Pseudogenes: Functions and Protocols

Description

Preface Contents Contributors Part I: Introductory Chapter Chapter 1: Pseudogenes: Four Decades of Discovery 1 Introduction 2 Pseudogene Discovery 3 Properties and Classes of Human Pseudogenes 3.1 Processed Pseudogenes 3.2 Duplicated Pseudogenes 3.3 Unitary Pseudogenes 3.4 Polymorphic Pseudogenes 4 Expressed Pseudogenes 5 Emerging Pseudogene Functions 5.1 Competition for microRNAs 5.2 Pseudogene RNA-Protein Interactions 5.3 Pseudogene Transcripts as Epigenetic Regulators 5.4 Processing of Pseudogenes into Small Interfering RNAs 5.5 Pseudogenes with Protein Coding Potential 6 Closing Remarks References Part II: Methods for Pseudogene Identification Chapter 2: Methods to Identify and Study the Evolution of Pseudogenes Using a Phylogenetic Approach 1 Introduction 2 Methods 2.1 Pseudogene Identification Using Phylogenetic Tools 2.2 Pseudogene Dating and Characterization Using Phylogenetic Tools 2.3 Pseudogene Analysis at the Nucleotide Level Using Phylogenetic Tools 2.4 Automation 2.5 Closing Remarks 3 Notes References Chapter 3: Computational Methods for Pseudogene Annotation Based on Sequence Homology 1 Introduction 2 Overview of Methods 2.1 Basic Procedures 2.2 Application of Sequence Homology-Based Methods to Genome-Scale Pseudogene Annotation 2.3 Further Developments 3 Checking for Suspected Pseudogenes in a Piece of Genomic DNA of Interest: Some General Points 4 Conclusions References Chapter 4: A Methodology to Study Pseudogenized lincRNAs 1 Introduction 1.1 Regulatory Roles of lincRNAs 1.2 The Biogenesis of lincRNAs 2 Materials and Methods 2.1 Scoring Matrix for Noncoding Sequence Alignments 2.2 Aligning lincRNAs Against Protein-Coding Genes 2.2.1 Define Query and Target Sequences 2.2.2 Find Alignment Blocks 2.2.3 Establishing the Best Alignment Block 2.2.4 Improving the Alignment Among Different ORFs 2.3 Alignment Visualization 2.3.1 Color Representation of the Alignments 2.3.2 Quality Representation of the Alignments 2.3.3 Additional Symbols Used 2.4 Comparing with tblastn 2.4.1 An Application of the Rost Curve to Find Remnants of Protein-Coding Sequences Within lincRNA 3 Concluding Remarks References Part III: Pseudogene Repositories Chapter 5: GENCODE Pseudogenes 1 Introduction 2 GENCODE Pseudogenes 2.1 General Considerations 2.2 Human GENCODE Pseudogenes 2.3 Mouse GENCODE Pseudogenes 3 GENCODE Pseudogene Resources 4 Conclusions References Part IV: Methods to Study Pseudogene-Related Alterations in Cancer Chapter 6: Comprehensive Detection of Pseudogenes Transcribed by Readthrough 1 Introduction 1.1 Transcription Readthrough 1.2 Detecting Transcription Readthrough from Transcriptome Profiles 2 Materials 2.1 Hardware and Software Requirements 2.2 Genome and Gene Annotations 2.3 Case Study and Dataset Resources 3 Methods 3.1 Computational Pipeline Overview 3.2 Case-Study: Detecting Pseudogenes Expressed by Transcription Readthrough in Renal Cancer 3.2.1 Step 1: RNA-seq Data Alignment 3.2.2 Step 2: Detection of Readthrough Transcripts Invading Pseudogenes 3.2.3 Step 3: Detection of Spliced Readthrough Transcripts Involving Pseudogenes 3.3 Applying the Framework to Several Samples 4 Notes References Part V: Methods to Study Transcribed Pseudogenes Chapter 7: Mathematical Modeling of ceRNA-Based Interactions 1 Introduction 2 Materials 2.1 Biological Prerequisites 2.2 Computational Tools Necessary for Mathematical Modeling 3 Methods 3.1 Defining Key Variables 3.2 Defining Model Structure: Drawing an Interaction Diagram 3.3 Defining Model Dynamics: Adding Equations to Edges of the Interaction Diagram 3.4 Parameter Estimation and Simulation 3.5 Stability Analysis and Analysis of End Behavior 3.6 Sensitivity Analysis 3.7 Validation 3.8 Supportive Evidence from Large Datasets 4 Notes References Chapter 8: Pseudogenes as Competitive Endogenous RNAs: Target Prediction and Validation 1 Introduction 2 Materials 2.1 Tissue Culture Components 2.2 siRNA, DNA, or Dual Transfection 2.3 RNA and Protein Analysis 2.4 Luciferase Assays 3 Methods 3.1 Bioinformatics Prediction of Putative Pseudogene ceRNAs 3.1.1 Input 3.1.2 The Null Hypothesis 3.1.3 The ceRNA Score The Dissimilarity Measure The Similarity Measure 3.1.4 Predicted ceRNA Partners 3.2 ceRNA Validation I: siRNA Silencing 3.3 ceRNA Validation II: 3′UTR Overexpression 3.4 ceRNA Validation III: Luciferase Reporter Assays 3.5 ceRNA Validation IV: microRNA Dependence 3.5.1 Dicer1 Activity-Deficient HCT116 Cells 3.5.2 Dicer1-Floxed Cells 3.5.3 Dicer1 Knockdown 4 Notes References Chapter 9: Pseudogenes as Competitive Endogenous RNAs: Testing miRNA Dependency 1 Introduction 2 Luciferase Assay for miRNA Reporter 2.1 Rationale 2.2 Materials 2.3 Method 2.4 Expected Results 3 Hybridization-Based RNA Pulldown 3.1 Rationale 3.2 Materials 3.2.1 Online Tools 3.2.2 Reagents and Kits 3.3 Method 3.3.1 Preparing Probes 3.3.2 Cross-Linking 3.3.3 Lysis 3.3.4 Hybridization 3.3.5 Precipitation 3.3.6 RNA Isolation 3.3.7 qPCR Analysis 3.4 Expected Results 4 MRE Mutation 4.1 Rationale 4.2 Materials 4.3 Method 4.4 Expected Results 5 Target Site Blockers 5.1 Rationale 5.2 Materials 5.3 Method 5.4 Expected Results 6 miRNA Mimics and Inhibitors 6.1 Rationale 6.2 Materials 6.3 Method 6.4 Expected Results 7 Droplet Digital PCR (ddPCR) 7.1 Rationale 7.2 Materials 7.3 Method 7.4 Expected Results 8 Notes References Chapter 10: An Overview of the Computational Models Dealing with the Regulatory ceRNA Mechanism and ceRNA Deregulation in Canc... 1 Introduction 2 ceRNA Mechanism 3 Dysregulation of ceRNAs in Cancer 4 Databases of miRNA-Target Interactions 5 Databases of ceRNA Interactions 6 Computational Methods for Identifying ceRNA Cross Talk in Cancer 6.1 Statistics-Based Methods 6.1.1 Methods Overview 6.1.2 Web Server/R Packages 6.2 Mathematical Modeling Methods References Chapter 11: Studying the Oncosuppressive Functions of PTENP1 as a ceRNA 1 Introduction 2 Materials 2.1 Cloning of PTENP1 into a Mammalian Expression Plasmid 2.1.1 Cloning. 2.1.2 Bacterial Growth 2.2 Plasmid Preparation 2.3 Tissue Culture (TC) 2.4 Transfection 2.5 Cell Proliferation Assays 2.5.1 CCK-8 Assay 2.5.2 EdU Assay 3 Methods 3.1 Cloning of PTENP1 into a Mammalian Expression Plasmid 3.1.1 Production, Purification, and Cloning of the PTENP1 PCR Product 3.1.2 Preparation and Sequencing of Plasmid DNA 3.2 Cell Culture and Maintenance of Cell Lines 3.3 Transfection and Determining Transfection Efficiency Using Flow Cytometry 3.4 CCK-8 Cell Proliferation Assay 3.4.1 Producing a Standard Curve 3.4.2 Cell Coloration 3.4.3 Data Analysis 3.5 EdU Cell Proliferation Assay 3.5.1 Prepreparation of Stock Solutions 3.5.2 Cell Labelling 3.5.3 Fixation and Permeabilization of Cells 3.5.4 EdU Detection 3.5.5 DNA/Nuclear Staining 3.5.6 Mounting of Coverslips 3.5.7 Fluorescence Imaging 3.5.8 Image Analysis 3.6 Additional Oncosuppressive Functions of PTENP1 4 Notes References Chapter 12: Methods to Study Protein-Binding to Pseudogene Transcripts 1 Introduction 2 PCR Amplification and Plasmid-Based Molecular Cloning of the Pseudogene Under Study 2.1 Materials 2.1.1 PCR Amplification 2.1.2 Electrophoresis and Purification of the PCR Product 2.1.3 Cloning of the PCR Product into PCDNA-3 Basic Vector 2.2 Method 2.2.1 PCR Amplification 2.2.2 Electrophoresis and Purification of the PCR Product 2.2.3 Cloning of the PCR Product into PCDNA-3 Basic Vector 3 Identification of the Pseudogene Transcript Under Study as RBP Target Using RNA-Electrophoretic Mobility Shift Assay (RNA-EM... 3.1 Materials 3.1.1 Cytoplasmic and Nuclear Protein Extraction 3.1.2 In Vitro Transcription and Biotin Labeling of RNA Probe 3.1.3 Binding Reaction 3.1.4 Gel Electrophoresis of the Binding Reaction, Transfer to Nylon Membrane, and Chemiluminescent Detection 3.2 Method 3.2.1 Cytoplasmic and Nuclear Protein Extraction 3.2.2 In Vitro Transcription and Biotin Labelling of RNA Probe 3.2.3 Gel Prerun 3.2.4 Binding Reaction 3.2.5 Gel Electrophoresis of the Binding Reaction 3.2.6 Electrophoretic Transfer of the Binding Reaction to Nylon Membrane and Chemiluminescent Detection of the Shifted Bands 3.3 Experimental Controls 3.4 Expected Results 4 Identification of the Transcripts That Bind an RBP of Interest 4.1 RNA Immunoprecipitation (RIP) 4.2 Crosslinking and Immunoprecipitation (CLIP) 5 Notes References Chapter 13: Chromatin Regulation at Parental Gene Promoters by Pseudogene Sense lncRNAs 1 Pseudogene Derived RNAs 2 Mechanisms of Parental Gene Expression Control by Sense Pseudogene lncRNAs 3 Oct4 Pseudogenes Display Controlled Expression and Have Acquired Multiple Biological Functions 4 mOct4P4 lncRNA-Mediated Chromatin Regulation, a Novel Paradigm of Parental Gene Expression Control In Trans 4.1 Structural Features of mOct4P4 lncRNA and Biological Relevance 4.2 mOct4P4 lncRNA Has Acquired New Elements with Defined Function 4.3 Chromatin Modulation of Parental Oct4 Promoter Triggered by mOct4P4 lncRNA 4.4 RNA:Protein Interaction Regulates mOct4P4 lncRNA Function 5 Mechanisms Underlying Target Specificity for Pseudogene lncRNAs That Modify Chromatin Structure In Trans 5.1 mOct4P4 lncRNA Recruitment by Factors Deposited at the Oct4 Promoter 5.2 Long-Distance Chromatin Interactions Between Oct4 and mOct4P4 Alleles 6 Fast Strategies to Address the Biological Relevance of Pseudogene-Encoded Sense lncRNAs 6.1 Individuating a Model System That Allows to Address the Biological Relevance of Pseudogene-Derived lncRNAs 6.2 Using Publicly Available Genomics Data and Genome Browsers to Get First Information on Pseudogenes 6.3 Design of Specific Primer Sets for PCR Amplification 6.4 Characterization of Key Features of the Pseudogene lncRNA 6.5 Finding First Evidence for Pseudogene lncRNA Mediated Control of Parental Gene Expression and Function 7 Dissecting Chromatin Regulation by Pseudogene lncRNAs Using the MS2 Tagging System 7.1 Identification and Validation of Target Chromatin Sites by ChIP 7.2 Identification and Validation of Protein Interactors by RIP 7.3 Identification of Functional Domains of Pseudogene lncRNA by Deletion Analysis 8 Future Outlook References Chapter 14: Pseudogenes: A Novel Source of Trans-Acting Antisense RNAs 1 Noncoding RNAs 2 AsRNA-Mediated Regulation of Gene Expression 2.1 Definition of Cis and Trans Acting asRNAs 2.2 Functions of Cis and Trans Acting asRNAs 3 Pseudogenes as a Source of asRNAs 3.1 Processed Pseudogenes Transcribed in Antisense 3.2 Unprocessed Pseudogenes Transcribed in Antisense 3.3 Genome-Wide Studies of asRNAs Transcribed from Pseudogenes 4 Pseudogenes with Characterized asRNA Transcription 4.1 nNOS 4.2 PTEN Pseudogene asRNA 4.3 OCT4 Pseudogene asRNA 5 Techniques for Identification and Analysis of asRNAs Transcribed from Pseudogenes 5.1 Identification of asRNAs Transcribed from Pseudogenes 5.1.1 Identification Using Genome-Wide Data Sets 5.1.2 Identification of Isoforms 5.1.3 Identification of Localization and Polyadenylation 5.2 Analysis of asRNAs Transcribed from Pseudogenes 5.2.1 Set Up of Pseudogene asRNA-Specific PCR 5.2.2 Profiling of Pseudogene asRNA Expression 5.2.3 Functional Analysis of Pseudogene asRNAs Knockdown Overexpression 6 Concluding Remarks References Part VI: Methods to Study Translated Pseudogenes Chapter 15: Methods to Study Translated Pseudogenes: Recombinant Expression and Complementation, Targeted Proteomics, and RNA ... 1 Introduction 2 Materials 2.1 PCR Amplification and Cloning 2.2 Purification of Fusion Protein from Bacteria 2.3 Analysis of Subcellular Localization of Fusion Protein 2.4 Targeted Proteomic Analysis 3 Methods 3.1 Specific PCR Amplification of the Pseudogenic Coding Region and Cloning by Recombination 3.1.1 Reverse Transcription 3.1.2 General Considerations About PCR Primer Design 3.1.3 PCR Amplification 3.2 Generation of a Fusion Pseudogenic Protein for Purification from Bacteria and Enzymatic Analysis 3.3 Generation of a Fusion Pseudogenic Protein for Subcellular Localization Analysis 3.4 Complementation Assay for the Pseudogenic Protein in Bacterial and Mammalian Cells 3.5 Targeted Proteomic Analysis of the Pseudogenic Protein 3.5.1 Cell Lysis and Protein Isolation (Day 1) 3.5.2 Filter Aided Sample Preparation (FASP) (Day 2) 3.5.3 Digestion of the Protein Sample (Day 2 and 3) 3.5.4 Purification of Digested Peptides (Day 3) 3.5.5 Global LC-MS/MS Analysis (Day 4) 3.5.6 Targeted LC-MS/MS Analysis for Low Abundance Proteins (Day 4) 3.6 Consideration of the Full Profile of the RNA Isoforms of the Pseudogenic Protein 4 Notes References Chapter 16: A Restriction Endonuclease-Based Assay to Distinguish NANOGP8 Retrogene from Parental NANOG 1 Introduction 2 Materials 3 Methods 3.1 Cell Culture 3.2 RNA Extraction 3.3 PCR Amplification of NANOG and NANOGP8 from cDNA 3.4 AlwNI Digestion of the Purified PCR Product 4 Notes References Part VII: CRISPR/CAS Technologies Applied to Pseudogenes Chapter 17: CRISPR/Cas Technologies Applied to Pseudogenes 1 Introduction 1.1 Pseudogenes 1.2 CRISPR/Cas System 2 CRISPR/Cas-Based Technologies for Negative and Positive Manipulation of Pseudogene Expression 2.1 Genome Editing 2.2 Negative and Positive Transcriptional Manipulation 2.3 Knock-Down of Endogenous Transcripts 2.4 RNA Editing 3 CRISPR/Cas System as RNA Binding Protein 3.1 RNA Immunoprecipitation (RIP) and RNA Pull-Down 3.2 Live Imaging Transcript Tracking 4 Why Use CRISPR/Cas System to Study Pseudogenes References Part VIII: In Vivo Models to Study Pseudogenes Chapter 18: Strategies to Study the Functions of Pseudogenes in Mouse Models of Cancer 1 Introduction 2 Pseudogene Conservation in Human and Mouse 3 Mouse Models of Cancer and Applications for Pseudogene Studies 3.1 Cell Line Transplantation Models 3.2 Genetically Engineered Mouse Models: Transgenic Approach 3.3 Genetically Engineered Mouse Models: Germline and Conditional Gene Targeting Approach 3.3.1 Knockin Approaches for Pseudogene Overexpression 3.3.2 Knockin and Knockout Approaches for Pseudogene Depletion 3.4 Embryonic Stem Cell-Genetically Engineered Mouse Models 4 Concluding Remarks References Part IX: Clinical Relevance of Pseudogenes Chapter 19: Pseudogene Profiling for Cancer Subtype Classification 1 Introduction 2 Materials 2.1 Pseudogene Annotation 2.1.1 Canonical Pseudogenes 2.1.2 Novel Pseudogenes 2.2 Pseudogene-Gene Relationships 2.2.1 1:1 Pseudogene-Parent Gene Relationships 2.2.2 Pseudogene-Gene (PGG) Families 2.3 Pseudogene Expression 2.4 Datasets with Clinical Information 3 Methods 3.1 Identification of Differentially Expressed Pseudogenes 3.2 Basic Steps of Pseudogene Profiling for Cancer Subtype Characterization and Classification 3.2.1 Cancer Subtype Characterization Using Differentially Expressed Pseudogenes 3.2.2 Cancer Subtype Classification Using Differentially Expressed Pseudogenes 3.3 Basic Steps of Pseudogene Profiling for Patients Stratification 4 Concluding Remarks References Chapter 20: Pseudogenes as Biomarkers and Therapeutic Targets in Human Cancers 1 Introduction 2 Panorama of Pseudogenes in Cancer 3 Pseudogenes as Biomarkers and Therapeutic Targets in Human Cancer 3.1 Pseudogenes as Diagnostic Markers 3.2 Pseudogenes as Prognostic Markers 3.3 Pseudogenes as Therapeutic Targets 4 Conclusions References Chapter 21: Methods for the Detection of Circulating Pseudogenes and Their Use as Cancer Biomarkers 1 Introduction 2 Materials 2.1 Cell Culture Media 2.1.1 Proliferation Medium for Human Neural Stem Cells (HNSCs) and Glioblastoma Multiforme (GBM) Primary Cells 2.1.2 Differentiation Medium for Human Neural Stem Cells (HNSCs) and Glioblastoma Multiforme (GBM) Primary Cells 2.1.3 Medium for Primary Umbilical Cord Blood-Derived AC133-Positive Endothelial Progenitor Cells 2.1.4 Medium for HEK293 Cell Line 2.2 Exosome Precipitation/Purification 2.2.1 PEG-Mediated Precipitation with Normal Centrifugation 2.2.2 One-Step Sucrose Cushion Ultracentrifugation 2.2.3 OptiPrep Density Gradient Ultracentrifugation 2.2.4 Immunoaffinity Purification of Preenriched Exosomes 2.3 Exosome Characterization 2.4 Exosomal NANOGP8 DNA Analysis 3 Methods 3.1 Exosome Precipitation/Purification 3.1.1 Ultracentrifugation 3.1.2 PEG-Mediated Precipitation with Normal Centrifugation 3.1.3 One-Step Sucrose Cushion Ultracentrifugation 3.1.4 OptiPrep Density Gradient Ultracentrifugation 3.1.5 Immunoaffinity Purification of Preenriched Exosomes 3.2 Exosome Characterization 3.2.1 Lipofectamine 2000-Mediated Transfection of HEK293 Cells and Precipitation/Purification of RFP-Packed Exosomes 3.2.2 DiO Staining of RFP-Packed HEK293 Exosomes 3.3 Qualitative Analysis of Exosomal NANOGP8 DNA 3.3.1 Primer Design for the Amplification of Exosomal NANOGP8 DNA 3.3.2 PCR Amplification of Exosomal NANOGP8 DNA 3.3.3 Gel Electrophoresis 3.3.4 Restriction Fragment Length Polymorphism (RFLP analysis) of the PCR Product 3.3.5 Sequence Analysis of Exosomal NANOGP8 DNA Cloning of NANOGP8 PCR Product in a Suitable Vector BLAST Analysis of NANOGP8 Clones Comparison of the Frequency of a 22 bp Insertion in Exosomal NANOGP8 DNA Obtained from Different Sources 3.4 Quantitative Analysis of Exosomal DNA: Amount of ssDNA and dsDNA 3.4.1 Quantification of Exosomal dsDNA Using XCFTM Exosomal DNA Isolation Kit 4 Notes References Part X: Methods to Avoid Pseudogene Detection Chapter 22: Dealing with Pseudogenes in Molecular Diagnostics in the Next Generation Sequencing Era 1 Introduction 2 Methods for Target Sequence Enrichment 2.1 Methods Based on Sequencing of PCR Amplicons 2.2 Methods Based on Capture of ONT Probes 2.3 Methods Based on Molecular Inversion Probes (MIPs) 2.4 Advantages and Disadvantages of the Different Enrichment Methods in the Context of the Analysis of Genes with Pseudogenes 2.4.1 Methods Based on Sequencing of PCR Amplicons 2.4.2 Methods Based on Capture of ONT Probes 2.4.3 Methods Based on Molecular Inversion Probes (MIPs) 3 Methods for Data Analysis 4 Examples of the Application of Next Generation Sequencing in a Medical Context When the Detection of Pseudogenes Needs to Be... 4.1 PKD1 and Its Six Pseudogenes 4.2 BRCA1 and PsiBRCA1 4.3 STRC and pSTRC 4.4 NF1 and Its 7 Pseudogenes 4.5 PMS2 and Its 15 Pseudogenes 5 Concluding Remarks References Index