Methods in Molecular Biology 1126 Klemens J. Hertel Editor Spliceosomal Pre-mRNA Splicing Methods and Protocols M ETHODS IN MOLECULAR BIOLOGY Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Spliceosomal Pre-mRNA Splicing Methods and Protocols Edited by Klemens J. Hertel Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, USA Editor Klemens J. Hertel Department of Microbiology & Molecular Genetics University of California Irvine , CA , USA ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-62703-979-6 ISBN 978-1-62703-980-2 (eBook) DOI 10.1007/978-1-62703-980-2 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2014931088 © Springer Science+Business Media, LLC 2014 This work is subject to copyright. 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Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Pref ace The splicing of nuclear pre-mRNAs is carried out by the spliceosome, which recognizes splicing signals and catalyzes the removal of noncoding intronic sequences to assemble protein coding sequences into mature mRNA prior to export and translation. Of the approximately 25,000 genes encoded by the human genome, more than 90 % are believed to produce transcripts that are alternatively spliced. Thus, alternative splicing of pre-mRNAs can lead to the production of multiple protein isoforms from a single pre-mRNA, signifi - cantly enriching the proteomic diversity of higher eukaryotic organisms. Because regulation of this process can determine the timing and location that a particular protein isoform is produced, changes in alternative splicing patterns modulate many cellular activities. Consequently, the process of splicing must occur with a high degree of specifi city and fi del- ity to ensure the appropriate expression of functional mRNAs. Mutations in RNA splicing regulatory elements or in genes encoding splicing regula- tors that bind splicing regulatory elements can cause or modify the severity of disease. Early estimates, based on the identifi cation of mutations within splice sites, suggested that ~15 % of all single base mutations change splicing patterns. However, it is now clear that many more mutations affect splicing by disrupting other important RNA elements, such as splic- ing enhancers or silencers binding sites. New estimates suggest that up to 60 % of known mutations could cause disease through changes in pre-mRNA splicing. A signifi cant step towards identifying some of these disease-causing mutations has been made recently by combining novel high-throughput experimental and bioinformatic approaches to defi ne splicing patterns and splicing regulatory elements. The advent of novel methods to analyze the activities of the spliceosome has led to the merging of different analytical disciplines. The goal of this book is to provide the reader with a guide to classical experimental approaches to decipher splicing mechanisms and to provide experimental strategies that rely on novel multidisciplinary approaches. This book was written with graduate and medical students, clinicians, and postdoctoral researchers in mind. It describes the theory of alternative pre-mRNA splicing in seven introductory chapters and then introduces protocols and their theoretical background rel- evant for a variety of experimental research. These protocol chapters cover basic methods to detect splicing events, analyses of alternative pre-mRNA splicing in vitro and in vivo, manipulation of splicing events, and high-throughput and bioinformatic analyses of alter- native splicing. Each chapter provides a theoretical introduction and a practical guide for molecular biologists, geneticists, clinicians, and every researcher interested in alternative splicing. In general, the protocols require a basic knowledge of molecular biology and/or RNA methods. The protocols in this book are a collection of commonly used methods in the fi eld of alternative splicing. These protocols should be viewed as guides for experiments that allow investigators to understand basic procedures. It is hoped that the chapters will allow readers v vi Preface to quickly fi nd the experimental tools necessary for their projects and that it will stimulate their interest in trying out other techniques. As such, I hope that this compendium of methods and protocols will help newcomers and seasoned molecular biologists to under- stand the fascinating world of alternative splicing with the ultimate goal of paving the way for many new discoveries to come. Irvine , CA, USA Klemens J. Hertel Contents Preface. v Contributors . ix PART I INTRODUCTORY CHAPTERS 1 The Pre-mRNA Splicing Reaction. 3 Somsakul Pop Wongpalee and Shalini Sharma 2 Diversity and Evolution of Spliceosomal Systems. 13 Scott William Roy and Manuel Irimia 3 Mechanisms of Spliceosomal Assembly . 35 Ni-ting Chiou and Kristen W. Lynch 4 Alternative Pre-mRNA Splicing. 45 Stacey D. Wagner and J. Andrew Berglund 5 Regulation of Alternative Pre-mRNA Splicing . 55 Miguel B. Coelho and Christopher W.J. Smith 6 Introduction to Cotranscriptional RNA Splicing . 83 Evan C. Merkhofer, Peter Hu, and Tracy L. Johnson 7 Chromatin and Splicing . 97 Nazmul Haque and Shalini Oberdoerffer PART II METHODS CHAPTERS 8 Preparation of Splicing Competent Nuclear Extracts . 117 Chiu-Ho T. Webb and Klemens J. Hertel 9 Preparation of Yeast Whole Cell Splicing Extract . 123 Elizabeth A. Dunn and Stephen D. Rader 10 Efficient Splinted Ligation of Synthetic RNA Using RNA Ligase. 137 Martha R. Stark and Stephen D. Rader 11 In Vitro Assay of Pre-mRNA Splicing in Mammalian Nuclear Extract . 151 Maliheh Movassat, William F. Mueller, and Klemens J. Hertel 12 Kinetic Analysis of In Vitro Pre-mRNA Splicing in HeLa Nuclear Extract . 161 William F. Mueller and Klemens J. Hertel 13 In Vitro Systems for Coupling RNAP II Transcription to Splicing and Polyadenylation. 169 Eric G. Folco and Robin Reed vii viii Contents 14 Isolation and Accumulation of Spliceosomal Assembly Intermediates. 179 Janine O. Ilagan and Melissa S. Jurica 15 Complementation of U4 snRNA in S. cerevisiae Splicing Extracts for Biochemical Studies of snRNP Assembly and Function . 193 Martha R. Stark and Stephen D. Rader 16 Expression and Purification of Splicing Proteins from Mammalian Cells . 205 Eric Allemand and Michelle L. Hastings 17 Single Molecule Approaches for Studying Spliceosome Assembly and Catalysis . 217 Eric G. Anderson and Aaron A. Hoskins 18 Cell-Based Splicing of Minigenes. 243 Sarah A. Smith and Kristen W. Lynch 19 Quantifying the Ratio of Spliceosome Components Assembled on Pre-mRNA . 257 Noa Neufeld, Yehuda Brody, and Yaron Shav-Tal 20 Antisense Methods to Modulate Pre-mRNA Splicing . 271 Joonbae Seo, Eric W. Ottesen, and Ravindra N. Singh 21 Using Yeast Genetics to Study Splicing Mechanisms . 285 Munshi Azad Hossain and Tracy L. Johnson 22 Medium Throughput Analysis of Alternative Splicing by Fluorescently Labeled RT-PCR. 299 Ryan Percifield, Daniel Murphy, and Peter Stoilov 23 Chromatin Immunoprecipitation Approaches to Determine Co-transcriptional Nature of Splicing. 315 Nicole I. Bieberstein, Korinna Straube, and Karla M. Neugebauer 24 Computational Approaches to Mine Publicly Available Databases . 325 Rodger B. Voelker, William A. Cresko, and J. Andrew Berglund 25 Approaches to Link RNA Secondary Structures with Splicing Regulation . 341 Mireya Plass and Eduardo Eyras 26 Methods to Study Splicing from High-Throughput RNA Sequencing Data . 357 Gael P. Alamancos, Eneritz Agirre, and Eduardo Eyras 27 Global Protein–RNA Interaction Mapping at Single Nucleotide Resolution by iCLIP-Seq . 399 Chengguo Yao, Lingjie Weng, and Yongsheng Shi 28 Predicting Alternative Splicing . 411 Yoseph