Methods in Molecular Biology 1823 Ulf Andersson Vang Ørom Editor miRNA Biogenesis Methods and Protocols M M B ethods in olecular iology Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 miRNA Biogenesis Methods and Protocols Edited by Ulf Andersson Vang Ørom Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark Editor Ulf Andersson Vang Ørom Department of Molecular Biology and Genetics Aarhus University Aarhus, Denmark ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-8623-1 ISBN 978-1-4939-8624-8 (eBook) https://doi.org/10.1007/978-1-4939-8624-8 Library of Congress Control Number: 2018947780 © Springer Science+Business Media, LLC, part of Springer Nature 2018 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms 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. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Printed on acid-free paper This Springer imprint is published by the registered company Springer Science+Business Media, LLC part of Springer Nature. The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A. Preface miRNAs are well-studied regulatory small RNAs affecting the translation and stability of numerous mRNAs. miRNAs are emerging through a complex biogenesis pathway going from primary transcripts that can be as long as 30 kb to mature miRNA of approximately 21 nts. miRNA biogenesis is a sophisticated series of events assuring a finely tuned regula- tion of miRNA expression and activity. Understanding miRNA biogenesis better could expand our knowledge of molecular events, contribute to extended molecular networks in gene regulation, and lead to development of new therapeutic interventions. It is my hope that this protocol series will contribute to the further development of knowl- edge and application of miRNA biogenesis, both in the basic research lab and in the clinic. Aarhus, Denmark Ulf Andersson Vang Ørom v Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 High-Throughput Characterization of Primary microRNA Transcripts . . . . . . . . 1 Tsung-Cheng Chang and Joshua T. Mendell 2 Identifying Pri-miRNA Transcription Start Sites . . . . . . . . . . . . . . . . . . . . . . . . . 11 Georgios Georgakilas, Nikos Perdikopanis, and Artemis G. Hatzigeorgiou 3 Metabolic Pulse-Chase RNA Labeling for pri-miRNA Processing Dynamics . . . . 33 Annita Louloupi and Ulf Andersson Vang Ørom 4 In Vitro System for Coupling RNAP II Transcription to Primary microRNA Processing and a Three-Way System for RNAP II Transcription/Splicing/microRNA Processing . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Shanye Yin, Alexander Iocolano, Yong Yu, Jaya Gangopadhyay, and Robin Reed 5 Purification of Microprocessor-Associated Factors . . . . . . . . . . . . . . . . . . . . . . . 51 Ross A. Cordiner and Sara Macias 6 Inhibiting Pri-miRNA Processing with Target Site Blockers . . . . . . . . . . . . . . . . 63 Annita Louloupi and Ulf Andersson Vang Ørom 7 MicroRNA Analysis Using the Quantitative Real-Time PCR Reaction . . . . . . . . 69 Marta Kotlarek, Anna Kubiak, Krystian Jażdżewski, and Anna Wójcicka 8 MicroRNA Analysis Using Next-Generation Sequencing . . . . . . . . . . . . . . . . . . 87 Monika Kolanowska, Anna Kubiak, Krystian Jażdżewski, and Anna Wójcicka 9 Identification of microRNA Precursor-Associated Proteins . . . . . . . . . . . . . . . . . 103 Thomas Treiber, Nora Treiber, and Gunter Meister 10 Analysis of 3′ End Modifications in microRNAs by High-T hroughput Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Madalena M. Reimão-Pinto, Angela M. Rodrigues-Viana, and Stefan L. Ameres 11 Uncovering the Stability of Mature miRNAs by 4-Thio-U ridine Metabolic Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Matteo J. Marzi and Francesco Nicassio 12 Detection of microRNA-Target Interactions by Chimera PCR (ChimP) . . . . . . . 153 James P. Broughton and Amy E. Pasquinelli 13 High-Quality Overlapping Paired-End Reads for the Detection of A-to-I Editing on Small RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Josephine Galipon, Rintaro Ishii, Soh Ishiguro, Yutaka Suzuki, Shinji Kondo, Mariko Okada-Hatakeyama, Masaru Tomita, and Kumiko Ui-Tei vii viii Contents 14 Targeting miRNA for Therapeutics Using a Micronome Based Method for Identification of miRNA- mRNA Pairs and Validation of Key Regulator miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Parveen Bansal, Ashish Kumar, Sudhir Chandna, Malika Arora, and Renu Bansal 15 Method for Detection of miRNAs in Non-Model Organisms with Unreported Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Parveen Bansal, Ashish Kumar, Sudhir Chandna, Malika Arora, and Renu Bansal 16 Detection and Verification of Mammalian Mirtrons by Northern Blotting . . . . . 209 Mohammad Farid Zia and Alex S. Flynt 17 Detecting Agotrons in Ago CLIPseq Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 Thomas B. Hansen Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 Contributors Stefan L. amereS • IMBA—Institute of Molecular Biotechnology, Vienna Biocenter, Vienna, Austria maLika arora • Multidisciplinary Research Unit, Indian Council of Medical Research, GGSMCH, Faridkot, Punjab, India Parveen BanSaL • University Centre of Excellence in Research, Baba Farid University of Health Sciences, Faridkot, Punjab, India renu BanSaL • Department of Microbiology, GGSMCH, Faridkot, Punjab, India JameS P. Broughton • Department of Dermatology, Stanford University School of Medicine, Stanford, CA, USA Sudhir Chandna • Division of Natural Radiation Response Mechanisms, Institute of Nuclear Medicine and Allied Sciences, Timarpur, Delhi, India tSung-Cheng Chang • Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA roSS a. Cordiner • Centre for mRNP Biogenesis and Metabolism, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark aLex S. fLynt • Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, MS, USA JoSePhine gaLiPon • Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan; Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa, Japan; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Jaya gangoPadhyay • Department of Cell Biology, Harvard Medical School, Boston, MA, USA georgioS georgakiLaS • Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA; Institute for Immunology, University of Pennsylvania, Philadelphia, PA, USA; Epigenetics Institute at Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA thomaS B. hanSen • Department of Molecular Biology and Genetics (MBG) and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark artemiS g. hatzigeorgiou • DIANA-Lab, Hellenic Pasteur Institute, Athens, Greece; Department of Electrical and Computer Engineering, University of Thessaly, Volos, Greece aLexander ioCoLano • Department of Cell Biology, Harvard Medical School, Boston, MA, USA Soh iShiguro • Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan; Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa, Japan; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan rintaro iShii • Department of Computational Biology, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-shi, Chiba-ken, Japan ix x Contributors kryStian JażdżewSki • Warsaw Genomics INC, Warsaw, Poland; Genomic Medicine, Medical University of Warsaw, Warsaw, Poland; Centre of New Technologies, University of Warsaw, Warsaw, Poland monika koLanowSka • Warsaw Genomics INC, Warsaw, Poland; Genomic Medicine, Medical University of Warsaw, Warsaw, Poland; Centre of New Technologies, University of Warsaw, Warsaw, Poland ShinJi kondo • Inter-University Research Institute Corporation, Research Organization of Information and Systems, Tachikawa, Tokyo, Japan marta kotLarek • Warsaw Genomics INC, Warsaw, Poland; Genomic Medicine, Medical University of Warsaw, Warsaw, Poland anna kuBiak • Warsaw Genomics INC, Warsaw, Poland; Genomic Medicine, Medical University of Warsaw, Warsaw, Poland; Centre of New Technologies, University of Warsaw, Warsaw, Poland aShiSh kumar • Texas Biomedical Research Institute, San Antonio, TX, USA annita LouLouPi • Max Planck Institute for Molecular Genetics, Berlin, Germany; Free University of Berlin, Berlin, Germany Sara maCiaS • Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, UK matteo J. marzi • Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia (IIT), Milan, Italy gunter meiSter • Laboratory for RNA Biology, Biochemistry Center Regensburg (BZR), University of Regensburg, Regensburg, Germany JoShua t. mendeLL • Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA; Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA; Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA franCeSCo niCaSSio • Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia (IIT), Milan, Italy mariko okada-hatakeyama • Laboratory for Integrated Cellular Systems, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Kanagawa, Japan; Laboratory of Cell Systems, Institute for Protein Research, Osaka University, Suita-shi, Osaka-fu, Japan uLf anderSSon vang Ørom • Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark amy e. PaSquineLLi • Division of Biology, University of California, San Diego, La Jolla, CA, USA nikoS PerdikoPaniS • DIANA-Lab, Hellenic Pasteur Institute, Athens, Greece; Department of Electrical and Computer Engineering, University of Thessaly, Volos, Greece roBin reed • Department of Cell Biology, Harvard Medical School, Boston, MA, USA madaLena m. reimão-Pinto • IMBA—Institute of Molecular Biotechnology, Vienna Biocenter, Vienna, Austria angeLa m. rodrigueS-viana • IMBA—Institute of Molecular Biotechnology, Vienna Biocenter, Vienna, Austria Contributors xi yutaka Suzuki • Department of Computational Biology, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-shi, Chiba-ken, Japan maSaru tomita • Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan; Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa, Japan nora treiBer • Laboratory for RNA Biology, Biochemistry Center Regensburg (BZR), University of Regensburg, Regensburg, Germany thomaS treiBer • Laboratory for RNA Biology, Biochemistry Center Regensburg (BZR), University of Regensburg, Regensburg, Germany kumiko ui-tei • Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; Department of Computational Biology, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-shi, Chiba-ken, Japan anna wóJCiCka • Warsaw Genomics INC, Warsaw, Poland; Genomic Medicine, Medical University of Warsaw, Warsaw, Poland Shanye yin • Department of Cell Biology, Harvard Medical School, Boston, MA, USA yong yu • Department of Cell Biology, Harvard Medical School, Boston, MA, USA mohammad farid zia • Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, MS, USA