Methods in Molecular Biology 1095 Christoph Arenz Editor miRNA Maturation Methods and Protocols www.ketabdownload.com 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 www.ketabdownload.com www.ketabdownload.com miRNA Maturation Methods and Protocols Edited by Christoph Arenz Institute for Chemistry, Humboldt-Universität zu Berlin, Berlin, Germany www.ketabdownload.com Editor Christoph Arenz Institute for Chemistry Humboldt-Universität zu Berlin Berlin, Germany ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-62703-702-0 ISBN 978-1-62703-703-7 (eBook) DOI 10.1007/978-1-62703-703-7 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2013949785 © Springer Science+Business Media New York 2014 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifi cally for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) www.ketabdownload.com Pref ace The last decade has seen a dramatic development in the fi eld of micro RNAs (miRNAs). Starting with a small set of small noncoding regulatory RNAs in D. melanogaster and C. elegans miRNAs are now regarded as important regulatory components in many eukaryotic species including humans. The number of reported miRNAs exceeds 1,000 and many of these miRNAs have been implicated in important biological processes and human diseases. In this volume of “Methods in Molecular Biology” we concentrate on the pathway of microRNA maturation. After its synthesis, the primary miRNA transcript (pri-miRNA) is cleaved by an endonuclease called Drosha to yield the precursor miRNA (pre-miRNA). After being transported to the cytoplasm, the latter is further cleaved by another nuclease called Dicer to yield the mature double-stranded miRNA. This mature miRNA consists of the active guide strand which remains bound to the miRNA effector complex, whereas the passenger strand is degraded. These few relatively simple steps (although the cellular machinery promoting this pathway is rather complex) are common to most miRNAs and thus are highly important to study. In this book we concentrate on three important aspects of this maturation pathway: First of all, we give an overview over the current knowledge of the pathway of miRNA maturation (Chapter 1 ) and how this pathway relates to human disease (Chapter 2 ). In the third review, established and novel approaches to manipulate miRNA maturation and activity are described. During the last 5 years many correlations between the levels of certain miRNAs and various human malignancies have been identifi ed, and in some cases the causative role of certain miRNAs in disease formation has been shown. Specifi c miRNAs can regulate certain proteins causing or preventing disease formation. These miRNA–disease correlations call for easy and reliable methods for quantifying specifi c miRNA species from biological sam- ples, a topic which is only partially covered in this book. While miRNAs today have some- how lost their “exotic” touch with many standard methods established in laboratories around the world, novel methods with the potential to expand the view on miRNAs are currently being developed. Such innovation can arise from improving existing methods or by the development of completely new methods allowing for addressing questions other than previous ones. In this book, established methods (qRT-PCR of miRNA maturation components, qRT-PCR of miRNAs) are completed with fl uorescent and nonfl uorescent methods for homogenous assays of Dicer-mediated miRNA maturation or an in vivo assay for Drosha activity. Since miRNAs appear to be emerging drug targets, anti-miRs are already commer- cially available. Less common is the use of biologically stable PNA as anti-miRs or even miRNA maturation inhibitors, doubtlessly adding to the arsenal of current oligonucleotide approaches to manipulate miRNA activity. Apart from oligonucleotides affecting miRNA activity, which are already under clinical investigation, there is a huge interest in fi nding v www.ketabdownload.com vi Preface small molecules with equivalent effects. Specially adapted luciferase assays have proven as powerful tools for identifying candidate small molecule miRNA effectors. I am convinced that the collection of methods of this book is suitable to widen the view on miRNA as biological mediators and potential drug targets and thus stimulate future research in this highly dynamic and thrilling topic. Berlin , Germany Christoph Arenz www.ketabdownload.com Contents Preface . v Contributors . ix PART I REVIEWS 1 The Pathway of miRNA Maturation . 3 Michael Sand 2 MicroRNA Maturation and Human Disease . 11 Marlen Hesse and Christoph Arenz 3 Approaches to the Modulation of miRNA Maturation. 27 Valerie T. Tripp, Jaclyn R. McKenna, and Douglas D. Young PART II METHODS 4 Expression Profiling of Components of the miRNA Maturation Machinery . 61 Michael Sand and Marina Skrygan 5 Primary MicroRNA Processing Assay Reconstituted Using Recombinant Drosha and DGCR8 . 73 Ian Barr and Feng Guo 6 In Vivo Processing Assay Based on a Dual-Luciferase Reporter System to Evaluate DROSHA Enzymatic Activity. 87 Vera Bilan, Danilo Allegra, Florian Kuchenbauer, and Daniel Mertens 7 Assaying Dicer-Mediated miRNA Maturation by Means of Fluorescent Substrates . 95 Marlen Hesse, Brian P. Davies, and Christoph Arenz 8 A Fluorescence Correlation Spectroscopy-Based Enzyme Assay for Human Dicer . 103 Eileen Magbanua and Ulrich Hahn 9 Detection of microRNA Maturation Using Unmodified pre-microRNA and Branched Rolling Circle Amplification . 109 Saskia Neubacher and Christoph Arenz 10 Quantitative RT-PCR Specific for Precursor and Mature miRNAs. 121 Hannah Zöllner, Stephan A. Hahn, and Abdelouahid Maghnouj vii www.ketabdownload.com viii Contents 11 Cellular MicroRNA Sensors Based on Luciferase Reporters . 135 Colleen M. Connelly and Alexander Deiters 12 Identification of Inhibitors of MicroRNA Function from Small Molecule Screens. 147 Colleen M. Connelly and Alexander Deiters 13 Inhibition of miRNA Maturation by Peptide Nucleic Acids . 157 Concetta Avitabile, Enrica Fabbri, Nicoletta Bianchi, Roberto Gambari, and Alessandra Romanelli 14 Molecular Methods for Validation of the Biological Activity of Peptide Nucleic Acids Targeting MicroRNAs . 165 Eleonora Brognara, Enrica Fabbri, Nicoletta Bianchi, Alessia Finotti, Roberto Corradini, and Roberto Gambari 15 Lentiviral Overexpression of miRNAs . 177 Hannah Zöllner, Stephan A. Hahn, and Abdelouahid Maghnouj Index . 191 www.ketabdownload.com Contributors DANILO ALLEGRA • Internal Medicine III, University of Ulm, Ulm, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany CHRISTOPH ARENZ • Institute for Chemistry, Humboldt-Universität zu Berlin , Berlin , Germany CONCETTA AVITABILE • Dipartimento delle Scienze Biologiche , Università di Napoli “Federico II” , Napoli , Italy IAN BARR • Department of Biological Chemistry, University of California Los Angeles, Los Angeles, CA, USA NICOLETTA BIANCHI • Department of Life Sciences and Biotechnology , Ferrara University , Ferrara , Italy ; Laboratory for the Development of Pharmacological and Pharmacogenomic Therapy of Thalassaemia, Biotechnology Center , Ferrara University , Ferrara , Italy VERA BILAN • Internal Medicine III , University of Ulm , Ulm , Germany ELEONORA BROGNARA • Department of Life Sciences and Biotechnology , Ferrara University , Ferrara , Italy COLLEEN M. CONNELLY • Department of Chemistry , North Carolina State University , Raleigh , NC , USA ROBERTO CORRADINI • Department of Organic Chemistry , Parma University , Parma , Italy BRIAN P. DAVIES • Institute for Chemistry, Humboldt Universität zu Berlin, Germany ALEXANDER DEITERS • Department of Chemistry