Book review: “Alternative pre-mRNA splicing – Theory and Protocols” – the elements for a code and the recipes to gain access to them
Article first published online: 25 APR 2012
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Special Issue: Focus: Imaging technology
Volume 7, Issue 5, pages 589–591, May 2012
How to Cite
(2012), Book review: “Alternative pre-mRNA splicing – Theory and Protocols” – the elements for a code and the recipes to gain access to them. Biotechnology Journal, 7: 589–591. doi: 10.1002/biot.201200118
- Issue published online: 25 APR 2012
- Article first published online: 25 APR 2012
Alternative pre-mRNA splicing – Theory and Protocols Stefan Stamm, Chris Smith, Reinhard Luhrmann (eds.), Wiley-VCH, Weinheim, February 2012, 660 pages, ISBN 978-3-527-32606-8
Dr. Laurent Corcos firstname.lastname@example.org*, Marine Pesson*, * Inserm U1078 – Faculty of Medicine, Brest, France
The book edited by Stefan Stamm, Chris Smith and Reinhard Lührmann, “Alternative pre-mRNA splicing, theory and Protocols”, has the ambition to provide a very large compendium of both theory and applications in pre-mRNA splicing analysis for all biology and biochemistry investigators and those who wish to enter this wide field. The 622 page book is divided into two main parts: Theory and Protocols – with six sub-parts (Basic Methods, Detection of Splicing Events, Analysis of Splicing in vitro, Analysis of Splicing in vivo, and Manipulation of Splicing Events and Bioinformatic Analysis of Splicing). The book was initiated by the European Community within the EURASNET framework that comprises 43 research groups form Europe, Israel and Argentina.
For younger investigators, who may not be familiar with the so-called “bible” of molecular biology, this new book resembles the book Molecular Cloning edited by Tom Sambrook and Tom Maniatis, which provides both, some theory about molecular biology as well as experimental protocols. Since the evolution of molecular techniques to study pre-mRNA splicing has dramatically increased over the past few years, a specific book to document both the theory, based on data from several organisms and tissues, and the detailed protocols that will be of use for the experimental study of various aspects of pre-mRNA splicing was still needed and is provided by the current book by Stamm, Smith and Lührmann.
Alternative pre-mRNA splicing is now considered as a rule rather than an exception. It is estimated that 92% of all human genes undergo alternative pre-mRNA splicing, which leads to the assembly of tens, hundreds or even thousands of distinct mRNA species (e.g. in flies). Alternative pre-mRNA splicing accounts for a large part of the diversity of the proteome and fills the gap that exists between the numbers of genes (20000–25000) and proteins (100000) in a typical mammalian cell. Alternative pre-mRNA splicing is affected in pathological states, either in association with a disease, or even causing the disease itself. This includes genetic diseases in which the RNA sequence may be altered by mutation or vary through polymorphism, preventing the proper interaction with regulatory proteins involved in splicing control. In addition, trans-acting proteins may show alterations, which hamper their ability to direct correct splicing. Furthermore, alternative pre-mRNA splicing is deteriorated in cancer cells, which relates to strong alterations occurring in the splicing machinery. All of this takes place under the control of higher order mechanisms involving, for example, epigenetic mark-ups in tight coordination with the onset of transcription, capping and 3'-end pre-mRNA processing. These aspects, and more, are extensively discussed in this book. Importantly, since high-throughput technologies have been developed to analyze pre-mRNA splicing events at a large scale that may occur at the whole transcriptome level, bioinformatic tools are also presented.
Each chapter of the Theory part of the book starts with a brief outline of the key concepts that are covered in the respective chapter. This part accounts for many aspects of RNA biochemistry, RNA species and roles in metabolism and, of course, splicing determinants. This last aspect is further documented with analyses on RNA binding proteins and the spliceosome, a macromolecular structure that contains more than 150 proteins and 5 snRibo Nucleo Proteins (U1, U2, U4/U6 ad U5). Splicing in yeast, as a model organism amenable to straightforward genetic approaches, is presented, followed by a comparative analysis of plant and mammalian alternative splicing. Finally, the mechanisms of alternative splice site selection, the integration of splicing in nuclear and cellular events, the alterations of splicing in disease and the ways to go from the bedside (the pathological state) back to the bench end this Theory part of the book. Taken together, this first part is most useful for people who are not familiar with the topic of pre-mRNA splicing. It provides most of the clues to embark on experimental analyses such as those described in the subsequent parts of the book.
The Protocols part comprises 75% of the book, showing that the authors mainly aimed at providing several methods described in depth. All chapters start with a one page graphical outline of the procedures (see figure), followed by a short theoretical background and end with a troubleshooting section, which is presented in a rational and user-oriented way. All these chapters are clearly illustrated and include a protocol, the description of an example experiment and a short discussion of the results. Several of the technical chapters propose more than one protocol to follow, offering the possibility to use alternative methods in case of experimental difficulties or for reasons of convenience. Diagrams or gel analyses, in direct relation with the protocols used, are shown.
The second part of the book, Basic methods, describes the common analysis of splicing problems, the purification of spliceosomes, the chemical synthesis of RNA, RNA interference, the purification of splicing proteins, the detection of RNA-protein complexes, the identification of sequences that drive exon inclusion or exclusion, the identification of splicing cis-acting elements, and the SELEX method to identify RNA targets of RNA binding proteins.
The third part discusses the Detection of splicing events, including the quantification of alternative splice variants, the high-throughput analysis of splicing by RT-PCR, splice monitoring in plants, microarray analyses, the CLIP method to study protein-RNA interactions, the identification of proteins bound to RNA, the detection of splicing events in live cells. The fourth part deals with the Analysis of splicing in vitro, with the preparation of cell nuclear extracts, in vitro splicing assays, the assembly and isolation of spliceosomal complexes, the analysis of site-specific RNA-protein interactions, the immunoprecipitation of nuclear proteins, the quantitative analysis of Protein-RNA complexes by mass spectrometry. The fifth part addresses the Analysis of splicing in vivo, with the cloning of splicing reporter minigenes, the analysis of splicing assays, coupled promoter-splicing systems, the engineering of stable cell lines with splicing reporters, the detection of splicing and splicing factors by chromatin immunoprecipitation, yeast, HIV, plant or drosophila splicing. The sixth part describes the Manipulation of splicing events, with antisense methodologies, the screening for splicing modulators and changes induced to the spliceosome. The seventh and last part discusses the Bioinformatic analysis of splicing, the analysis of transcripts by high-throughput RNA sequencing, that of pre-mRNA secondary structures in relation with alternative pre-mRNA splicing, the structure prediction of proteins synthesized from alternatively spliced pre-mRNAs, and the comparative genomics methods for the prediction of small RNA-binding sites.
Although the grouping of some of the chapters within the same or different parts could have been somewhat different, and a few chapters from the introductory part are not strictly related to pre-mRNA alternative splicing, all the information reported should certainly satisfy the vast majority of the readers and especially newcomers to the field. Importantly, the investigators are encouraged to get directly in contact with the authors and editors of the book who are certainly among the most prominent specialists in their respective fields. The readers will, however, miss the email addresses.
This book, prepared within the context of the EURASNET framework, is the first of its kind and, as such, an important piece of scientific literature, which will inspire more investigators to enter the rising field of alternative pre-mRNA splicing. One can but hope that an updated version will be prepared in the coming years to account for the increase of knowledge in this field, thanks to both the constant evolution of technology and to that of concepts that will undoubtedly occur.
Dr. Laurent Corcos and Marine Pesson
Inserm U1078 – Faculty of Medicine, Brest, France
About the book editors
Stefan Stamm is Associate Professor in the Department for Molecular and Cellular Biochemistry at the University of Kentucky, USA. He studied Biochemistry in Hannover (Germany) and did the practical work for his PhD as well as postdoctoral work at the Cold Spring Harbor Laboratory, NY, USA. His research focuses on mechanisms and regulation of alternative splicing, with the aim to apply the findings to the Prader-Willi Syndrome and Spinal Muscular Atrophy.
Chris Smith is Professor of RNA Molecular Biology in the Department of Biochemistry at the University of Cambridge, UK. His PhD research in Biochemistry was carried out at the University of London, UK, followed by postdoctoral research at Harvard Medical School, USA. His research interests encompass the mechanisms, regulation and function of alternative splicing.
Reinhard Luhrmann is Director at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, where he is head of the Department of Cellular Biochemistry. He has studied chemistry at the University of Münster, Germany, where he also received his PhD. He worked as a postdoctoral fellow at the Max Planck Institute for Molecular Genetics in Berlin, Germany. His research focuses on the structure and mechanism of the spliceosome.