RNA Processing: Disease and Genome-wide Probing

Preface
Contents
Contributors
Chapter 1: Experimental and Computational Considerations in the Study of 
RNA-Binding Protein-RNA Interactions
	1 Background
	2 What Is an 
RNA-Binding Protein?
	3 Identification of RNA-Binding Protein Binding Sites In 
Vivo
	4 Challenges of Peak Finding for RNA-Binding Proteins Compared to 
DNA Binding Proteins
	5 RBP-Responsive RNA Targets
	6 Choosing Between Depletion Versus 
Over-Expression Experiments
	7 Quantitation of 
RNA Isoform Abundance
	8 Identification of Altered RNA Splicing Events by 
Microarray
	9 Quantifying Alternative Splicing by 
High-Throughput Sequencing
	10 Identification of Novel Alternative Splicing Events by RNA-Seq and 
Microarray
	11 Alternative Polyadenylation Sites
	12 Transcriptome-wide Measurement of RNA Stability
	13 Global Quantification of Ribosome Occupancy
	14 Challenges of 
Scale
	15 Learning Predictive RNA Processing Networks
	16 Integrating Target Information to Generate Regulatory Maps for 
Individual RBPs
	17 Integration of 
Multiple RBP Datasets
	18 Conclusion
	References
Chapter 2: Genome-Wide Approaches for 
RNA Structure Probing
	1 Introduction
	2 Classical RNA Structure Probing Methodologies
		2.1 Nuclease-Based Approaches
		2.2 Base Modification-Based Approaches
		2.3 Backbone Modification-Based Methods
	3 Nuclease-Based High-Throughput Approaches
		3.1 Parallel Analysis of 
RNA Structures (PARS)
			3.1.1 PARS Analysis in Saccharomy
ces cerevisiae
			3.1.2 PARS with 
Temperature Elevation
			3.1.3 PARS Analysis of the Human Transcriptome, In Vitro and Native Dep
roteinized
		3.2 FragSeq
			3.2.1 FragSeq on€Mouse Nuclear RNA
		3.3 ds/ssRNA-Seq
			3.3.1 dsRNA-Seq in€Arabidopsis thaliana RDR6 Mutants
			3.3.2 ds/ssRNA-Seq in€Drosophila melanogaster and€Caenorhabditis elegans
			3.3.3 ds/ssRNA-Seq in€Arabidopsis
			3.3.4 Native Deproteinized ds/ssRNA-Seq in€Arabidopsis
	4 Chemical-Based High-Throughput Approaches
		4.1 Base Modification-Based High-Throughput Approaches
			4.1.1 DMS-Seq in€Yeast and€Human Cells
			4.1.2 Structure-Seq in€Arabidopsis
			4.1.3 Mod-Seq in€Yeast
			4.1.4 CIRS-Seq in€Mouse Embryonic Cells
		4.2 SHAPE-Based High-Throughput Approaches
	5 Conclusions and€Outlook
	References
Chapter 3: Tethered Function Assays as Tools to Elucidate the€Molecular Roles of 
RNA-Binding Proteins
	1 Introduction
	2 Designing and€Performing Tethered Function Assays and€Interpreting Their Results
		2.1 Constructs
		2.2 Position and€Number of€the€Tethering Sites
		2.3 Limitations, Controls, and€Interpretation
		2.4 Follow-Up and Validation
	3 Tethering Systems
		3.1 MS2
		3.2 λN
		3.3 PP7
		3.4 Iron Responsive Protein (IRP)
		3.5 Qβ, GA, Tat/TAR, and U1A
	4 Applications of€Tethered Function Assays
		4.1 mRNA Stability
		4.2 Translation
		4.3 Pre-mRNA Splicing
		4.4 RNA Transport and€Localization
	5 Conclusions and€Outlook
	References
Chapter 4: Single Molecule Approaches in 
RNA-Protein Interactions
	1 Introduction
	2 Single Molecule Investigation of€RNA-Protein Interactions
		2.1 Why Look at Single Molecules?
		2.2 Using Fluorescence Microscopy for€Single Molecule Imaging
	3 Single Molecule Co-localization Spectroscopy (CoSMoS)
		3.1 CoSMoS Instrumentation
		3.2 CoSMoS: Practical Considerations
		3.3 Fluorescent Proteins
		3.4 SNAP and 
CLIP Tags
		3.5 HaloTag
		3.6 DHFR Tag
	4 CoSMoS Data Analysis
	5 Conclusions
	References
Chapter 5: RNA Dynamics in the Control of 
Circadian Rhythm
	1 Introduction
	2 Circadian Regulation of 
Transcription Initiation
	3 Circadian Regulation of 
Transcription Termination
	4 Evidence for the Relevance of Posttranscriptional Events on 
Circadian Gene Expression
	5 Circadian Regulation of 
Alternative Splicing
	6 Circadian Polyadenylation
	7 Regulation at Translation Initiation and 
Ribosome Biogenesis
	8 RNA-binding Proteins Regulating mRNA Stability and Translational Efficiency Are Important for Oscillation of 
Core Clock Components
	9 Conclusion
	References
Chapter 6: Roles of RNA-binding Proteins and  Post- transcriptional Regulation in Driving Male Germ Cell Development in 
the Mouse
	1 Introduction
	2 Male Germ Cell Development
		2.1 Overview of 
Pathway
		2.2 Embryonic Stages of 
Germ Cell Development
		2.3 Postnatal Germ Cell Development
			2.3.1 Spermatogonia Proliferation, Renewal, and 
Differentiation
			2.3.2 Meiosis
			2.3.3 Spermiogenesis (Spermatid Differentiation)
	3 Complexity and Post-transcriptional Regulation of the 
Developing Germ Cell Transcriptome
		3.1 Modulating Gene Output Via Alternative Splicing and 
Polyadenylation
		3.2 Functional Consequences of Alternative Processing of 
Germ Cell mRNAs
			3.2.1 LIG3, SOX17, and 
CREM
			3.2.2 Different Fates for 
Alternatively Polyadenylated Germ Cell mRNAs
		3.3 Translational Control: Global and 
Message-Specific
		3.4 Post-transcriptional Control Through 
PolyA Tail Length Regulation
	4 Roles of RNA-binding Proteins in 
Germ Cell mRNA Regulation
		4.1 Elavl1/HuR
		4.2 CELF (CUGBP, ELAV-Like Family) Proteins
		4.3 Sam68
		4.4 PTB (Polypyrimidine Tract Binding) Family of 
RBPs
		4.5 τ-Cstf64
		4.6 Y-Box Proteins
		4.7 CPEB (Cytoplasmic Polyadenylation Element Binding Protein)
		4.8 Pumilio and 
Nanos
		4.9 Dazl
	5 Conclusion
	References
Chapter 7: Regulation of Stem Cell Self-Renewal and 
Oncogenesis by RNA-Binding Proteins
	1 Stem Cell Systems in Tissue Homeostasis and 
Oncogenesis
		1.1 Hematopoietic Stem Cells
		1.2 Intestinal Stem Cells
		1.3 Cancer Stem Cells
	2 Developmental Signals Regulating Stem Cell Self-Renewal
	3 RNA-binding Proteins: The Emerging Players in 
Stem Cell Regulatory Network
		3.1 Heterogeneous Ribonucleoprotein E2 (hnRNP E2)
		3.2 IGF2BP/IMP Family
		3.3 Lin28
		3.4 Musashi Family
		3.5 HuR/Elav Family
		3.6 FET Family
		3.7 Eukaryotic Translation Initiation Factor eIF4E
		3.8 PUF Family
	4 Conclusion
	References
Chapter 8: Controlling the Editor: The Many Roles of  RNA-Binding Proteins in 
Regulating A-to-I RNA Editing
	1 Introduction to 
RNA Editing
		1.1 Influence of ADAR Protein Domains on 
A-to-I Editing
	2 Identification of 
RNA Editing Sites
		2.1 Transcriptome-Wide Identification of 
RNA Editing Sites
	3 Regulation of 
ADAR Editing Activity
	4 RNA-binding Proteins that Regulate RNA Editing
		4.1 RNA-binding Proteins that Alter Editing and 
Splicing
		4.2 Double-Stranded RNA-binding Proteins that Influence RNA Editing
		4.3 Disease-Associated RNA-binding Proteins that Regulate RNA Editing
	5 Conclusions
	References
Chapter 9: Splicing Factor Mutations in 
Cancer
	1 Introduction
	2 Discovery of  Splicing Factor Mutations in 
Hematologic Malignancies
		2.1 SF3B1
		2.2 U2AF1
		2.3 SRSF2
		2.4 ZRSR2
	3 Additional Splicing Factors
	4 Splicing Inhibitors
	5 Summary
	References
Chapter 10: Regulation of Tissue-Specific Alternative Splicing: C. elegans as 
a Model System
	1 Introduction
		1.1 Importance of Alternative Splicing in Generating Diversity, Specialization and 
Regulation
	2 Mechanisms of Splicing
	3 Regulation of  Splice-Site Recognition: Auxiliary cis- Elements and 
Regulatory Factors
	4 Splicing Factors and 
Tissue Specific Alternative Splicing
	5 Importance of Alternative Splicing in Defining and 
Functionalizing Tissues
	6 Constitutive and Alternative Splicing in 
C. elegans
	7 Monitoring Alternative Splicing in 
C. elegans
	8 Tissue Specific Alternative Splicing in 
C. elegans
		8.1 Observing Tissue-Specific Alternative Splicing
		8.2 Assigning Function to Tissue-Specific Isoforms
	9 Cell-Specific Alternative Splicing Within Tissues
		9.1 Observing Cell-Type Specific Alternative Splicing
	10 RNA-binding Proteins and cis-Elements Controlling Cell Specific Alternative Splicing in 
C. elegans
	11 Evolution of Tissue-specific Alternative Splicing in 
C. elegans
		11.1 Evolutionary Characteristics of Alternative Splicing from 
Worm to Human
		11.2 Evolution of Tissue-Specific Alternative Splicing
		11.3 Conservation of 
Tissue-Specific Splicing Factors
	12 Perspectives and 
Future Goals
		12.1 Dynamic Regulation of Alternative Splicing in Response to 
Environmental Stimuli
		12.2 Splicing Regulation at the Level of Single Cells During 
Animal Development
	References
Chapter 11: RNA Granules and Diseases: A Case Study of Stress Granules in 
ALS and FTLD
	1 A Cellular World of 
RNA Granules
	2 Stress Granules
		2.1 Molecular Composition
		2.2 Proposed Functions
	3 RNA Granule Formation via Multivalency and 
Intrinsically Disordered Regions
		3.1 Multivalency
		3.2 Intrinsically Disordered Regions
		3.3 SG Assembly
			3.3.1 Regulation by Chaperones
			3.3.2 Regulation by Post Translational Modifications
	4 ALS and 
FTLD: When Aggregation Goes Awry
		4.1 SGs and 
Cytoplasmic Pathological Inclusions
		4.2 FUS, TDP-43 and 
Neurodegenerative Pathogenesis
			4.2.1 Mislocalization of FUS from the Nucleus to the 
Cytoplasm
			4.2.2 Prionogenicity of FUS and 
TDP-43
			4.2.3 Aggregation-Prone Mutations in FUS and 
TDP-43
			4.2.4 Mislocalization Without Aggregation
		4.3 Aggregation, Toxicity, and 
a Role for SGs
			4.3.1 Neuron-Specific Toxicity
			4.3.2 Stress and 
Inclusion Solubility
			4.3.3 Disruptions to 
Endogenous TDP-43/FUS Function
		4.4 TDP-43, FUS, and 
Oxidative Stress
	5 Concluding Remarks
	Notes
	References
Chapter 12: Post-Translational Modifications and 
RNA- Binding Proteins
	1 Introduction
	2 PTM-Mediated Regulation of 
Pre-mRNA Processing
		2.1 Transcription
		2.2 Splicing
		2.3 Alternative Splicing
		2.4 mRNA 5′ G-Capping and Decapping
		2.5 RNA Editing
	3 PTM Regulation of 
Subcellular Localization
		3.1 Nuclear/Cytoplasmic Shuttling
		3.2 RNA Granules, P-Bodies and 
Nuclear Speckles
		3.3 Exosome
	4 PTM Regulation of 
Translation
	5 PTM Regulation of RNA Stability and 
Destruction
		5.1 miRNA Related Repression
		5.2 RNA Decay
	6 Conclusion
	References
Index