeast Cell Growth and Imaging Dish Preparation 3.4 Probing the Dynamic Behavior of Condensates 4 Notes References Chapter 19: Analysis of Oligonucleotide Biodistribution and Metabolization in Experimental Animals 1 Introduction 2 Materials 2.1 General Points 2.2 Isolation and Processing of Animal Tissues 2.3 Analysis of Tissue Biodistribution by CL-qPCR 2.3.1 Primer Design and Synthesis 2.3.2 CL-qPCR 2.4 Metabolite Identification by LC-MS 3 Methods 3.1 General Points 3.2 Isolation and Processing of Animal Tissues 3.3 Analysis of Tissue Biodistribution by CL-qPCR 3.4 Metabolite Identification by LC-MS 4 Notes References Index