Methods in Molecular Biology 2166 Manfred Heinlein Editor RNA Tagging Methods and Protocols M ETHODS IN M OLECULAR B IOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK For further volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by- step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed. RNA Tagging Methods and Protocols Edited by Manfred Heinlein Institut de Biologie Moléculaire des Plantes (IBMP-CNRS), Université de Strasbourg, Strasbourg, France Editor Manfred Heinlein Institut de Biologie Mole´culaire des Plantes (IBMP-CNRS) Universite´ de Strasbourg Strasbourg, France ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-0711-4 ISBN 978-1-0716-0712-1 (eBook) https://doi.org/10.1007/978-1-0716-0712-1 © Springer Science+Business Media, LLC, part of Springer Nature 2020 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, expressed 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. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A. Preface RNA molecules play diverse roles in the cell owing to their secondary structure and interaction with various proteins and nucleic acids. To study the composition, localization, dynamics, and function of these functional RNA complexes in vitro or in live cells and tissues of animals and plants requires the specific tagging of the RNA or of RNA-binding proteins with suitable reporter molecules. This book provides a compendium of state-of-the-art methods for the labeling, detection, and purification of RNA and RNA–protein complexes and thereby constitutes an important toolbox for researchers interested in understanding the complex roles of RNA molecules in development, signaling, and disease. In addition to studying the natural function of RNA molecules within cells, numerous labs have developed methods to actually apply RNA molecules as guides for the sequence-specific cleavage, modification, or imaging of RNA and DNA. Therefore, this book also includes protocols that apply RNA molecules as sequence-specific guide molecules. The protocol chapters of this book are organized in six parts. Part I provides protocols for the in situ detection of RNA molecules using fluorescent in situ hybridization (FISH) techniques, whereas Part II provides protocols for the tagging of RNA molecules for specific detection in live cells by fluorescent RNA-binding proteins. A dedicated review article describes the imaging of RNA molecules with fluorogens that bind to RNA targets tagged with specific light-up RNA aptamers. Part III contains protocols for monitoring RNA uptake by cells or for addressing RNA transport between cells. Important protocols useful for the characterization of RNA-binding proteins and protein complexes are presented in Part IV. Part V is dedicated to protocols for the application of RNA molecules as guides in RNA-mediated editing and imaging of chromosome loci. Starting with an overview article about CRISPR guide RNA design for genome editing, this part continues by presenting protocols in which RNA molecules are applied to guide nuclease-deactivated CRISPR- associated protein 9 (dCas9) for the imaging of specific genome loci. Another interesting protocol in this part of the book allows the in vivo monitoring of transcribed loci by imaging tagged nascent messenger RNA molecules emerging during gene transcription. The final Part VI provides advanced protocols for the functional analysis of RNA molecules. These include the detection of small RNAs involved in RNA silencing by Northern blot hybridiza- tion, the monitoring of miRNA-mediated RNA silencing events with an in vivo reporter system, the isolation and characterization of RNA molecules associated with polyribosomes, a protocol for transfection of RNA molecules into plant protoplasts, and also the biochemi- cal in vitro modification and tagging of RNA molecules for imaging and structural analysis. The experimental protocols are provided by leading experts with hands-on experience in the respective method. I hope that the provision of these protocols will further stimulate research in RNA biology and that the research community interested in this field will accept this book as an important reference. I am very grateful to all the authors who contributed to this book. I also thank the series editor, John M. Walker, for his continuous support in developing this volume. Strasbourg, France Manfred Heinlein v Contents Preface . ................................................................... v Contributors................................................................. xi PART IIN SITU DETECTION OF RNA MOLECULES USING FISH TECHNIQUES 1 Tagging and Application of RNA Probes for Sequence-Specific Visualization of RNAs by Fluorescent In Situ Hybridization . ............... 3 Thomas Dresselhaus and Andrea Bleckmann 2 Quantitative Fluorescence In Situ Hybridization Detection of Plant mRNAs with Single-Molecule Resolution . ....................... 23 Kun Huang, Mona Batish, Chong Teng, Alex Harkess, Blake C. Meyers, and Jeffrey L. Caplan 3 Visualization of Endoplasmic Reticulum-Associated mRNA in Mammalian Cells . .................................................... 35 Jingze J. Wu and Alexander F. Palazzo 4 Simultaneous Detection of mRNA and Protein in S. cerevisiae by Single-Molecule FISH and Immunofluorescence . ....................... 51 Evelina Tutucci and Robert H. Singer PART II IN VIVO IMAGING OF RNA TRANSPORT AND LOCALIZATION 5 Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More . ..................................... 73 Michael Ryckelynck 6 Visualization of Transiently Expressed mRNA in Plants Using MS2 . ........ 103 Eduardo Jose´ Pen˜a and Manfred Heinlein 7 New Generations of MS2 Variants and MCP Fusions to Detect Single mRNAs in Living Eukaryotic Cells .................................. 121 Xavier Pichon, Marie-Ce´cile Robert, Edouard Bertrand, Robert H. Singer, and Evelina Tutucci 8 In Vivo Imaging of Mobile mRNAs in Plants Using MS2.................... 145 Kai-Ren Luo, Nien-Chen Huang, and Tien-Shin Yu 9 RNA Imaging with RNase-Inactivated Csy4 in Plants and Filamentous Fungi.................................................................. 157 David Burnett, Alexander Lichius, and Jens Tilsner PART III IMAGING AND ANALYSIS OF RNA UPTAKE AND TRANSPORT BETWEEN CELLS 10 Utilizing Potato Virus X to Monitor RNA Movement....................... 181 Zhiming Yu, Sung Ki Cho, Pengcheng Zhang, Yiguo Hong, and David J. Hannapel vii viii Contents 11 A Protocol for Non-biased Identification of RNAs Transferred Between Heterologous Mammalian Cell Types Using RNA Tagging, Cell Sorting, and Sequencing............................................. 195 Sandipan Dasgupta and Jeffrey E. Gerst 12 Synthesizing Fluorescently Labeled dsRNAs and sRNAs to Visualize Fungal RNA Uptake ......................................... 215 Rachael Hamby, Ming Wang, Lulu Qiao, and Hailing Jin 13 Labeling of dsRNA for Fungal Uptake Detection Analysis ................... 227 Matteo Galli, Jafargholi Imani, and Karl-Heinz Kogel PART IV IDENTIFICATION AND ANALYSIS OF RNA-BINDING PROTEINS 14 Using RNA Affinity Purification Followed by Mass Spectrometry to Identify RNA-Binding Proteins (RBPs) . .............................. 241 Mengge Shan and Brian D. Gregory 15 Plant Individual Nucleotide Resolution Cross-Linking and Immunoprecipitation to Characterize