Note: Alternative pre-mRNA Splicing: Theory and Protocols -- Contents -- Preface -- List of Abbreviations -- List of Contributors -- Part One: Theory -- 1 Splicing in the RNA World -- 1.1 Introduction: The Fascination of Alternative Pre-mRNA Splicing -- 1.2 RNA Can Adopt a Flexible Conformation -- 1.3 Enzymatic RNAs and the RNA World -- 1.4 Common Classes of Eukaryotic RNA -- 1.5 Alternative Pre-mRNA Splicing as a Central Element of Gene Expression -- 1.6 Increasing Numbers of Human Diseases are Associated with .Wrong. Splice Site Selection -- References -- 2 RNPs, Small RNAs, and miRNAs -- 2.1 Introduction -- 2.2 Ribonuclease P (RNase P) -- 2.3 Small Nucleolar RNAs (snoRNAs) -- 2.4 Small Regulatory RNAs -- 2.4.1 Short Interfering RNAs (siRNAs) -- 2.4.2 MicroRNAs (miRNAs) -- 2.4.3 Piwi-Interacting RNAs (piRNAs) -- 2.5 7SL RNA -- 2.6 7SK RNA -- 2.7 U-Rich Small Nuclear RNAs (U snRNAs) -- References -- 3 RNA Elements Involved in Splicing -- 3.1 Introduction -- 3.2 Splice Site Sequence -- 3.3 Intron/Exon Architecture -- 3.4 Splicing Regulatory Elements (SREs) -- 3.5 RNA Secondary Structure -- 3.6 Coupling between Transcription and RNA Processing -- 3.7 Combinatorial Effects of Splicing Elements -- References -- 4 A Structural Biology Perspective of Proteins Involved in Splicing Regulation -- 4.1 Introduction -- 4.2 The RRM: A Versatile Scaffold for Interacting with Multiple RNA Sequences and also Proteins -- 4.2.1 RRM-RNA Interaction and Splicing Regulation -- 4.2.1.1 RNA Binding by Splicing Factors Containing a Single RRM -- 4.2.1.2 RNA Binding by Splicing Factors Containing Multiple RRMs -- 4.2.2 RRM-RRM and RRM-Protein Interactions in Splicing Regulation -- 4.2.2.1 RRM-Protein Interactions Without RNA Binding -- 4.2.2.2 RRM-Protein Interactions Allowing RNA Binding -- 4.2.2.3 Impact of RRM-RRM Interactions on Splicing Mechanism. , 4.3 The Zinc Finger Domain -- 4.4 The KH Domain -- 4.5 Conclusions and Perspectives -- References -- 5 The Spliceosome in Constitutive Splicing -- 5.1 Introduction -- 5.2 The Mechanism of Splicing -- 5.3 The Stepwise Assembly Pathway of the Spliceosome -- 5.4 Dynamics of the Spliceosomal RNA-RNA Rearrangements -- 5.5 Splice-Site Recognition and Pairing Involves the Coordinated Action of RNA and Proteins -- 5.6 Driving Forces and Molecular Switches Required During the Spliceosome.s Activation and Catalysis -- 5.7 A Conformational Two-State Model for the Spliceosome.s Catalytic Center -- 5.8 Compositional Dynamics and Complexity of the Spliceosome -- 5.9 Reconstitution of Both Steps of S. cerevisiae Splicing with Purified Spliceosomal Components -- 5.10 Evolutionarily Conserved Blueprint for Yeast and Human Spliceosomes -- 5.11 Concluding Remarks -- References -- 6 The Use of Saccharomyces cerevisiaeto Study the Mechanism of pre-mRNA Splicing -- 6.1 Introduction -- 6.2 The Basics of Splicing -- 6.3 Yeast Intron-Exon Organization -- 6.4 The Yeast Spliceosome -- 6.5 Defining the Constellation of Yeast Splicing Factors: Primary Screens and Genomic Inspection -- 6.6 Reporter Genes as Readouts of Splicing Efficiency -- 6.7 Genetic Interaction: Dosage Suppression or Antagonism -- 6.8 Extragenic Suppressors -- 6.9 Synthetic Lethality -- 6.10 Systematic Approaches to Define the Interactome -- References -- 7 Challenges in Plant Alternative Splicing -- 7.1 Introduction -- 7.2 Plant Introns -- 7.3 The Plant Spliceosome -- 7.4 Plant Spliceosomal Proteins -- 7.5 Alternative Splicing in Plants -- References -- 8 Alternative Splice Site Selection -- 8.1 Introduction -- 8.2 The Players: Splicing Regulators -- 8.3 The Stage: The Splicing Complex Assembly and Exon Definition -- 8.4 Switching Splicing Patterns. , 8.5 SrcN1 Exon: A Model of Combinatorial Splicing Regulation -- 8.6 The Global View: Towards a Splicing Code -- References -- 9 Integration of Splicing with Nuclear and Cellular Events -- 9.1 Introduction -- 9.2 Overview -- 9.3 Nuclear Structure and Distribution of Splicing Factors -- 9.3.1 Cajal Bodies (CBs) -- 9.3.2 Splicing Factor Compartments (SFCs)/Speckles -- 9.3.3 Paraspeckles -- 9.4 Integration of Splicing with Nuclear and Cellular Processes -- 9.4.1 Splicing and Transcription -- 9.4.2 Splicing and mRNA Capping -- 9.4.3 Splicing and 30 End Processing -- 9.4.4 Splicing and Export -- 9.4.5 Splicing and Translation -- 9.4.6 Splicing and Nonsense-Mediated Decay (NMD) -- 9.4.7 Splicing and Chromatin Structure -- References -- 10 Splicing and Disease -- 10.1 Introduction -- 10.2 Splicing and Disease -- 10.3 Therapeutic Approaches -- 10.4 The Generation of Aberrant Transcripts -- 10.5 Exon Skipping -- 10.6 Cryptic Splice Site Activation -- 10.7 Intron Retention -- 10.8 Pseudoexon Inclusion -- 10.9 Unexpected Splicing Outcomes Following the Disruption of Classical Splicing Sequences -- 10.10 Conclusions -- References -- 11 From Bedside to Bench: How to Analyze a Splicing Mutation -- 11.1 Introduction -- 11.2 From Clinical Evaluation to Mutation Testing -- 11.3 An Example of an Uncertain Diagnosis -- 11.4 Mutation Testing Procedures -- 11.4.1 In-VitroSplicing -- 11.4.2 Minigene Splicing -- 11.5 Concluding Remarks -- References -- Part Two: Basic Methods -- 12 Analysis of Common Splicing Problems -- 12.1 Introduction -- 12.2 Is a Mutation Causing a Change in AS? -- 12.3 How is a Splicing Event Regulated, and How Can it be Influenced? -- 12.4 Is There a Difference in Alternative pre-mRNA Processing Between Two Cell Populations? -- References -- 13 Ultracentrifugation in the Analysis and Purification of Spliceosomes Assembled In Vitro. , 13.1 Theoretical Background -- 13.2 Protocol -- 13.2.1 Preparation of the Gradient -- 13.2.1.1 Manual Gradient Formation -- 13.2.1.2 Automatic Gradient Formation with the Gradient Master -- 13.2.2 Preparing the Run -- 13.2.2.1 Loading the Sample -- 13.2.2.2 Sedimentation Markers -- 13.2.3 The Ultracentrifuge Run -- 13.2.4 Harvesting the Gradient -- 13.3 Example Experiment -- 13.3.1 Purification of the Spliceosomal B Complex -- 13.3.1.1 Preliminaries -- 13.3.1.2 Preparation of the Spliceosomal B Complex -- 13.4 Troubleshooting -- References -- 14 Chemical Synthesis of RNA -- 14.1 Theoretical Background -- 14.1.1 RNA Solid-Phase Synthesis -- 14.1.2 RNA Modifications -- 14.1.2.1 RNA Modification During Solid-Phase Synthesis -- 14.1.2.2 Post-Synthetic RNA Modification -- 14.1.3 Combined Chemical and Enzymatic Strategies -- 14.2 Representative Protocols -- Protocol 1: Incorporation of Modified Phosphoramidites During Solid-Phase Synthesis -- Protocol 2: Coupling of Biophysical Probes to Aliphatic Amino Groups on RNA -- Protocol 3: Enzymatic Ligation of RNA fragments using T4 RNA or T4 DNA Ligase -- 14.3 Troubleshooting -- References -- 15 RNA Interference (siRNA, shRNA) -- 15.1 Theoretical Background -- 15.1.1 RNAi -- 15.1.2 siRNAs and shRNAs -- 15.1.3 Lentiviral-Mediated RNAi -- 15.2 Protocol -- 15.2.1 Map of pLKO.1 Puro -- 15.2.2 Oligonucleotide Design -- 15.2.2.1 Determining the Optimal 21-mer Targets in the Gene -- 15.2.2.2 Ordering Oligos Compatible with pLKO.1 -- 15.2.3 Generating the pLKO.1 Puro with a shRNA Construct -- 15.2.3.1 Annealing of the Oligonucleotides -- 15.2.3.2 Preparation of pLKO.1 TRC for Cloning -- 15.2.3.3 Ligating and Transforming into Bacteria -- 15.2.3.4 Screening for Inserts -- 15.2.4 Production of Lentiviral Particles -- 15.2.5 Lentiviral Infection -- 15.3 Example Experiment -- 15.4 Troubleshooting -- References. , 16 Expression and Purification of Splicing Proteins -- 16.1 Theoretical Background -- 16.2 Protocol 1: The Preparation of Total HeLa SR Proteins -- 16.2.1 Example Experiment -- 16.2.2 Troubleshooting and Important Points -- 16.3 Protocol 2: The Purification of Individual SR Proteins -- 16.3.1 Expression of SR Proteins in Escherichia coliand Purification -- 16.3.2 Preparation of SR Proteins Using a Baculovirus System -- 16.3.3 Example Experiment -- 16.3.4 Troubleshooting and Important Points -- 16.3.5 Production and Purification of Individual SR Proteins in Mammalian Cells -- References -- 17 Detection of RNA-Protein Complexes by Electrophoretic Mobility Shift Assay -- 17.1 Theoretical Background -- 17.1.1 Choice of RNA Substrate -- 17.1.2 Detection and Quantitation of Binding -- 17.1.3 Fluorescence -- 17.1.4 Chromogenic and Chemiluminescent Detection Methods -- 17.1.5 Stability of RNA-Protein Complexes During Electrophoresis -- 17.1.6 Competing Nucleic Acids and Polyanions -- 17.1.7 Binding Stoichiometry -- 17.1.8 Measurement of Binding Activity -- 17.1.9 Measurement of Dissociation Constants -- 17.1.10 Binding Competition -- 17.2 Protocol -- 17.2.1 Equipment -- 17.2.2 Reagents -- 17.2.3 Gel Preparation -- 17.2.4 Pre-Electrophoresis -- 17.2.5 Sample Preparation -- 17.2.6 Electrophoresis and Imaging -- 17.3 Example Experiment -- 17.4 Troubleshooting -- References -- 18 Functional Analysis of Large Exonic Sequences Through Iterative In VivoSelection -- 18.1 Theoretical Background -- 18.1.1 Spinal Muscular Atrophy -- 18.2 Protocol -- 18.2.1 Minigene, Cell Culture, Transfection, and In VivoSplicing Assay -- 18.2.2 Generation of a Partially Random Exon -- 18.2.3 In vivo Selection -- 18.2.4 Analysis of Sequences -- 18.3 Example Experiment -- 18.3.1 Generating the Initial Pool of Splicing Cassettes -- 18.3.2 In VivoSelection Procedure. , 18.4 Troubleshooting.