Alternative pre-mRNA Splicing E-Book

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 1414

Veröffentlichungsjahr: 2012

Beschreibung

This book was written for graduate and medical students, as well as clinicians and postdoctoral researchers. It describes the theory of alternative pre-mRNA splicing in twelve introductory chapters and then introduces protocols and their theoretical background relevant for experimental research. These 43 practical chapters cover: Basic methods, Detection of splicing events, Analysis of alternative pre-mRNA splicing in vitro and in vivo, Manipulation of splicing events, and Bioinformatic analysis of alternative splicing. A theoretical introduction and practical guide for molecular biologists, geneticists,clinicians and every researcher interested in alternative splicing. Website: www.wiley-vch.de/home/splicing

Leseprobe

Contents

Cover

Related Titles

Title Page

Copyright

Preface

List of Abbreviations

List of Contributors

Part One: Theory

Chapter 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

Acknowledgments

References

Chapter 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

2.5 7SL RNA

2.6 7SK RNA

2.7 U-Rich Small Nuclear RNAs (U snRNAs)

References

Chapter 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

Acknowledgments

References

Chapter 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

4.3 The Zinc Finger Domain

4.4 The KH Domain

4.5 Conclusions and Perspectives

Acknowledgments

References

Chapter 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

Acknowledgments

References

Chapter 6: The Use of Saccharomyces cerevisiae to 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

Acknowledgments

References

Chapter 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

Acknowledgments

References

Chapter 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 Src N1 Exon: A Model of Combinatorial Splicing Regulation

8.6 The Global View: Towards a Splicing Code

Acknowledgments

References

Chapter 9: Integration of Splicing with Nuclear and Cellular Events

9.1 Introduction

9.2 Overview

9.3 Nuclear Structure and Distribution of Splicing Factors

9.4 Integration of Splicing with Nuclear and Cellular Processes

References

Chapter 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

Conclusions

Acknowledgments

References

Chapter 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

11.5 Concluding Remarks

References

Part Two: Basic Methods

Chapter 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

Chapter 13: Ultracentrifugation in the Analysis and Purification of Spliceosomes Assembled In Vitro

13.1 Theoretical Background

13.2 Protocol

13.1 Example Experiment

Troubleshooting

References

Chapter 14: Chemical Synthesis of RNA

14.1 Theoretical Background

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

Troubleshooting

References

Chapter 15: RNA Interference (siRNA, shRNA)

15.1 Theoretical Background

15.2 Protocol

15.3 Example Experiment

15.4 Troubleshooting

References

Chapter 16: Expression and Purification of Splicing Proteins

16.1 Theoretical Background

16.2 Protocol 1: The Preparation of Total HeLa SR Proteins

16.3 Protocol 2: The Purification of Individual SR Proteins

References

Chapter 17: Detection of RNA–Protein Complexes by Electrophoretic Mobility Shift Assay

17.1 Theoretical Background

17.2 Protocol

17.3 Example Experiment

17.4 Troubleshooting

Acknowledgments

References

Chapter 18: Functional Analysis of Large Exonic Sequences Through Iterative In Vivo Selection

18.1 Theoretical Background

18.2 Protocol

18.3 Example Experiment

18.4 Troubleshooting

18.5 Acknowledgments

References

Chapter 19: Identification of Splicing cis-Elements Through an Ultra-Refined Antisense Microwalk

19.1 Theoretical Background

19.2 Protocol

19.3 Example Experiment

19.4 Troubleshooting

Acknowledgments

References

Chapter 20: Genomic SELEX to Identify RNA Targets of Plant RNA-Binding Proteins

20.1 Introduction

20.2 Protocols

Protocol 1: Genomic Library Construction

Protocol 2: Affinity Selection of RNA Targets

20.3 Example Experiment

20.4 Troubleshooting

Acknowledgments

References

Part Three: Detection of Splicing Events

Chapter 21: Quantification of Alternative Splice Variants

21.1 Theoretical Background

21.2 Protocol

21.3 Example Experiment: Microarray Validation of PTB-Regulated Events

21.4 Troubleshooting

Acknowledgments

References

Chapter 22: High-Throughput Analysis of Alternative Splicing by RT-PCR

22.1 Theoretical Background

22.2 Protocol

22.3 Example Experiment

22.4 Troubleshooting

Acknowledgments

References

Chapter 23: Monitoring Changes in Plant Alternative Splicing Events

23.1 Theoretical Background

23.2 Protocols

Protocol 1: Multiple RT-PCR AS Panel Using 96-Well Plates

Protocol 2: Cloning and Characterization of Novel Alternatively Spliced Transcripts

Protocol 3: Assessing AS in Genes of Interest by Cloning Full-Length cDNA Transcripts

23.3 Example Experiments

23.4 Troubleshooting

Acknowledgments

References

Chapter 24: Array Analysis

24.1 Theoretical Background

24.2 Protocol

24.3 Example Experiment

24.4 Troubleshooting

Acknowledgments

References

Chapter 25: The CLIP Method to Study Protein–RNA Interactions in Intact Cells and Tissues

25.1 Theoretical Background

25.2 Protocols

Protocol 1: UV Crosslinking

Protocol 2: Immunoprecipitation

Protocol 3: 3′ RNA Adapter Ligation

Protocol 4: RNA Purification

Protocol 5: 5′ RNA Adapter Ligation

Protocol 6: Reverse Transcription

Acknowledgments

References

Chapter 26: RNA–Protein Crosslinking and Immunoprecipitation (CLIP) in Schizosaccharomyces pombe

26.1 Introduction

26.2 Protocol

26.3 Example Experiment

26.4 Troubleshooting

Acknowledgments

References

Chapter 27: Identification of Proteins Bound to RNA

27.1 Theoretical Background

27.2 Protocol

27.3 Example Experiment

27.4 Troubleshooting

References

Chapter 28: Single-Cell Detection of Splicing Events with Fluorescent Splicing Reporters

28.1 Theoretical Background

28.2 Protocols

Protocol 1: Two-Step PCR Amplification of attB-DNA Fragments

Protocol 2: BP Clonase II Reaction and Selection of Appropriate “Entry” Clones

Protocol 3: LR Clonase II Plus Reaction and Selection of Appropriate “Expression” Clones

Protocol 4: RT-PCR Analysis of Minigene-Derived mRNAs

28.3 Example Experiments

28.4 Troubleshooting

Acknowledgments

References

Part Four: Analysis of Splicing In Vitro

Chapter 29: The Preparation of HeLa Cell Nuclear Extracts

29.1 Theoretical Background

29.2 Protocols

Protocol 1: Large-Scale Growth of HeLa Cells

Protocol 2: Small-Scale Preparation of Nuclear Extracts

29.3 Example Experiment

29.4 Troubleshooting

References

Chapter 30: In Vitro Splicing Assays

30.1 Theoretical Background

30.2 Protocols

Protocol 1: In Vitro Splicing Reactions with Nuclear Extracts

Protocol 2: Analysis of Splicing Products by Denaturing PAGE

30.3 Example Experiment

30.4 Troubleshooting

References

Chapter 31: Assembly and Isolation of Spliceosomal Complexes In Vitro

31.1 Theoretical Background

31.2 Protocols

Protocol 1: The Core Splicing Reaction

Protocol 2: Agarose Gel Electrophoresis of Splicing Complexes

Protocol 3: MS2–MBP Purification

Example Experiment

31.4 Troubleshooting

References

Chapter 32: Analysis of Site-Specific RNA–Protein Interactions

32.1 Theoretical Background

32.2 Protocols

Protocol 1: UV Crosslinking

Protocol 2: Immunoselection of Digested Crosslinked Products

Protocol 3: In Vitro Probing of RNA Structure and RNA/Protein Complexes

Protocol 4: Probing of Yeast RNAs Modified In Vivo by DMS Treatment

Protocol 5: Supershift Experiment

32.3 Example Experiments

32.4 Troubleshooting

Acknowledgments

References

Chapter 33: Immunoprecipitation and Pull-Down of Nuclear Proteins

33.1 Theoretical Background

33.2 Protocols

Protocol 1: Immunoprecipitation of GFP-Tagged Proteins

Protocol 2: GST Pull-Down of Nuclear Proteins

33.3 Example Experiments

33.4 Troubleshooting

Acknowledgments

References

Chapter 34: Analysis of Protein (-RNA) Complexes by (Quantitative) Mass Spectrometric Analysis

34.1 Theoretical Background

34.2 Protocols

Protocol 1: Standard Proteome Analysis

34.4 Protocol 2: Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Protocol 3: iTRAQ Labeling of In-Gel-Digested Proteins

34.3 Example Experiment

34.4 Troubleshooting

References

Chapter 35: Fast Cloning of Splicing Reporter Minigenes

35.1 Theoretical Background

35.2 Protocol

35.3 Example Experiment

35.4 Troubleshooting

Acknowledgments

References

Chapter 36: In Vivo Analysis of Splicing Assays

36.1 Theoretical Background

36.2 Protocol

Protocol 1: Transfection (see also Figure 36.2)

Protocol 2: RNA Extraction

Protocol 3: RT-PCR

36.3 Example Experiment

36.4 Troubleshooting

Acknowledgments

References

Chapter 37: Coupled Promoter Splicing Systems

37.1 Theoretical Background

37.2 Protocol

37.3 Example Experiment

37.4 Troubleshooting

References

Chapter 38: Stable Cell Lines with Splicing Reporters

38.1 Theoretical Background

38.2 Protocol

38.3 Example Experiment

38.4 Troubleshooting

References

Chapter 39: Splicing Factor ChIP and ChRIP: Detection of Splicing and Splicing Factors at Genes by Chromatin Immunoprecipitation

39.1 Theoretical Background

39.2 Protocol

39.3 Example Experiment

39.4 Troubleshooting

Acknowledgments

References

Chapter 40: Yeast Genetics to Investigate the Function of Core Pre-mRNA Splicing Factors

40.1 Theoretical Background

40.2 Protocol

Protocol 1: Transformation of Yeast Cells

40.3 Example Experiment

40.4 Troubleshooting

References

Chapter 41: Analysis of HIV-1 RNA Splicing

41.1 Theoretical Background

41.2 Protocols

41.3 Example Experiment

41.4 Troubleshooting

Acknowledgments

References

Chapter 42: In Vivo Analysis of Plant Intron Splicing

42.1 Theoretical Background

42.2 Protocols

Protocol 1: Transfection of Tobacco Leaf Protoplasts

Protocol 2: Transfection of Arabidopsis Cell Suspension Protoplasts

Protocol 3: Agrobacterium-Mediated Infiltration of Nicotiana benthamiana Leaves

42.3 Example Experiment

42.4 Troubleshooting

Acknowledgments

References

Chapter 43: Modification State-Specific Antibodies

43.1 Theoretical Background

43.2 Protocol

43.3 Example Experiment

43.4 Troubleshooting

References

Chapter 44: Analysis of Alternative Splicing in Drosophila Genetic Mosaics

44.1 Theoretical Background

44.2 Protocol

44.3 Example Experiment

44.4 Troubleshooting

References

Part Six: Manipulation of Splicing Events

Chapter 45: Antisense Derivatives of U7 Small Nuclear RNA as Modulators of Pre-mRNA Splicing

45.1 Theoretical Background

45.2 Protocols

Protocol 1: PCR-Based Introduction of Functional Sequences into U7 SmOPT

Protocol 2: Transfer of a U7 Cassette into a Lentiviral Vector for Stable Integration into the Genome

45.3 Example Experiment

45.4 Troubleshooting

Acknowledgments

References

Chapter 46: Screening for Alternative Splicing Modulators

46.1 Theoretical Background

46.2 Protocols

46.3 Example Experiment

46.4 Troubleshooting

References

Chapter 47: Use of Oligonucleotides to Change Splicing

47.1 Theoretical Background

47.2 Protocol

47.3 Example Experiment

47.4 Troubleshooting

References

Chapter 48: Changing Signals to the Spliceosome

48.1 Theoretical Background

48.2 Protocols

48.3 Example Experiment

48.4 Troubleshooting

Acknowledgments

References

Part Seven: Bioinformatic Analysis of Splicing

Chapter 49: Overview of Splicing Relevant Databases

49.1 Theoretical Background

49.2 Protocol

49.3 Example Experiment

49.4 Troubleshooting

References

Chapter 50: Analysis of RNA Transcripts by High-Throughput RNA Sequencing

50.1 Theoretical Background

50.2 Protocol

50.3 Example Experiment

50.4 Troubleshooting

References

Chapter 51: Identification of Splicing Factor Target Genes by High-Throughput Sequencing

51.1 Theoretical Background

51.2 Protocol

51.3 Example Experiment

51.4 Troubleshooting

Acknowledgments

References

Chapter 52: Bioinformatic Analysis of Splicing Events

52.1 Theoretical Background

52.2 Protocol

52.3 Example Experiment

Troubleshooting

Acknowledgments

References

Chapter 53: Analysis of Pre-mRNA Secondary Structures and Alternative Splicing

53.1 Theoretical Background

53.2 Protocol

53.3 Example Experiment

53.4 Troubleshooting

Acknowledgments

References

Chapter 54: Structure Prediction for Alternatively Spliced Proteins

54.1 Theoretical Background

54.2 Protocol

54.3 Example Experiment

54.4 Troubleshooting (see Table 54.1)

Acknowledgments

References

Chapter 55: Comparative Genomics Methods for the Prediction of Small RNA-Binding Sites

55.1 Theoretical Background

55.2 Protocol

55.3 Example Experiment

55.4 Troubleshooting

Acknowledgments

References

Appendix A1: Yeast Nomenclature Systematic Open Reading Frame (ORF) Designations

A1.1 Protein-Coding Genes

A1.2 Recombinant Derivatives

A1.3 Proteins

A1.4 Noncoding Genes, Genes Not Encoded by Nuclear Chromosomal DNA, and other Chromosomal Features

A1.5 Yeast Strains

References

Appendix A2: Glossary

Index

Preface

The sequencing and analysis of numerous genomes, and more recently transcriptomes, has shown that RNA is much more than a recording tape used to transmit genetic information from DNA to proteins. Although most human DNA is transcribed into RNA, only 1–2% of the DNA contains information directly encoding proteins, the biomolecules that orchestrate most of the cell's functions. Owing to its chemical properties and reactivity, and the lack of a fully complementary strand, RNA is very versatile: as a container for genetic information, as a tool that can regulate gene expression, as a catalyst for chemical reactions and even as a scaffold for protein complexes. RNA is therefore central not only to the basic processes of gene expression (DNA → RNA → protein), but it also has widespread roles in controlling gene expression at all levels.

Of the RNA-based mechanisms to regulate gene expression, alternative splicing has the most direct influence on the proteins formed. Alternative splicing enables the vast majority of human genes to encode more than one protein. In some instances a single gene can even create thousands of functionally distinct proteins. Much progress has been made in understanding the basic mechanism of pre-mRNA splicing and the rules that control alternative exon selection. The study of model organisms has demonstrated clearly that the correct regulation of alternative splicing is important for the proper functioning of cells and organisms, and this is underscored by evidence from the clinic. Changes in alternative splicing, caused either by mutations or alterations of regulatory factors can result in human diseases. An ever increasing catalogue of splicing-related pathologies attests to the importance of alternative splicing for human health.

The past few years have seen an encouraging start towards meeting major challenges such as predicting from genomic DNA sequence alone how splice sites will be recognized and regulated in different tissues, or the therapeutic manipulation of splicing in animal models of human disease. But, despite this considerable progress, much exciting work remains to be done before we can fully comprehend alternative pre-mRNA splicing.

As alternative pre-mRNA splicing impacts upon many areas in biology and medicine, scientists working on the subject come from different disciplines, ranging from medical geneticists to biochemists, molecular biologists and bioinformaticians. To bring these groups together, a European Alternative Splicing Network (EURASNET) was initiated in 2005 and continued until its formal winding-up in early 2011. EURASNET was a consortium of 43 research groups from Europe, Israel and Argentina, funded by the European Commission through its VIth Framework programme from 2006 to 2011. EURASNET has contributed to the integration of research efforts and dissemination of knowledge in the field of alternative pre-mRNA splicing. Its network activities were not restricted to research, but were also aimed at informing other scientists of alternative splicing. EURASNET organized numerous workshops and practical ‘hands-on’ courses all over Europe. Many of the protocols described in this book were tested in these settings.

One of the key missions of EURASNET was to reach out across scientific disciplines and make people aware of the importance of alternative splicing. In this spirit, we encourage post-doctoral fellows and graduate students to contact the authors of the various chapters, listed in the list of contributors, if they see any opportunity for collaboration. A simple Internet search or a visit to the EURASNET site (www.eurasnet.info) will reveal their current e-mail addresses.

Despite the importance of alternative splicing as a fundamental biological phenomenon and as a physiological mechanism which impacts on human health, to date there has been no single volume summarizing the current state of knowledge about alternative splicing, as well as detailing the experimental protocols for its analysis. This book aims to make good this deficit, giving an overview of both the theory and practice of alternative splicing. Its intendedreadership includes graduate students, post-doctoral fellows working in life sciences, medical practitioners who encounter aberrant alternative splicing in patients, and established investigators from other fields.

The book consists of two major parts: The first one provides a brief theoretical introduction that gives a short overview of alternative splicing and cites key papers in the field for more in-depth information. The second part is a collection of experimental protocols that are used in the field of alternative splicing. We envisage the protocols not as ‘cookbook-recipes’, but as guides for experiments that allow investigators to understand the procedures. Therefore, each protocol has a theoretical introduction explaining the background of the experiments, a list for troubleshooting and an example of an experiment. Each protocol is preceded by a graphic outline of the procedure that concisely summarizes the method and lists theexpected outcome of the experiment and the scientific questions that can be asked. We hope that this feature will allow readers to quickly find the experimental tools necessary for their projects and that it will stimulateinterest in looking at other techniques. The protocolsare arranged into six groups according to the scientific question that they address, which together with the theoretical introduction subdivide the book into seven parts. To help in orientation, these parts are marked by sidebars in Isaac Newton's rainbow colors.

The protocols are generally advanced, and a basic knowledge of molecular biology and RNA methods is necessary. Excellent textbooks that cover these topics are listed in Chapter 12 (Stamm), which can serve as a further entry point.

We would like to thank the European Commission for their vision and support, which has promoted collaboration and the establishment of durable links that have changed the landscape of research in alternative splicing and its medical impact. We are also grateful to the members of the scientific advisory board of EURASNET for their constant encouragement and critical input: Joan Steitz, Mariano-Garcia Blanco, James Dahlberg, Witek Filipowicz, Adrian Krainer, Michael Rosbash and Robert Singer.

Many thanks to Anne Chassin du Guerny, Andrea Zschaege, Nitin Vashisht and Andreas Sendtko from Wiley for their excellent support in publishing this book.

Finally, we are indebted to Jason Scroggin and Akari Takebayashi who teach at the University of Kentucky College of Design and are principals of Design Office Takebayashi Scroggin (http://dots-ky.com) for unifying the graphic design of the book, for getting into molecular biology and for their patience with endless revisions.

We hope that the book will help newcomers, especially medically oriented investigators, to understand the fascinating world of alternative splicing. We envisage that the protocols will allow experiments that push the field forward and that the background information will stimulate the improvement of existing protocols and development of new procedures.

October 2011

Stefan Stamm, Lexington, KY, USA Chris Smith, Cambridge, United Kingdom Reinhard Lührmann, Göttingen, Germany

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty can be created or extended by sales representatives or written sales materials. The Advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

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List of Abbreviations

µCEmicrocapillary electrophoresisα-MEMalpha-modified Eagle's medium2,4-D2,4-dichlorophenoxyacetic acid2-Dtwo-dimensional2-D-LCtwo-dimensional liquid chromatography2OMePS2′-O-methyl phosphorothioate3′ UTR3′ untranslated region3′SS3′ splice site4Risoform containing four C-terminal microtubule-binding repeat domains5′ UTR5′ untranslated region5′SS5′ splice site5-FOA5-fluoroorotic acidAAVadenovirus-associated virusACEbis(2-acetoxyethoxy)methyl orthoesterAgoArgonauteAhx2-aminohexanoic acidamuatomic mass unitAONantisense oligonucleotideAPOBapo-lipoprotein BAPOB100apo-lipoprotein B full-length isoformAQUAabsolute quantificationASalternative splicingASAPAlternative Splicing Annotation ProjectASDBAlternative Splicing DataBaseASEalternative splicing eventASOantisense oligonucleotideASTDAlternative Splicing and Transcript Diversity DatabaseASTRAAlternative Splicing and Transcription ArchivesATMataxia telangiectasia mutated geneATPadenosine 5′ triphosphateattattachment siteattBattachment site BattPattachment site PBACBacterial artificial chromosomeBAP6-benzylaminopurineBBSborate-buffered salineBcl-xB-cell lymphoma X geneBcl-xLB-cell lymphoma X large isoformBcl-xSB-cell lymphoma X small isoformbpbase pairsBPSbranchpoint sequenceBRCA1breast cancer 1 geneBSbranch siteBSAbovine serum albuminCaMVcauliflower mosaic virusccdcontrolled cell deathcDNAcomplementary DNACFTRcystic fibrosis transmembrane conductance regulator geneChIPchromatin immunoprecipitationChRIPchromatin RNA immunoprecipitationcICATcleavable isotope coded affinity tagCIDcollision-induced dissociationCMVcytomegalovirusc-myccellular myelocytomatosis viral oncogenecog-1Connection of Gonad defective family member 1COL7A1collagen type VII alpha 1COX2cyclooxygenase 2Ctcycle thresholdCTDC-terminal domainDAMDNA adenine methylasedammutant for DNA adenine methylaseDAmPDecreased Abundance by mRNA PerturbationDBHSDrosophila melanogaster behavior, human splicingdddouble-distilledDDBJDNA Data Bank of JapanDEPCdiethylpyrocarbonateDMDDuchenne muscular dystrophyDMEMDulbecco's modified Eagle mediumDMFdimethylformamideDMSdimethylsulfateDMSOdimethylsulfoxideDMT4,4,′-dimethoxytritylDNAdeoxyribonucleic aciddsDNAdouble-stranded DNAdsRBDdouble-stranded RNA-binding domainDTEdithioerythritolDTTdithiothreitolE33exon number 33ECDelectron capture dissociationECLenhanced chemiluminescenceEDIExtra Domain IEDTAEthylenediaminetetraacetic acidEGFPenhanced green fluorescent proteinEJCexon junction complexEMBLEuropean Molecular Biology LaboratoryEMSAelectrophoretic mobility shift assayENCODEENCyclopedia Of DNA ElementsESEexonic splicing enhancerESIelectrospray ionizationESSExon splicing silencerESTexpressed sequence tagExinctextended inhibitory contextFAST DBFriendly Alternative Splicing and Transcripts DataBaseFBSfetal bovine serumFCSfetal calf serumflcDNAfull-length complementary DNAFlp-InFlippase IntegraseFOLH1folate hydrolase (prostate-specific membrane antigen) 1FRTFlippase Recognition TargetGFglucose/fructoseGFPgreen fluorescent proteinGMglucose-mannitolGSHglutathioneGSPgene-specific primerGSSGenome Survey SequenceGSTglutathione S-transferaseHBSHEPES-buffered salineHDhomology domainH-DBASHuman-transcriptome Database for Alternative SplicingHEBheparin elution bufferHexim1hexamethylene bis-acetamide-inducible mRNA1HGPSHutchison–Gilford progeria syndromeHIVhuman immune deficiency virusHLAhuman leukocyte antigenhnRNPheterogeneous nuclear ribonucleoproteinHRPhorseradish peroxidaseHTGShigh-throughput genomic sequenceICPLisotope-coded protein labelIgGimmunoglobulin GIHFintegration host factorIL-5RαInterleukin-5 receptor alpha geneIPGimmobilized pH gradientIPTGisopropyl-beta-D-thiogalactopyranosideISEintronic splicing enhancerISSintronic splicing silencerISS-N1intronic splicing silencer N1iTRAQisobaric tags for relative and absolute quantificationKLHkeyhole limpet hemocyaninK-turnkink-turnLMPTPlow-molecular-weight phosphotyrosine phosphataseLQTSlong QT syndromeLsmlike SmMAASEManually Annotated Alternatively Spliced Events DatabaseMALDImatrix-assisted laser desorption/ionizationMALDI-ToF MSmatrix-assisted laser desorption ionization time-of-flight mass spectrometryMAPTmicrotubule-associated protein tau geneMBNL1muscleblind-like 1MBPmaltose-binding proteinMCMmethylmalonylCoA mutaseMCSmulticloning siteMdxmouse dystrophy X-linkedMES2[N-morpholino]ethanesulfonic acidMHCmajor histocompatibility complexMIDmiddle domainMIDASmicroarray detection of alternative splicingMIPSMunich Information Center for Protein SequencesmiRNAmicroRNAMOE2′-O-methoxy ethylMRImagnetic resonance imagingmRNAmessenger RNAMSmass spectrometryMuD-PITmultidimensional protein separation technologyMuMLVmurine leukemia virusMUTmethylmalonyl coenzyme A mutaseMyD88myeloid differentiation primary response gene (88)MyD88Lmyeloid differentiation primary response gene (88) large isoformMyD88Smyeloid differentiation primary response gene (88) small isoformNAA1-naphthaleneacetic acidNaOAcsodium acetatencRNAnoncoding RNAncRNAnon-protein-coding RNANEnuclear extractNF1neurofibromatosis type 1NGSNext Generation SequencingNHSN-hydroxysuccinimideNMDnonsense-mediated (mRNA) decayNMRnuclear magnetic resonanceNova2Neuro-oncological ventral antigen 2nPTBneural polypyrimidine binding proteinntnucleotideNTCnineteen complexOAocular albinismOA1ocular albinism type 1ORFopen reading framePABPN1poly(A) binding protein 1PACpattern-based correlationPAGEpolyacrylamide gel electrophoresisPALSdbPutative Alternative Splicing DataBasePBSphosphate-buffered salinePCCpropionyl coenzyme A carboxylasePCCApropionyl coenzyme A carboxylase APCCBpropionyl coenzyme A carboxylase BPCIphenol/chloroform/iso-amylalcoholPCRpolymerase chain reactionPEGpolyethylene glycolPEIpolyethyleneiminePESEputative exonic splicing enhancerPESSputative exonic splicing silencerPIPhosphorImagerPIP3phosphatidylinositol-3,4,5-trisphosphatepiRNAPiwi-interacting RNAPiwiP-element induced wimpy testis in DrosophilaPMLpromyelocytic leukemiaPMOphosphorodiamidate morpholino oligomerPMSFphenylmethanesulfonylfluoridePol IIpolymerase IIPPIasepeptidyl-prolyl (cis/trans) isomerasepre-mRNAprecursor messenger RNAΨpseudouridinepsnoRNAprocessed small nucleolar RNAPTBpolypyrimidine track binding proteinPTCpremature termination codonPTCH1patched homolog 1 genep-TEFbpositive transcription elongation factor bPTMpost-translational modificationPUprobability of being unpairedPVDFpolyvinylidene fluoridePVPpoly(vinyl pyrrolidone)PWSPrader–Willi syndromeQCquality controlqPCRquantitative PCRQ-ToFquadrupole time-of-flightQUA2Quaking homology 2RBPRNA-binding proteinRCFrelative centrifugal forceRFPred fluorescent proteinRGASPRNA-seq Genome Annotation Assessment ProjectRINRNA integrity numberRIPAradioimmunoprecipitation assayRISCRNA-induced silencing complexRITSRNA-induced transcriptional silencingRNAaRNA activationRNAiRNA interferenceRNaseribonucleaseRNA-Seqwhole transcriptome shotgun sequencingRNPribonucleoproteinRPreversed phaseRPKMreads per kilobase per millionRP-LCreversed phase liquid chromatographyRRMRNA recognition motifrRNAribosomal RNARSarginine–serine-richRTreverse transcriptase/transcriptionRT-PCRreverse transcription and polymerase chain reactionSBsonication bufferscaRNAsmall Cajal body RNAscaRNPCajal body-specific RNPSCXstrong cation-exchange chromatographysdRNAsnoRNA derived RNASDSsodium dodecyl sulfateSELEXSystematic Evolution of Ligands through Exponential enrichmentSF1/BBP proteinsplicing factor 1/branch binding proteinSF2splicing factor 2SF-ChIPsplicing factor ChIPSGAsynthetic generic arraySGDSaccharomyces Genome DatabaseshRNAshort hairpin RNASILACstable isotope labeling by amino acids in cell culturesiRNAshort interference RNASKIPSki oncogene interacting proteinSLAMsynthetic-lethal analysis by microarraySMAspinal muscular atrophySMaRTspliceosome-mediated RNA trans-splicingSMNsurvival of motoneuronsnoRNAsmall nucleolar RNAsnoRNPsmall nucleolar ribonucleoprotein particleSNPsingle nucleotide polymorphismSNRsignal-to-noise ratiosnRNAsmall nuclear RNAsnRNPsmall nuclear ribonucleoproteinSR proteinserine and arginine-rich proteinSREsplicing regulatory elementsRNAsnoRNA like RNASSsplice sitessDNAsingle-stranded DNASTP4-sulfonyl-tetrafluorophenylSTSsequence-tagged siteTAIRThe Arabidopsis Information ResourceTaqThermus aquaticusTARtransactivating responseTBDMStert-butyldimethylsilylTBSTris-buffered salineTCAtrichloroacetic acidTEVtobacco etch virusTMTtandem mass tagTNFαtumor necrosis factor alphaTOTetracycline ONTOM2′-O-triisopropylsilyloxymethylTPA12-O-tetradecanoylphorbol-13-acetatetra2transformer2TRBPHIV-1 TAR RNA-binding proteinT-RexTetracycline Repressor XTREXTRanscription EXporttRNAtransfer RNAUUridineUASupstream activator sequenceUBA1aUBP1-associated protein 1aUCSCUniversity of California, Santa CruzUHMU2AF homology motifULMUHM-ligand motifUPFup-frameshiftUTRuntranslated regionUVultravioletWBwash bufferWT1Wilms tumor protein 1 geneZFPzinc finger protein

List of Contributors

Annemieke Aartsma-Rus Leiden University Medical Center Department of Human Genetics Albinusdreef 22333 ZA Leiden The Netherlands

Frédéric H.-T. Allain ETH Zü;rich Institute for Molecular Biology and Biophysics Schafmattstr. 20, HPK G18 8093 Zurich Switzerland

Olga Bannikova Medical University of Vienna Max F. Perutz Laboratories Dr. Bohrgasse 9/3 1030 Vienna Austria

Diana Baralle University of Southampton Human Genetics Division Duthie Building (Mailpoint 808) Southampton General Hospital Tremona Road Southampton SO16 6YD UK

Francisco E. Baralle International Centre of Genetic Engineering and Biotechnology (ICGEB) Department of Molecular Pathology Padriciano 99 34012 Trieste Italy

Marco Baralle International Center of Genetic Engineering and Biotechnology (ICGEB) Department of Molecular Pathology Padriciano 99 34012 Trieste Italy

Nicole Bardehle Max Planck Institute of Molecular Cell Biology and Genetics Pfotenhauerstraße 108 01307 Dresden Germany

Andrea Barta Medical University of Vienna Max F. Perutz Laboratories Dr. Bohrgasse 9/3 1030 Vienna Austria

Jean D. Beggs University of Edinburgh Wellcome Trust Centre for Cell Biology King's Buildings, Mayfield Road Edinburgh EH9 3JR UK

Isabelle Behm-Ansmant Nancy University Faculté de Médecine Laboratoire AREMS CNRS UMR 7214 BP 70184 54506 Vandoeuvre-les-Nancy Cedex France

Michaela Beitzinger Universitä;t Regensburg Fakultä;t fü;r Biologie und Vorklinische Medizin Lehrstuhl fü;r Biochemie I Universitä;tsstraße 31 93053 Regensburg Germany

Natalya Benderska University of Kentucky Department of Molecular and Cellular Biochemistry B278 Biomedical/Biological Sciences Research Building 741 South Limestone Street Lexington, KY 40536-0298 USA

Eric A. Berg 21st Century Biochemicals, Inc. 260 Cedar Hill Street Marlborough, MA 01752 USA

Cyril F. Bourgeois University of Strasbourg Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) INSERM U964, CNRS UMR 7104 1 rue Laurent Fries, BP 10142 67404 Illkirch France

and

INSERM U1052, CNRS UMR 5286 Centre de Recherche en Cancérologie de Lyon (CRCL) Centre Léon Bérard 28 Rue Laë;nnec 69008 Lyon France

Christiane Branlant Nancy University Faculté de Médecine Laboratoire AREMS CNRS UMR 7214 BP 70184 54506 Vandoeuvre-les-Nancy Cedex France

John W.S. Brown University of Dundee at SCRI College of Life Sciences Division of Plant Sciences Dundee DD2 5DA UK

and

Scottish Crop Research Institute Genetics Programme Dundee DD2 5DA UK

Janusz M. Bujnicki International Institute of Molecular and Cell Biology in Warsaw Laboratory of Bioinformatics and Protein Engineering ul. Ks. Trojdena 4 02-109 Warsaw Poland

and

Adam Mickiewicz University Institute of Molecular Biology and Biotechnology ul. Umultowska 89 61-614 Poznan Poland

Emanuele Buratti International Centre of Genetic Engineering and Biotechnology (ICGEB) Department of Molecular Pathology Padriciano 99 34012 Trieste Italy

Daphne S. Cabianca San Raffaele Scientific Institute Division of Regenerative Medicine Via Olgettina 58 20132 Milan Italy

and

Università Vita-Salute San Raffaele Via Olgettina 58 20132 Milan Italy

Benoit Chabot Université de Sherbrooke Faculté de médecine et des sciences de la santé Département de microbiologie et d'infectiologie Laboratoire de génomique fonctionnelle de l'Université de Sherbrooke Sherbrooke, Québec Canada J1H 5N4

Sean Chapman Crop Research Institute Plant Pathology Programme Dundee DD2 5DA UK

Min Chen University of Kentucky Department of Biology 335A T.H. Morgan Building Lexington, KY 40506-0225 USA

Antoine Cléry ETH Zü;rich Institute for Molecular Biology and Biophysics Schafmattstr. 20, HPK G18 8093 Zurich Switzerland

Alan Cochrane University of Toronto Department of Molecular Genetics 1 King's College Circle Toronto, Ontario Canada M5S-1A8

Miguel B. Coelho University of Cambridge Department of Biochemistry 80 Tennis Court Road Cambridge CB2 1QW UK

Denise R. Cooper The James A. Haley Veterans Hospital Research Service 13000 Bruce B. Downs Blvd. Tampa, FL 33612 USA

and

University of South Florida College of Medicine Department of Molecular Medicine 12901 Bruce B. Downs Blvd. Tampa, FL 33612 USA

Christian Kroun Damgaard University of Aarhus Department of Molecular Biology C.F. Mllers Alle, Bldg 1130 8000 Aarhus C Denmark

Diane Davidson Scottish Crop Research Institute Genetics Programme Dundee DD2 5DA UK

Pierre de la Grange GenoSplice technology Centre Hayem Hôpital Saint-Louis 1 Avenue Claude Vellefaux 75010 Paris France

Manuel de la Mata Universidad de Buenos Aires Facultad de Ciencias Exactas y Naturales IFIBYNE-CONICET Departamento de Fisiologìa, Biologìa Molecular y Celular Laboratorio de Fisiologìa y Biologìa Molecular Pabellón 2, piso 2 C1428EHA Buenos Aires Argentina

and

Friedrich Miescher Institute for Biomedical Research, PO Box 2543 4002 Basel Switzerland

Simon Duffy University of Toronto Department of Molecular Genetics 1 King's College Circle Toronto, Ontario Canada M5S-1A8

Sherif Abou Elela Université de Sherbrooke Faculté de médecine et des sciences de la santé Département de microbiologie et d'infectiologie Laboratoire de génomique fonctionnelle de l'Université de Sherbrooke Sherbrooke, Québec Canada J1H 5N4

Patrizia Fabrizio Max Planck Institute for Biophysical Chemistry Department of Cellular Biochemistry Am Fassberg 11 37077 Göttingen Germany

Jordan B. Fishman 21st Century Biochemicals, Inc. 260 Cedar Hill Street Marlborough, MA 01752 USA

Michael G. Fried University of Kentucky Center for Structural Biology Department of Molecular and Cellular Biochemistry B278 Biomedical/Biological Sciences Research Building 741 South Limestone Street Lexington, KY 40536-0298 USA

John D. Fuller University of Dundee at SCRI Division of Plant Sciences Dundee DD2 5DA UK

Davide Gabellini San Raffaele Scientific Institute Division of Regenerative Medicine Via Olgettina 58 20132 Milan Italy

and

Dulbecco Telethon Institute Via Olgettina 58 20132 Milan Italy

Roderic Guigó Universitat Pompeu Fabra Centre de Regulació Genòmica Dr. Aiguader 88 08003 Barcelona Spain

Masatoshi Hagiwara Kyoto University Graduate School of Medicine Department of Anatomy and Developmental Biology Kyoto Japan

Klaus Hartmuth Max Planck Institute for Biophysical Chemistry Department of Cellular Biochemistry Am Fassberg 11 37077 Göttingen Germany

Klemens J. Hertel University of California, Irvine Department of Microbiology and Molecular Genetics B252 Medical Sciences I Irvine, CA 92697-4025 USA

Michael Hiller Stanford University Department of Developmental Biology Beckman Center B-321B 279 Campus Drive West (MC 5329) Stanford CA 94305-5329 USA

Claudia Höbartner Max Planck Institute for Biophysical Chemistry Research Group of Nucleic Acid Chemistry Am Fassberg 11 37077 Göttingen Germany

Rym Kachouri-Lafond University of Basel and Swiss Institute of Bioinformatics Biozentrum Klingelbergstraße 50–70 4056 Basel Switzerland

Maria Kalyna Medical University of Vienna Max F. Perutz Laboratories Dr. Bohrgasse 9/3 1030 Vienna Austria

Berthold Kastner Max Planck Institute for Biophysical Chemistry Department of Cellular Biochemistry Am Fassberg 11 37077 Göttingen Germany

Olga Kelemen University of Kentucky Department of Molecular and CellularBiochemistry B278 Biomedical/Biological SciencesResearch Building 741 South Limestone Street Lexington, KY 40536-0298 USA

Amit Khanna University of Kentucky Department of Molecular and Cellular Biochemistry B278 Biomedical/Biological Sciences Research Building 741 South Limestone Street Lexington, KY 40536-0298 USA

Jrgen Kjems University of Aarhus Department of Molecular Biology C.F. Mllers Alle, Bldg 1130 8000 Aarhus C Denmark

Roscoe Klinck Université de Sherbrooke Faculté de médecine et des sciences de la santé Département de microbiologie et d'infectiologie Laboratoire de génomique fonctionnelle de l'Université de Sherbrooke Sherbrooke, Québec Canada J1H 5N4

Alberto R. Kornblihtt Universidad de Buenos Aires Facultad de Ciencias Exactas y Naturales IFIBYNE-CONICET Departamento de Fisiologìa, Biologìa Molecular y Celular Laboratorio de Fisiologìa y Biologìa Molecular Pabellón 2, piso 2 C1428EHA Buenos Aires Argentina

Lukasz Kozlowski International Institute of Molecular and Cell Biology in Warsaw Laboratory of Bioinformatics and Protein Engineering ul. Ks. Trojdena 4 02-109 Warsaw Poland

Adrian R. Krainer Cold Spring Harbor Laboratory Cold Spring Harbor, NY 11724 USA

Hidehito Kuroyanagi Tokyo Medical and Dental University Graduate School of Biomedical Science Laboratory of Gene Expression Tokyo 113-8510 Japan

and

Tokyo Medical and Dental University Medical Research Institute Department of Functional Genomics Tokyo 113-8510 Japan

and

Japan Science and Technology Agency (JST) Precursory Research for Embryonic Science and Technology (PRESTO) Kawaguchi, Saitama Japan

Branislav Kusenda Medical University of Vienna Max F. Perutz Laboratories Dr. Bohrgasse 9/3 1030 Vienna Austria

Vincent Lacroix Université de Lyon Laboratoire de Biométrie et Biologie Evolutive 43 bd du 11 novembre 1918 69622 Villeurbanne France

Dominika Lewandowska Scottish Crop Research Institute Genetics Programme Dundee DD2 5DA UK

Michele Liney Scottish Crop Research Institute Genetics Programme Dundee DD2 5DA UK

Imke Listerman Max Planck Institute of Molecular Cell Biology and Genetics Pfotenhauerstraße 108 01307 Dresden Germany

Miriam Llorian University of Cambridge Department of Biochemistry Tennis Court Road Cambridge CB2 1QW UK

Isabel C. López-Mejìa Institut de Génétique Moléculaire de Montpellier UMR 5535 CNRS 1919 route de Mende 34293 Montpellier Cedex 5 France

and

Université Montpellier 2 Place Eugène Bataillon 34095 Montpellier Cedex 5 France

and

Université Montpellier 1 5 Bd Henry IV 34967 Montpellier Cedex 2 France

Reinhard Lü;hrmann Max Planck Institute for Biophysical Chemistry Department of Cellular Biochemistry Am Fassberg 11 37077 Göttingen Germany

Sren Lykke-Andersen University of Aarhus Department of Molecular Biology C.F. Mllers Alle, Bldg 1130 8000 Aarhus C Denmark

Sujatha Manthri University of Dundee at SCRI Division of Plant Sciences Dundee DD2 5DA UK

Nathalie Marmier-Gourrier Nancy University Faculté de Médecine Laboratoire AREMS CNRS UMR 7214 BP 70184 54506 Vandoeuvre-les-Nancy Cedex France

Monika Maronova Medical University of Vienna Max F. Perutz Laboratories Dr. Bohrgasse 9/3 A-1030 Vienna Austria

Yamile Marquez Medical University of Vienna Max F. Perutz Laboratories Dr. Bohrgasse 9/3 1030 Vienna Austria

William Mattox University of Texas M.D. Anderson Cancer Center Department of Genetics 1515 Holcombe Blvd. Houston, TX 77030 USA

and

University of Texas Graduate School of Biomedical Sciences Genes and Development Graduate Program Houston, TX USA

Akila Mayeda Fujita Health University Institute for Comprehensive Medical Science (ICMS) Division of Gene Expression Mechanism Toyoake, Aichi 470-1192 Japan

Gunter Meister Universitä;t Regensburg Fakultä;t fü;r Biologie und Vorklinische MedizinLehrstuhl fü;r Biochemie I Universitä;tsstraße 31 93053 Regensburg Germany

and

Max-Planck-Institute of Biochemistry Center for Integrated Protein Science Munich Am Klopferspitz 18 82152 Martinsried/Munich Germany

Kathrin Meyer Universitä;t Bern Institut fü;r Zellbiologie Baltzerstraße 4 3012 Bern Switzerland

William F. Mueller University of California, Irvine Department of Microbiology and Molecular Genetics B252 Medical Sciences I Irvine, CA 92697-4025 USA

Manuel J. Muñoz Universidad de Buenos Aires Facultad de Ciencias Exactas y Naturales IFIBYNE-CONICET Departamento de Fisiologìa, Biologìa Molecular y Celular Laboratorio de Fisiologìa y Biologìa Molecular Pabellón 2, piso 2 C1428EHA Buenos Aires Argentina

Karla M. Neugebauer Max Planck Institute of Molecular Cell Biology and Genetics Pfotenhauerstraße 108 01307 Dresden Germany

Takayuki Nojima Tokyo Medical and Dental University Graduate School of Biomedical Science Laboratory of Gene Expression Tokyo 113-8510 Japan

Diana O'Day University of Texas M.D. Anderson Cancer Center Department of Genetics 1515 Holcombe Blvd. Houston, TX 77030 USA

and

University of Texas Graduate School of Biomedical Sciences Genes and Development Graduate Program Houston, TX USA

Peter Odenwä;lder Max Planck Institute for Biophysical Chemistry Department of Cellular Biochemistry Am Fassberg 11 37077 Göttingen Germany

Fernando Carrillo Oesterreich Max Planck Institute of Molecular Cell Biology and Genetics Pfotenhauerstraße 108 01307 Dresden Germany

Raymond T. O'Keefe The University of Manchester Faculty of Life Sciences Manchester M13 9PT UK

Jerzy Orlowski International Institute of Molecular and Cell Biology in Warsaw Laboratory of Bioinformatics and Protein Engineering ul. Ks. Trojdena 4 02-109 Warsaw Poland

Marta Pabis Max Planck Institute of Molecular Cell Biology and Genetics Pfotenhauerstraße 108 01307 Dresden Germany

Niketa A. Patel The James A. Haley Veterans Hospital Research Service 13000 Bruce B. Downs Blvd. Tampa, FL 33612 USA

and

University of South Florida College of Medicine Department of Molecular Medicine 12901 Bruce B. Downs Blvd. Tampa, FL 33612 USA

Paolo Ribeca Centro Nacional de Análisis Genómico (CNAG) Baldiri Reixac 4 08028 Barcelona Spain

Maurizio Romano International Centre of Genetic Engineering and Biotechnology (ICGEB) Padriciano 99 34012 Trieste Italy

Tanja Rösel Justus-Liebig-University of Gießen Institute of Biochemistry Heinrich-Buff-Ring 58 35392 Gießen Germany

Brian Rymond University of Kentucky Department of Biology 335A T.H. Morgan Building Lexington, KY 40506-0225 USA

Michael Sammeth Centro Nacional de Análisis Genómico (CNAG) Baldiri Reixac 4 08028 Barcelona Spain

Aparna K. Sapra Max Planck Institute of Molecular Cell Biology and Genetics Pfotenhauerstraße 108 01307 Dresden Germany

Carla Schmidt Max Planck Institute for Biophysical Chemistry Bioanalytical Mass Spectrometry Group Am Fassberg 11 37077 Göttingen Germany

Daniel Schü;mperli Universitä;t Bern Institut fü;r Zellbiologie Baltzerstraße 4 3012 Bern Switzerland

Jason Scroggin (graphics work) Design Office Takebayashi Scroggin (D.O.T.S.) 231 North Broadway Floor 3 Lexington, KY 40507 USA

Joonbae Seo Iowa State University College of Veterinary Medicine Department of Biomedical Sciences Ames, IA 50011-1250 USA

Manli Shen University of Kentucky Center for Structural Biology Department of Molecular and Cellular Biochemistry B278 Biomedical/Biological Sciences Research Building 741 South Limestone Street Lexington, KY 40536-0298 USA

Craig G. Simpson Scottish Crop Research Institute Genetics Programme Dundee DD2 5DA UK

Natalia N. Singh Iowa State University College of Veterinary Medicine Department of Biomedical Sciences Ames, IA 50011-1250 USA

Ravindra N. Singh Iowa State University College of Veterinary Medicine Department of Biomedical Sciences Ames, IA 50011-1250 USA

Christopher W.J. Smith University of Cambridge Department of Biochemistry Tennis Court Road Cambridge CB2 1QW UK

Stefan Stamm University of Kentucky Department of Molecular and Cellular Biochemistry B278 Biomedical/Biological Sciences Research Building 741 South Limestone Street Lexington, KY 40536-0298 USA

James Stévenin University of Strasbourg Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) INSERM U964, CNRS UMR 7104 1 rue Laurent Fries, BP 10142 67404 IllkirchFrance

Peter Stoilov West Virginia University Department of Biochemistry 1 Medical Center Drive Morgantown, WV 26506 USA

Shihuang Su University of Texas M.D. Anderson Cancer Center Department of Genetics 1515 Holcombe Blvd. Houston, TX 77030 USA

Chiranthani Sumanasekera University of Kentucky Department of Molecular and Cellular Biochemistry B278 Biomedical/Biological Sciences Research Building 741 South Limestone Street Lexington, KY 40536-0298 USA

Naeem Hasan Syed University of Dundee at SCRI Division of Plant Sciences Dundee DD2 5DAUK

Akari Takebayashi (graphics work) Design Office Takebayashi Scroggin (D.O.T.S.) 231 North Broadway Floor 3 Lexington, KY 40507 USA

Akihide Takeuchi Kyoto University Graduate School of Medicine Department of Anatomy and Developmental Biology Kyoto Japan

Jamal Tazi Institut de Génétique Moléculaire de Montpellier UMR 5535 CNRS 1919 route de Mende 34293 Montpellier Cedex 5 France

and

Université Montpellier 2 Place Eugène Bataillon 34095 Montpellier Cedex 5 France

and

Université Montpellier 1 5 Bd Henry IV 34967 Montpellier Cedex 2 France

James Tollervey MRC-Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK

Jernej Ule MRC-Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK

Henning Urlaub Max Planck Institute for Biophysical Chemistry Bioanalytical Mass Spectrometry Group Am Fassberg 11 37077 Göttingen Germany

Judith C.T. van Deutekom Prosensa Therapeutics Wassenaarseweg 72 2333 AL Leiden The Netherlands

Maria A. van Santen Max Planck Institute for Biophysical Chemistry Department of Cellular Biochemistry Am Fassberg 11 37077 Göttingen Germany

Audrey Vautrin Nancy University Faculté de Médecine Laboratoire AREMS CNRS UMR 7214 BP 70184 54506 Vandoeuvre-les-Nancy Cedex France

Shanzhi Wang University of Texas M.D. Anderson Cancer Center Department of Genetics 1515 Holcombe Blvd. Houston, TX 77030 USA

and

University of Texas Graduate School of Biomedical Sciences Genes and Development Graduate Program Houston, TX USA

Mihaela Zavolan University of Basel and Swiss Institute of Bioinformatics Biozentrum Klingelbergstraße 50–70 4056 Basel Switzerland

Chaolin Zhang The Rockefeller University Howard Hughes Medical Institute Laboratory of Molecular Neuro-Oncology 1230 York Avenue New York, NY 10021 USA

Michael Q. Zhang The University of Texas at Dallas Center for Systems Biology Department of Molecular and Cell Biology 800 West Campbell Road, RL11 Richardson, TX 75080-3021 USA

and

Tsinghua University TNLIS MOE Key Laboratory of Bioinformatics & Bioinformatics Division Beijing 100084 China

Zhaiyi Zhang University of Kentucky Department of Molecular and Cellular Biochemistry B278 Biomedical/Biological Sciences Research Building 741 South Limestone Street Lexington, KY 40536-0298 USA

e-mails are available at www.eurasent.info/book and www.wiley-vch.de/home/splicing

Related Titles

Hartmann, R. K., Bindereif, A., Schön, A., Westhof, E. (eds.)Handbook of RNA Biochemistry Student Edition 2009 ISBN: 978-3-527-32534-4

Miller, L. W. (ed.)Probes and Tags to Study Biomolecular Function for Proteins, RNA, and Membranes 2008 ISBN: 978-3-527-31566-6

Smith, H. C. (ed.)RNA and DNA Editing Molecular Mechanisms and Their Integration into Biological Systems 2008 ISBN: 978-0-470-10991-5

Part One

Theory

Chapter 1

Splicing in the RNA World

Emanuele Buratti, Maurizio Romano, and Francisco E. Baralle

Key Concepts

Alternative splicing is a key element of eukaryotic gene expression.Almost all polymerase II transcripts are alternatively spliced.RNA is chemically and structurally more flexible than DNA, and can act as a catalyst.RNA is an active player in mediating genetic information, not just a static messenger.Almost the entire human genome is transcribed into RNA and new classes of noncoding RNA molecules are emerging.The number of diseases known to be associated with alternative splicing is steadily increasing.

1.1 Introduction: The Fascination of Alternative Pre-mRNA Splicing

The genetic information is stored in DNA, which is transferred from one generation to the next. During the life of a cell, this DNA information is retrieved as RNA. Whereas DNA is chemically very stable and therefore well suited to archive the genetic information, RNA is chemically more reactive, and thus unstable. Therefore, with the exception of RNA viruses, RNA does not store the genetic information but rather acts as an intermediate between DNA and proteins.

However, RNA does not simply copy the genetic information, as the primary RNA transcript generated from DNA undergoes processing. Most human polymerase II transcripts contain exonic sequences that are finally exported into the cytoplasm (exons, for exported sequence), whereas the intervening sequences (introns) remain in the nucleus. The removal of the introns and the joining of the exons is known as pre-mRNA splicing [1–3]. Almost all human protein-coding genes undergo alternative splicing (AS; see Chapter 3 Hertel) [4]; this means that, depending on the cellular conditions, an alternative exon can be either included or removed from the final messenger RNA (mRNA). For example, the protein kinase CβII gene contains an alternative exon encoding a protein part that regulates the subcellular localization and substrate specificity of the kinase. In skeletal muscle, the inclusion of this exon is promoted by insulin, via a phosphatidylinositol 3-kinase-dependent pathway [5, 6]. This example shows how the readout of the genetic information is regulated by AS in response to a daily activity, such as the eating of a meal. The carbohydrates in the food trigger an insulin response; the insulin binds to receptors on muscle cells that initiate a phosphorylation cascade which modulates the splicing machinery to use only certain parts of the genetic information, which in turn generates a regulatory protein with altered properties (see Chapter 48 Patel for signaling and splicing). This shows that the type of information transferred from the genome to the cell depends on inputs that the cell receives, which implies that the output of a gene is only defined in the context of the cellular state.

RNA is therefore more than just a copy of the genetic information: RNA can “interpret” the genetic information depending on environmental cues that the cell receives. Alternative splicing is a central mechanism in this interpretation process, as it allows the expression of selected parts of genetic information.

Due to its role as a flexible “interpreter,” AS strongly enhances the number of proteins that can be encoded by the genome. For example, by combining one exon out of four alternatively spliced regions that contain 12, 48, 33, and 2 alternative exons each, the Drosophila Dscam gene can generate 38 016 protein isoforms (12 × 48 × 33 × 2) [7]. Deep sequencing results (see Chapters 50 Guigó, 51 Zhang for this method) indicate that the fly actually generates this large number of isoforms. Alternative splicing can thus generate from a single gene a number of protein isoforms that is larger than the total number of protein coding genes in Drosophila.

The ability to change the output of the genetic information depending on cellular states, and the ability to expand the information content of the genome, makes AS a central element in gene expression. About 30 years after the discovery of splicing [1, 2], we are now beginning to understand on a molecular level how AS can be such a fascinating biological process (see Chapters 3 Hertel, 5 Lührmann, and 8 Smith).

1.2 RNA Can Adopt a Flexible Conformation

RNA molecules can be represented by a linear sequence of four classical bases: adenine and guanine (A/G, both purines); and cytosine and uracil (C/U, both pyrimidines). These bases can be subjected to more than 100 post-transcriptional modifications that are currently listed in the RNA modification database [8] http://library.med.utah.edu/RNAmods (see also Chapter 14 by Höbartner for synthetic available bases). In the RNA molecule, each of these bases (schematically represented in Figure 1.1a) is bound to the 1′ position of a ribose sugar that, through its 3′ position, utilizes a phosphate group to link with the 5′ position of the next ribose. The most important features that distinguish RNA (ribonucleic acid) from DNA (deoxyribonucleic acid) is the presence of a hydroxyl group (−OH) in the 2′ position of the ribose sugar (Figure 1.1b). The 2′ hydroxyl group is chemically reactive, which not only makes RNA more vulnerable to degradation but also it to participate in chemical reactions.

Figure 1.1 The structure of RNA. (a) Schematic representation of the four major bases of the ribonucleotides: adenosine (AMP) and guanine (GMP) that are both purines, and uracil (UMP) and cytosine (CMP) that belong to the pyrimidines; (b) The structure of RNA.

Although RNA molecules are described as a single-stranded sequence, most RNA molecules exhibit a high degree of double-helical character, as complementary segments of the RNA fold back on each other. The base-pairing of RNA is more flexible than that of DNA. In addition to the canonical Watson–Crick base pairs (cytosine with guanine, adenine with uracil), there are “noncanonical” base pairs, such as G–U pairing, and numerous other base pairings are possible [9]. Since the areas of complementarity in an RNA molecule are short, RNA molecules show local regions of base-pairing, which is referred to as “secondary structure.” RNA secondary structures are locally confined, which is in contrast to the extended double-stranded DNA helix.

As RNAs do not form a long-range double-stranded structure, the short RNA helices themselves can interact with each other to form what is known as the “tertiary structure” [10]. The rules that exactly define the final outcome of these folding processes, and the various factors that influence them, remain the subject of many active studies. In contrast to proteins, it is currently not possible to predict in vivo RNA tertiary structures accurately [10] (see Chapter 54 Bujnicki for structure prediction of splicing proteins). X-ray crystallography experiments have clearly shown defined tertiary structures for metabolically stable RNAs, such as transfer RNAs (tRNAs) or ribosomal RNAs (rRNAs) [11]. In contrast, structures in pre-mRNAs that form the substrate of the spliceosome can currently be predicted only indirectly by mutagenesis or bioinformatic analyses, as structures on pre-mRNA will most likely be formed only transiently (see Chapter 53, Hiller).

1.3 Enzymatic RNAs and the RNA World

The lack of a complementary strand, and the presence of the 2′ hydroxyl (OH) group, which confers chemical reactivity, combined with the ability to fold into complex tertiary structures, allow RNA to perform catalytic reactions [12]. The first example of an RNA with catalytic activity was the self-splicing pre-ribosomal RNA from the ciliate Tetrahymena [13], followed by the discovery of RNase P, a ribonucleoprotein complex that cleaves tRNAs [14]. The general catalytic mechanism of these RNA enzymes is an activation of the 2′-OH by a base, followed by a nucleophilic attack of the activated 2′-O− oxygen on the cleavable 3′-O-phosphobond. The outcome of this attack differs between the RNA classes: self-cleaving RNA, such as the hammerhead ribozymes, forms 2′,3′ cyclic phosphates, whereas group I, II and spliceosomal introns undergo splicing via a transesterification (see Chapter 5 Lührmann, see Figure 5.1 for the mechanism). In most cases, the base that activates the −OH group is a metal ion. Since these RNA molecules act as enzymes, they were named “ribozymes”; similarly to their protein relatives, ribozymes form specific three-dimensional (3-D) conformations that form solvent-protected active sites and undergo sterical changes during the reaction.

It has been demonstrated that RNA catalyzes peptide bond formation in the ribosome [15], and it has been proposed that RNA is responsible for catalysis in the human spliceosome [16], which in turn raises the question of the function of proteins in ribonucleoproteins (RNPs). The study of RNase P showed the importance of protein components associating with ribozymes. RNase P is an RNA–protein complex that cleaves tRNA precursors. In bacteria, the catalytic activity resides within the RNA [14], but in human mitochondria RNase P catalyzes the reaction without RNA, demonstrating that proteins can substitute for RNA functions [17]. The question then is, since similar biological functions can be performed by either RNA or protein complexes, why did evolution select RNPs such as ribosomes and spliceosomes for protein synthesis and pre-mRNA processing?

One possibility is that proteins facilitate the conformational changes of RNA that are necessary for catalysis. The spliceosome catalyzes the reaction between two structurally different substrates, which necessitates large spatial rearrangements during the reaction (see Chapter 5, Lührmann), which could be stabilized by additional RNA–protein interactions in the spliceosome. In fact, the spliceosome is an excellent example of an RNP machine, where the degree of interdependence between RNA and protein for catalytic function is such that it is justified to consider it a veritable RNP enzyme.

The discovery of the enzymatic activity of RNAs led to the concept of a primitive “RNA World” which could have existed before the appearance of modern proteins and DNA [18]. A schematic depiction of the RNA World hypothesis is shown in Figure 1.2. It is impossible to prove the existence of a pure RNA World in the prehistoric Earth, and RNA could have been coexisting with small peptides. However, the RNA World concept has been useful in analyzing the mechanism of RNA-based machines, such as the ribosome or spliceosome, as it points to RNA as the catalytic moiety [19]. One of the predictions of the RNA World hypothesis is that the core catalytic activity should be conserved in spliceosomes from different phyla. The comparison of spliceosomes between human (Chapter 5 Lührman), yeast (Chapter 6, Rymond), and plants (Chapter 7 Barta) shows which is the case, and suggests that they derived from a common precursor. Another echo of the RNA World is the fact that most of the human genome is transcribed into noncoding RNAs [20], which further suggests a larger regulatory role of RNA.

Figure 1.2 The RNA World hypothesis. The genesis of a hypothetical RNA World includes a series of events, summarized schematically in this figure, that include the abiotic synthesis of RNA monomers from common inorganic/organic molecules and their assembly into oligomers (in the likely presence of metal catalysts) that would have had to serve as templates for their own spontaneous copying or replication. At this stage, a set of catalytic RNAs may have evolved (among them, RNA-based RNA copying molecules), and this would have sustained their exponential growth in the prebiotic environment.

1.4 Common Classes of Eukaryotic RNA

Only about 1.2% of the human genome encodes proteins. The ENCODE project, which carefully analyzed gene expression in 1% of the human genome, showed that at least 93% of the human genome in this region is transcribed [20]. It is not clear however, whether other genomic regions are transcribed in a similarly active manner, as recent RNAseq data have suggested that most transcription is associated with known genes [21]. Most of the RNA expression consists of short, nuclear, non-protein-coding RNAs (ncRNAs) (Figure 1.3), although it is not clear whether these RNAs simply represent noise or have functions. As a large fraction of the ncRNAs show cell type-specific expression and derive from evolutionary highly conserved promoter regions, it is likely that they represent a pool of sequences that can be recruited by evolution to regulate gene expression [22], possibly by yet unknown mechanisms.

Figure 1.3 Expression of RNA. A schematic view of the most common noncoding and coding RNA families currently described in the eukaryotic cell.

The most abundant cellular RNAs are the rRNAs, which are the core of the ribosomes – the ribonucleoprotein particles in charge of translating the information encoded in mRNAs into proteins. The ribosomes in eukaryotes are formed by two subunits – the 60S and the 40S – named according to their sedimentation coefficients (see Chapter 13 Hartmuth for sedimentation analysis). These subunits contain the 28S/5S rRNA and 18S rRNA tightly associated with proteins. The amino acids are brought to the ribosome by tRNAs, whereby each tRNA is associated with an amino acid and recognizes the mRNA through a three-nucleotide sequence known as a “codon.” Formation of the peptide bonds which connect the amino acids is performed by the RNA part of the ribosome, which acts as a ribozyme [15].

An additional class of relatively abundant small RNAs is formed by the small nucleolar RNAs (snoRNAs) [23]. As indicated by their name, these localize to the nucleolus and are mainly involved in rRNA maturation, although they also play important functions in protein translation, mRNA splicing, and genome stability. There are two classes of snoRNA (C/D and H/ACA box) that function as ribonucleoprotein (RNP) complexes to guide the enzymatic modification of target RNAs. Generally, the C/D box snoRNAs guide the methylation of target RNAs, while the H/ACA box snoRNAs guide pseudouridylation [24]. It has also been recently discovered that snoRNAs can be additionally processed to yield smaller molecules, termed sno-derived RNAs (sdRNAs), that are associated with Ago2 and may thus be linked to gene silencing and transcriptional repression processes [25]. One of the shorter sdRNAs has been demonstrated to regulate AS in neurons [26, 27].

Another well-characterized family of RNAs are the small nuclear RNAs (snRNAs). Based on sequence homology and common protein factors, the snRNAs can be divided in two classes – the Sm and Lsm (like Sm) classes. The name Sm is derived from Stephanie Smith, a patient with lupus erythematosus whose blood contained antibodies against snRNA-associated proteins [28, 29], and which were used for the purification of such proteins. The sequence of the U1 snRNA shows a complementarity to the 5′ splice site, which at an early stage led to the correct hypothesis that these RNAs function in splicing [30]. The Sm class is composed of U1, U2, U4, U4atac, U5, U7, U11, and U12, whereas U6 and U6atac are associated with the Lsm class of proteins. While U1, U2, U4, U5, and U6 are components of what is termed the “major spliceosome” (which splices introns with GU at the 5′ splice site and AG at the 3′ splice site), the U11, U12, U4atac, and U6atac RNAs are components of the so-called “minor spliceosome” (which splices introns that have AT–AC at their 5′ and 3′ ends) [31]. After assembly with small nuclear ribonucleoproteins (snRNPs), all of the resulting snRNP particles form the core of the spliceosome (major or minor), and catalyze the removal of introns from pre-mRNA (see Chapter 5 Lührmann). The only exception to this is represented by U7 snRNP, which functions in histone pre-mRNA 3′-end processing (see Chapter 45 Schümperli).

Other classes of ncRNAs are micro RNAs (miRNAs) and short interference RNAs (siRNAs). The miRNAs are 21- to 23-nucleotide (nt) RNAs that regulate gene expression through binding to mRNAs via an imperfect complementarity. The siRNAs recognize perfect complementary RNAs, and induce their cleavage and subsequent degradation. Both of these RNA classes are discussed in Chapter 2 Meister and summarized in Table 2.1), while the application of siRNAs to knockdown genes is described in Chapter 15 Gabellini.

Piwi-interacting RNAs (piRNAs) are another class of ncRNAs, which are expressed only in the germline of flies, fish, and mammals; here, the name Piwi (P-element induced wimpy testis in Drosophila) refers to a class of RNA-binding proteins in Drosophila. These proteins were observed to interact with a novel class of longer-than-average miRNAs (26–31 nt), termed piRNAs. The expression of both piRNAs and Piwi proteins is restricted to the male germline.