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Methods in
Molecular Biology 2666

Ren-Jang Lin Editor

RNA-Protein
Complexes
and Interactions
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY

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
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 RNA-Protein Complexes
and Interactions

Methods and Protocols

Second Edition

Edited by

Ren-Jang Lin
Beckman Research Institute, Center for RNA Biology and Therapeutics, Irell & Manella Graduate School of
Biological Sciences, Duarte, CA, USA
Editor
Ren-Jang Lin
Beckman Research Institute, Center for
RNA Biology and Therapeutics
Irell & Manella Graduate
School of Biological Sciences
Duarte, CA, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-0716-3190-4 ISBN 978-1-0716-3191-1 (eBook)
https://doi.org/10.1007/978-1-0716-3191-1
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Dedication

To my family, for their unwavering love and support, and to City of Hope, for the resources
and freedom that make this project a reality.

v
Preface
RNA: Because without protein, we’d just be a bunch of lonely nucleotides—
Unknown

RNA-protein complexes play a crucial role in many cellular processes. The study of these
complexes and the understanding of the interactions between RNAs and proteins and the
dynamics of these interactions have been a central focus of biological research. The identifi-
cation and characterization of RNA-protein complexes can provide insights into the molec-
ular mechanisms that govern cell function and development, which have led to the discovery
of new disease-causing mechanisms and therapeutic targets.
In recent years, advances in technologies and techniques have allowed for the study of
RNA-protein interactions at an unprecedented level of detail. This book presents a collec-
tion of methods and techniques used to study RNA-protein complexes, interactions, and
RNA localization. They are meticulously written with detailed step-by-step instructions to
aid researchers across multiple disciplines in biological research.
The 22 chapters in this book cover a wide range of techniques, including simple
methods for detecting modified RNAs, techniques for analyzing gene expression patterns
and RNA localization, methods for measuring interactions between tRNAs and their
modifying enzymes, and techniques for probing RNA structure and RNA-protein interac-
tions in vivo and in vitro. As we proceed through the book, we will see how these various
techniques are used to study different aspects of RNA-protein complexes and interactions,
and how they can be applied to different organisms and cell types.
The chapters in this book are organized to address topics such as RNA modification and
localization, RNA-protein interactions, RNP assembly and purification, and other related
topics. This organization allows for a clear progression of information and a deeper under-
standing of the field.
RNA modification and localization
Chapter 1 describes a simple method for the detection of Wybutosine-modified tRNA/
Phe_GAA as a readout of retrograde tRNA nuclear import and re-export, using
HCl/Aniline cleavage and non-radioactive Northern hybridization. Another important
aspect of RNA analysis is the localization of RNA in cells, which is covered in Chapter 2
that describes the analysis of gene expression patterns and RNA localization by fluores-
cence in situ hybridization in whole mount Drosophila testes.
RNA-protein interaction
Biophysical techniques such as electrophoretic mobility shift assay (EMSA) and microscale
thermophoresis (MST) are discussed in Chapter 3, while biochemical methods such as
Fe(II)-EDTA cleavage and nuclease footprinting are covered in Chapter 4. Chapters 5,
6, and 7 describe the use of SHAPE to probe RNA structure and RNA-protein inter-
actions in vitro and in vivo. Additionally, Chapters 8 and 9 describe the use of native
RNA immunoprecipitation (RIP) and in vivo cross-linking and co-immunoprecipitation
to study protein-RNA interactions.

vii
viii Preface

RNP assembly and purification

Chapter 14 describes the in vitro reconstitution of pseudouridylation catalyzed by human
box H/ACA ribonucleoprotein particles, and Chapter 15 describes the arresting of
spliceosome intermediates at various stages of the splicing pathway. Additionally,
Chapter 16 describes the streamlined purification of RNA-protein complexes using
UV crosslinking and RNA antisense purification, and Chapter 17 describes the
MS2-MBP based affinity purification of nucleus- or cytoplasm-localized lncRNA-pro-
tein complexes formed in vivo.
R-loop, chromatin, extracellular vesicles, and SELEX
The book also covers a variety of other topics related to RNA biology, including R-loop
formation, RNA-chromatin interactions, and the isolation and characterization of
extracellular vesicles and exosomes. Chapter 19 describes the detection of R-loop
formation using a plasmid-based in vitro transcription assay, and Chapter 20 describes
methods to study RNA-chromatin interactions. Lastly, Chapters 21 and 22 cover the
challenges for studying and isolating extracellular vesicles from cell-conditioned media,
and the evolution of cell-type-specific RNA aptamers via live cell-based SELEX.
The methods and protocols are useful for studying and analyzing the various issues
commonly encountered by researchers in the field of RNA. For example:
RNA-protein complexes formed inside the cell
One method for purifying these complexes is UV crosslinking followed by antisense RNA
oligos. Crosslinking fixes the complexes and preserves the interactions between the
RNA and protein before purification. Another method is to tag the endogenous protein
with MS2-MBP and to purify by amylose affinity. This technique uses a specific tag to
pull out the protein of interest, along with any associated RNA. Tag with FLAG is
another alternative.
Cellular trafficking and localization
One method is through biochemical separation of different cellular compartments. Another
method is through the detection of location-specific RNA modification marks. In situ
RNA-PLA and in situ hybridization are also techniques that can be used to study cellular
localization of RNA-protein interactions.
Measurement of RNA-protein interactions
This can be done through various techniques such as electrophoretic mobility shift assay
(EMSA) and microscale thermophoresis (MST), providing information on the strength
and specificity of the interactions. Fe(II)-EDTA cleavage and nuclease footprinting,
SHAPE, RIP, CLIP, CLASH, ribosome profiling, and RNA-PLA are other techniques
that can be used to measure RNA-protein interactions.
RNA-protein co-immunoprecipitation
One application of this technique is for tRNA localization, which can provide information
on the location of the tRNA within the cell. Another application of this technique is for
the detection of native RIP, which can be used to identify and quantify specific
RNA-protein interactions. Additionally, the technique can be used to study the inter-
actions between microRNA and Ago protein.
 Preface ix

Assembly of RNA-protein complexes in lysates

One example of an assembly is the H/ACA snoRNP, which is a complex involved in the
modification of RNA. Another example is the spliceosomes, which are complexes
involved in the removal of introns from RNA.
RNA interactions with cellular components
RNA-chromatin interaction, the detection of R-loop formation, the analysis of extracellular
vesicles and exosomes, and cell-based SELEX to identify cell-type-specific RNA apta-
mers are also covered in the book.
It is important to note that the field is constantly evolving, and new techniques are
continuously being developed. This book can only cover a selection of the methods, but I
hope that the techniques presented here will prove useful in your own research and that it
will encourage further exploration of the fascinating world of RNA-protein interactions.
I would like to extend my heartfelt appreciation to the authors of this book for their
invaluable contributions. Their hard work and dedication have resulted in a valuable
resource for researchers and students in the field of RNA-protein complexes and interac-
tions. I am particularly grateful to the editor of the Methods in Molecular Biology series,
John M. Walker, for his trust and support in inviting me to be an editor for the third time,
following two previous method books on the same topic.
I would also like to acknowledge the publisher staff for their patience and tireless efforts
in bringing this book to fruition. Their support and guidance throughout the process have
been invaluable in ensuring that the book is of the highest quality. Their dedication to the
project has made it possible to deliver a comprehensive and informative book on this topic.
Special thanks to ChatGPT for providing suggestions and assistance in writing the preface.
Finally, I would like to express my gratitude to the readers of this book for taking the
time to engage with the material. It is through their interest and curiosity that books like this
can come to life and contribute to the advancement of knowledge in the field of
RNA-protein complexes and interactions.

Center for RNA Biology and Therapeutics, Beckman Ren-Jang Lin

Research Institute, Duarte, CA, USA
Contents

Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
1 A Simple Method for the Detection of Wybutosine-Modified
tRNAPheGAA as a Readout of Retrograde tRNA Nuclear Import
and Re-export: HCl/Aniline Cleavage and Nonradioactive
Northern Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Regina T. Nostramo and Anita K. Hopper
2 Analysis of Gene Expression Patterns and RNA Localization by
Fluorescence in Situ Hybridization in Whole Mount Drosophila Testes . . . . . . . . 15
Jaclyn M. Fingerhut and Yukiko M. Yamashita
3 Electrophoretic Mobility Shift Assay (EMSA) and Microscale
Thermophoresis (MST) Methods to Measure Interactions Between
tRNAs and Their Modifying Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Andrzej Chramiec-Gła˛bik, Michał Rawski,
Sebastian Glatt, and Ting-Yu Lin
4 Mapping of RNase P Ribozyme Regions in Proximity with a Human
RNase P Subunit Protein Using Fe(II)-EDTA Cleavage and
Nuclease Footprint Analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Phong Trang, Adam Smith, and Fenyong Liu
5 SHAPE to Probe RNA Structure and RNA–Protein Interactions
In Vitro. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Kaushik Saha and Gourisankar Ghosh
6 Chemical Probing of RNA Structure In Vivo Using SHAPE-MaP
and DMS-MaP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Kaushik Saha and Gourisankar Ghosh
7 Analysis of RNA-Protein Interaction Networks Using RNP-MaP . . . . . . . . . . . . . 95
Kaushik Saha and Gourisankar Ghosh
8 Native RNA Immunoprecipitation (RIP) for Precise Detection
and Quantification of Protein-Interacting RNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Mai Baker, Rami Khosravi, and Maayan Salton
9 In Vivo Cross-Linking and Co-Immunoprecipitation Procedure
to Analyze Nuclear tRNA Export Complexes in Yeast Cells . . . . . . . . . . . . . . . . . . 115
Kunal Chatterjee and Anita K. Hopper
10 Identify MicroRNA Targets Using AGO2-CLASH (Cross-linking,
Ligation, and Sequencing of Hybrids) and AGO2-CLIP
(Cross-Linking and Immuno-Precipitation) in Cells with or
Without the MicroRNA of Interest Depleted. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Mitsuo Kato
11 Ribosomal Profiling by Gradient Fractionation of Cell Lysates . . . . . . . . . . . . . . . 149
Nimisha Bhattarai, Bo Cao, Shelya X. Zeng, and Hua Lu

xi
xii Contents

12 Global Assessment of Protein Translation in Mammalian Cells

Using Polysome Fractionation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Jingrong Zhao and Sika Zheng
13 Fluorescent In Situ Detection of RNA–Protein Interactions in
Intact Cells by RNA-PLA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Tianqi Li, Wei Zhang, and Mingyi Xie
14 In Vitro Reconstitution of Pseudouridylation Catalyzed by
Human Box H/ACA Ribonucleoprotein Particles . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Hironori Adachi, Jonathan L. Chen, Qiangzong Yin,
Pedro Morais, and Yi-Tao Yu
15 Arresting Spliceosome Intermediates at Various Stages of
the Splicing Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Chi-Kang Tseng and Soo-Chen Cheng
16 Streamlined Purification of RNA–Protein Complexes Using
UV Cross-Linking and RNA Antisense Purification . . . . . . . . . . . . . . . . . . . . . . . . . 213
Nhu Trang, Tong Su, Simone Hall, Nada Boutros, Bobby Kong,
Calvin Huang, and Colleen A. McHugh
17 MS2-MBP-Based Affinity Purification of Nucleus- or
Cytoplasm-Localized lncRNA–Protein Complexes Formed In Vivo . . . . . . . . . . . 231
Shuai Hou, Weijie Wang, Tian Hao, and Haixin Lei
18 RNA and Protein Interactomes of an RNA-Binding Protein
Tagged with FLAG Epitopes Using Combinatory Approaches of
Genome Engineering and Stable Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Sze Cheng, Meeyeon Park, and Jeongsik Yong
19 Detecting R-Loop Formation Using a Plasmid-Based In Vitro
Transcription Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Lei Shen and Yanzhong Yang
20 Methods to Study RNA–Chromatin Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
Kiran Sriram, Yingjun Luo, Naseeb K. Malhi,
Aleysha T. Chen, and Zhen Bouman Chen
21 Challenges for Studying and Isolating Extracellular Vesicles
from Cell-Conditioned Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Andrew R. Chin
22 Evolution of Cell-Type-Specific RNA Aptamers via Live
Cell-Based SELEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Alberto Herrera, Jiehua Zhou, Min-sun Song, and John J. Rossi

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Contributors

HIRONORI ADACHI • Department of Biochemistry and Biophysics, University of Rochester

Medical Center, Rochester, NY, USA
MAI BAKER • Department of Biochemistry and Molecular Biology, The Institute for Medical
Research Israel–Canada, Hebrew University–Hadassah Medical School, Jerusalem, Israel
NIMISHA BHATTARAI • Department of Biochemistry & Molecular Biology and Cancer Center,
Tulane University School of Medicine, New Orleans, LA, USA
NADA BOUTROS • Department of Chemistry and Biochemistry, University of California San
Diego, La Jolla, CA, USA
BO CAO • Xavier University of Louisiana College of Pharmacy, New Orleans, LA, USA
KUNAL CHATTERJEE • Department of Molecular Genetics, Center for RNA Biology, The Ohio
State University, Columbus, OH, USA; Department of Biology, Wittenberg University,
Springfield, OH, USA
ALEYSHA T. CHEN • Department of Diabetes Complications and Metabolism, Arthur Riggs
Diabetes Metabolism Research Institute and Beckman Research Institute, City of Hope,
Duarte, CA, USA
JONATHAN L. CHEN • Department of Biochemistry and Biophysics, University of Rochester
Medical Center, Rochester, NY, USA
ZHEN BOUMAN CHEN • Department of Diabetes Complications and Metabolism, Arthur
Riggs Diabetes Metabolism Research Institute and Beckman Research Institute, City of
Hope, Duarte, CA, USA; Irell and Manella Graduate School of Biological Sciences,
Beckman Research Institute, City of Hope, Duarte, CA, USA
SOO-CHEN CHENG • Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan,
Republic of China
SZE CHENG • Department of Biochemistry, Molecular Biology and Biophysics, University of
Minnesota Twin Cities, Minneapolis, MN, USA
ANDREW R. CHIN • Sean N. Parker Center for Allergy and Asthma Research at Stanford
University, Stanford, CA, USA
ANDRZEJ CHRAMIEC-GŁA˛BIK • Malopolska Centre of Biotechnology (MCB), Jagiellonian
University, Krakow, Poland
JACLYN M. FINGERHUT • Whitehead Institute for Biomedical Research, Massachusetts
Institute of Technology, Department of Biology, Cambridge, MA, USA; Howard Hughes
Medical Institute, Cambridge, MA, USA
GOURISANKAR GHOSH • Department of Chemistry and Biochemistry, University of California
San Diego, La Jolla, CA, USA
SEBASTIAN GLATT • Malopolska Centre of Biotechnology (MCB), Jagiellonian University,
Krakow, Poland
SIMONE HALL • Department of Chemistry and Biochemistry, University of California San
Diego, La Jolla, CA, USA
TIAN HAO • Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China
ALBERTO HERRERA • Center for RNA Biology and Therapeutics, Beckman Research Institute
of City of Hope, Duarte, CA, USA; Irell and Manella Graduate School of Biological
Sciences, Beckman Research Institute of City of Hope, Duarte, CA, USA

xiii
xiv Contributors

ANITA K. HOPPER • Department of Molecular Genetics, Center for RNA Biology, The Ohio
State University, Columbus, OH, USA
SHUAI HOU • Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China
CALVIN HUANG • Department of Chemistry and Biochemistry, University of California San
Diego, La Jolla, CA, USA
MITSUO KATO • Department of Diabetes Complications and Metabolism, Arthur Riggs
Diabetes & Metabolism Research Institute, Beckman Research Institute of City of Hope,
Duarte, CA, USA
RAMI KHOSRAVI • Department of Biochemistry and Molecular Biology, The Institute for
Medical Research Israel–Canada, Hebrew University–Hadassah Medical School,
Jerusalem, Israel
BOBBY KONG • Department of Chemistry and Biochemistry, University of California San
Diego, La Jolla, CA, USA
HAIXIN LEI • Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China
TIANQI LI • Department of Biochemistry and Molecular Biology, University of Florida,
Gainesville, FL, USA
TING-YU LIN • Malopolska Centre of Biotechnology (MCB), Jagiellonian University, Krakow,
Poland
FENYONG LIU • School of Public Health, University of California, Berkeley, CA, USA;
Program in Comparative Biochemistry, University of California, Berkeley, CA, USA
HUA LU • Department of Biochemistry & Molecular Biology and Cancer Center, Tulane
University School of Medicine, New Orleans, LA, USA
YINGJUN LUO • Department of Diabetes Complications and Metabolism, Arthur Riggs
Diabetes Metabolism Research Institute and Beckman Research Institute, City of Hope,
Duarte, CA, USA
NASEEB K. MALHI • Department of Diabetes Complications and Metabolism, Arthur Riggs
Diabetes Metabolism Research Institute and Beckman Research Institute, City of Hope,
Duarte, CA, USA
COLLEEN A. MCHUGH • Department of Chemistry and Biochemistry, University of
California San Diego, La Jolla, CA, USA
PEDRO MORAIS • ProQR Therapeutics, Leiden, The Netherlands; Research and Development,
Pharmaceuticals, Bayer AG, Wuppertal, Germany
REGINA T. NOSTRAMO • Department of Molecular Genetics, Center for RNA Biology, The
Ohio State University, Columbus, OH, USA
MEEYEON PARK • Department of Biochemistry, Molecular Biology and Biophysics, University
of Minnesota Twin Cities, Minneapolis, MN, USA
MICHAŁ RAWSKI • Malopolska Centre of Biotechnology (MCB), Jagiellonian University,
Krakow, Poland
JOHN J. ROSSI • Center for RNA Biology and Therapeutics, Beckman Research Institute of
City of Hope, Duarte, CA, USA; Irell and Manella Graduate School of Biological Sciences,
Beckman Research Institute of City of Hope, Duarte, CA, USA
KAUSHIK SAHA • Department of Chemistry and Biochemistry, University of California San
Diego, La Jolla, CA, USA
MAAYAN SALTON • Department of Biochemistry and Molecular Biology, The Institute for
Medical Research Israel–Canada, Hebrew University–Hadassah Medical School,
Jerusalem, Israel
LEI SHEN • Department of Cancer Genetics and Epigenetics, Beckman Research Institute,
City of Hope National Cancer Center, Duarte, CA, USA
 Contributors xv

ADAM SMITH • Program in Comparative Biochemistry, University of California, Berkeley,

CA, USA
MIN-SUN SONG • Center for RNA Biology and Therapeutics, Beckman Research Institute of
City of Hope, Duarte, CA, USA
KIRAN SRIRAM • Department of Diabetes Complications and Metabolism, Arthur Riggs
Diabetes Metabolism Research Institute and Beckman Research Institute, City of Hope,
Duarte, CA, USA; Irell and Manella Graduate School of Biological Sciences, Beckman
Research Institute, City of Hope, Duarte, CA, USA
TONG SU • Department of Chemistry and Biochemistry, University of California San Diego,
La Jolla, CA, USA
NHU TRANG • Department of Chemistry and Biochemistry, University of California San
Diego, La Jolla, CA, USA
PHONG TRANG • School of Public Health, University of California, Berkeley, CA, USA
CHI-KANG TSENG • Graduate Institute of Microbiology, National Taiwan University,
College of Medicine, Taipei, Taiwan, Republic of China
WEIJIE WANG • Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China;
Institute of Pediatrics, Children’s Hospital of Fudan University, Shanghai, China
MINGYI XIE • UF Health Cancer Center, University of Florida, Gainesville, FL, USA
YUKIKO M. YAMASHITA • Whitehead Institute for Biomedical Research, Massachusetts
Institute of Technology, Department of Biology, Cambridge, MA, USA; Howard Hughes
Medical Institute, Cambridge, MA, USA
YANZHONG YANG • Department of Cancer Genetics and Epigenetics, Beckman Research
Institute, City of Hope National Cancer Center, Duarte, CA, USA
QIANGZONG YIN • Department of Biochemistry and Molecular Pharmacology, University of
Massachusetts Medical School, Worcester, MA, USA
JEONGSIK YONG • Department of Biochemistry, Molecular Biology and Biophysics, University
of Minnesota Twin Cities, Minneapolis, MN, USA
YI-TAO YU • Department of Biochemistry and Biophysics, University of Rochester Medical
Center, Rochester, NY, USA
SHELYA X. ZENG • Department of Biochemistry & Molecular Biology and Cancer Center,
Tulane University School of Medicine, New Orleans, LA, USA
WEI ZHANG • Department of Genetics, Yale University School of Medicine, New Haven, CT,
USA
JINGRONG ZHAO • Division of Biomedical Sciences, Center for RNA Biology and Medicine,,
University of California, Riverside, CA, USA
SIKA ZHENG • Division of Biomedical Sciences, Center for RNA Biology and Medicine,,
University of California, Riverside, CA, USA
JIEHUA ZHOU • HitGen Inc., Chengdu, China
 Chapter 1

A Simple Method for the Detection of Wybutosine-Modified

tRNAPheGAA as a Readout of Retrograde tRNA Nuclear Import
and Re-export: HCl/Aniline Cleavage and Nonradioactive
Northern Hybridization
Regina T. Nostramo and Anita K. Hopper

Abstract
tRNAs are highly mobile molecules that are trafficked back and forth between the nucleus and cytoplasm by
several proteins. However, characterization of the movement of tRNAs and the proteins mediating these
movements can be difficult. Here, we describe an easy and cost-effective assay to discover genes that are
involved in two specific tRNA trafficking events, retrograde nuclear import and nuclear re-export for yeast,
Saccharomyces cerevisiae. This assay, referred to as the hydrochloric acid (HCl)/aniline assay, identifies the
presence or absence of a unique modification on tRNAPheGAA called wybutosine (yW) that requires mature,
spliced tRNAPheGAA to undergo retrograde nuclear import and subsequent nuclear re-export for its
addition. Therefore, the presence/absence of yW-modified tRNAPheGAA serves as a readout of retrograde
nuclear import and nuclear re-export. This simple assay can be used to determine the role of any gene
product in these previously elusive tRNA trafficking events.

Key words Hydrochloric acid, Aniline, Northern blot, tRNA retrograde nuclear import, tRNA
re-export, Wybutosine, Yeast

1 Introduction

In eukaryotic cells, tRNAs are trafficked back and forth between the
nucleus and the cytoplasm. These movements are required for the
maturation of primary tRNA transcripts into mature tRNAs, which
function as essential adaptor molecules in protein synthesis. The
trafficking of tRNAs within the cell consists of three main steps
(Fig. 1) [reviewed in [1]]. The first is primary export of tRNAs
from the nucleus, where they are transcribed, to the cytoplasm.
Second, tRNAs undergo retrograde nuclear import, which relo-
cates them back to the nucleus. Finally, tRNAs are re-exported back
to the cytoplasm. Understanding subcellular trafficking of tRNAs
and its regulation, as well as the proteins involved in these

Ren-Jang Lin (ed.), RNA-Protein Complexes and Interactions: Methods and Protocols,
Methods in Molecular Biology, vol. 2666, https://doi.org/10.1007/978-1-0716-3191-1_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

1
2 Regina T. Nostramo and Anita K. Hopper

Nucleus Cytoplasm

5’ Ex 3’ Ex Mitochondria
Primary Export

Intron
pre-tRNAPhe

Retrograde Import
Cm Spliced
Trm7 Gm tRNAPhe
Trm5
m1G
Re-export Tyw1
Tyw2
Tyw3
yW Tyw4

Fig. 1 Maturation of tRNAPheGAA in Saccharomyces cerevisiae. The maturation of

tRNAPheGAA in S. cerevisiae includes the sequential addition of several modifica-
tions. Since many of the enzymes that catalyze the addition of these modifica-
tions display either nuclear or cytoplasmic localization, tRNAPheGAA must undergo
trafficking between these two compartments to become fully mature. After
tRNAPheGAA is transcribed in the nucleus, it undergoes multiple processing events
and modifications (not shown), resulting in pre-tRNAPheGAA, which contains a 5′
and 3′ exon (black lines) separated by an intron (orange line). Pre-tRNAPheGAA is
exported to the cytoplasm in the first trafficking event called primary nuclear
export. Once in the cytoplasm, pre-tRNAPheGAA is transported to the surface of
the mitochondria where its intron is spliced, and the 5′ and 3′ exons are ligated
together. Spliced tRNAPheGAA is a substrate for the enzyme Trm7, which methy-
lates positions 32 (Cm) and 34 (Gm) (pink). Next, spliced tRNAPheGAA is trafficked
back to the nucleus in a step called retrograde nuclear import. Spliced tRNA-
Phe Phe
GAA (but not intron-containing tRNA GAA) is a substrate for the nuclear-
localized enzyme Trm5, which methylates G at position 37 (m1G) (green). In
the final event of its maturation, tRNAPheGAA is re-exported from the nucleus to
the cytoplasm where it is modified sequentially by the Tyw1-4 enzymes, result-
ing in the addition of wybutosine (yW) at position 37 (blue). Therefore, any
spliced tRNAPheGAA containing the yW modification must have undergone both
retrograde nuclear import and nuclear re-export and can thus serve as a readout
of these two trafficking events. (Reproduced from Ref. 4 with permission from
Oxford Journals)

processes, has and continues to shed light on the physiological and

pathophysiological roles of tRNAs in the cell [2–4].
The first tRNA trafficking event, primary nuclear export, is
perhaps the most well-studied of the three. This is because in
organisms like budding and fission yeast a subset of tRNAs possess
introns, which are spliced on the surface of the mitochondria
immediately following primary nuclear export by exporters such
as Los1 [5–7]. Therefore, any intron-containing tRNA that has had
HCl/Aniline Assay for tRNAPheGAA Nuclear Import and Re-export 3

its intron removed must have undergone primary export from the
nucleus to the cytoplasm. This would be reflected by a decrease in
the size of the tRNA, which can be easily detected by Northern blot
analysis [8]. To this end, a genome-wide screen using this approach
identified three novel proteins (Mex67, Mtr2, and Crm1) that are
involved in the primary export of tRNA in yeast, in addition to the
canonical tRNA exporter Los1 [2, 9].
Understanding of the two subsequent tRNA trafficking events,
specifically retrograde nuclear import and nuclear re-export, how-
ever, is not as complete. While the size of intron-containing tRNAs
decreases upon primary export and subsequent intron splicing,
there is no size change in tRNAs upon retrograde import and
re-export. Additionally, tracking tRNA movement by other means
like tagging can be complicated given the lack of a 3’ UTR, addi-
tion of an amino acid to the 3′ end, and the critical importance of
tRNA tertiary structure in mediating its function. Additionally,
fractionating the cytoplasm and nucleus in particular organisms,
such as Saccharomyces cerevisiae, is extremely difficult and not ame-
nable to genome-wide screens. Therefore, to address this issue, we
developed a simple assay that allows for the identification of gene
products involved in tRNA retrograde nuclear import and re-ex-
port. This assay takes advantage of a specific modification, wybuto-
sine (yW), that occurs exclusively on tRNAPheGAA in S. cerevisiae
and requires both retrograde nuclear import and nuclear re-export
for its addition [10].
The process of tRNAPheGAA maturation in S. cerevisiae, culmi-
nating in the addition of the yW modification, begins with its
transcription in the nucleus (Fig. 1). Once transcribed, this primary
tRNA undergoes 5′ leader and 3′ trailer removal and 3’ CCA
addition, resulting in the formation of pre-tRNAPheGAA. Pre--
tRNAPheGAA is then exported to the cytoplasm and localized to
the surface of the mitochondria for intron splicing by the splicing
endonuclease complex [3, 5, 6]. This spliced tRNAPheGAA is now a
substrate for the cytoplasmic methyltransferase, Trm7, which
methylates positions 32 (Cm32) and 34 (Gm34) [11]. These mod-
ifications, particularly Gm34, are prerequisites for the later addition
of the yW modification. The tRNA then undergoes retrograde
nuclear import, where it is now a substrate for the nuclear-localized
methyltransferase Trm5, which methylates G at position
37 (m1G37) [12]. Trm5 only recognizes spliced tRNAPheGAA and
not intron-containing tRNAPheGAA [13, 14], and therefore, the
presence of this modification is indicative of prior primary nuclear
export and retrograde nuclear import. Finally, the m1G37-modified
tRNAPheGAA is re-exported to the cytoplasm where it is sequentially
modified by the enzymes Tyw1, Tyw2, Tyw3, and Tyw4, resulting
in a wybutosine-modified tRNAPheGAA [15]. Thus, any tRNAPhe-
GAA containing the yW modification is evidence of a tRNA that has
undergone all three trafficking events.
4 Regina T. Nostramo and Anita K. Hopper

A.
yW-modified HCl Aniline
tRNAPhe

B.
tRNAPhe HCl Aniline
lacking yW

Fig. 2 The effects of sequential treatment of tRNAPheGAA with HCl and aniline. (a)
Treatment of fully mature tRNAPheGAA containing the wybutosine
(yW) modification (green) with HCl elicits the removal of the yW base, leaving
an abasic site (AP site; gray unfilled circle). Subsequent treatment with aniline
causes scission of the RNA chain at the AP site, resulting in the splitting of the
tRNA into a 5′ and 3′ half. (b) Treatment of tRNAPheGAA lacking the yW modifica-
tion with HCl does not elicit AP site formation, and therefore, no RNA chain
scission occurs in response to subsequent aniline treatment

Given the ability of the yW modification to be used as a readout

of retrograde nuclear import and re-export, we developed an easy,
cost-effective assay, herein called the hydrochloric acid (HCl)/ani-
line assay, to detect the presence of this modification [4]. The basis
for this assay stems from research conducted in the late 1960s and
1970s demonstrating that mild acid treatment of tRNAPheGAA
containing yW results in removal of the yW base without cleaving
the sugar-phosphate backbone, creating an abasic site
[16, 17]. Cleavage of the sugar-phosphate backbone at this abasic
site can then be achieved by incubation with aniline at a low pH via
a β-elimination reaction [18]. Given that the yW modification is
located at nucleotide position 37, the last nucleotide of the 5′ exon,
this results in cleavage of tRNAPheGAA into 5′ and 3′ half-sized
fragments (Fig. 2a). This drastic change in size can be visualized
by Northern blot analysis (Fig. 3, lanes 3 vs. 4). Thus, any mature
tRNAPheGAA that is cleaved following sequential HCl and aniline
treatment is indicative of a tRNA that has undergone both retro-
grade nuclear import and nuclear re-export. Conversely, any tRNA-
Phe
GAA lacking the yW modification would remain intact following
sequential HCl and aniline treatment (Fig. 2b). For example, cells
lacking Tyw1, the enzyme that catalyzes the first step in the con-
version of m1G37 of tRNAPheGAA to yW, do not contain the yW
modification and therefore do not display tRNA cleavage following
sequential HCl and aniline treatment when visualized by Northern
blot (Fig. 3, lanes 1 vs. 2).
The HCl/aniline assay can thus be used to detect gene pro-
ducts that play a role in the retrograde nuclear import or re-export
steps, as the lack of tRNAPheGAA cleavage following HCl and aniline
treatment in a strain deficient in a specific gene product would
indicate the role of this gene product in either tRNAPheGAA
HCl/Aniline Assay for tRNAPheGAA Nuclear Import and Re-export 5

tyw1Δ WT mtr10Δ
- + - + + HCl

+ + + + + Aniline

1 2 3 4 5

Fig. 3 Northern blot analysis of tRNAPheGAA treated with HCl and aniline. Small
RNAs were isolated from wild-type (WT), tyw1Δ, or mtr10Δ cells and subjected
to sequential treatment with HCl and aniline. Northern blot analysis was
performed using a DIG-labeled tRNAPheGAA 5′/3′ exon probe. For all strains, no
tRNAPheGAA strand cleavage was observed when treated with aniline only (lanes
1 and 3). Conversely, nearly all of the tRNAPheGAA was cleaved in WT cells
subjected to sequential HCl/aniline treatment (lane 4). This cleavage was absent
in tyw1Δ cells (lane 2), which lack the Tyw1 enzyme needed for the synthesis of
yW. In cells lacking Mtr10, a protein involved in the retrograde nuclear import of
tRNAPheGAA, cleavage of the tRNA was greatly inhibited as compared to WT cells
(lane 5). The detected RNAs correspond to mature (M; 76 nts), 5′ exon (5′ Ex;
37 nts), or 3′ exon (3′ Ex; 39 nts) tRNAPheGAA. Mature tRNAPheGAA migrates on the
gel as two bands. Since two of the ten copies of this gene differ by a single
nucleotide substitution in both the 5′ and 3′ exons, this dual migration pattern
may be due to these substitutions or to under-modification of the tRNA. 5S rRNA
levels (121 nts) were measured by Northern blot and serve as a loading control

maturation (as with tyw1Δ cells) or the retrograde nuclear import or

re-export processes. For example, Mtr10 is a member of the
β-importin family and has previously been shown to function in
the import of tRNAs under conditions of amino-acid deprivation
[19]. Using the HCl/aniline assay, Mtr10 was also shown to func-
tion in the constitutive retrograde nuclear import of tRNAPheGAA,
as mature, uncleaved tRNAPheGAA levels are increased in mtr10Δ
cells following sequential HCl and aniline treatment, as compared
to wild-type cells (Fig. 3, lanes 4 vs. 5) [4].
6 Regina T. Nostramo and Anita K. Hopper

When using the HCl/aniline assay for the identification of

novel proteins involved in the retrograde nuclear import or
re-export of tRNAPheGAA, a second step, such as fluorescence in
situ hybridization (FISH) analysis, will be needed to differentiate
between these two processes. For gene products that are involved in
retrograde nuclear import of tRNAPheGAA, cytoplasmic accumula-
tion of the tRNA would be observed upon gene deletion/inactiva-
tion. Conversely, nuclear accumulation would be observed if the
defect occurred in the nuclear re-export step [19].
Overall, the value the HCl/aniline assay will add to the field of
tRNA biology is immense. Currently, this assay has been used to
identify a constitutive role of Mtr10 in the retrograde import of
tRNAPheGAA and to confirm a role of Ssa2 in this process under
conditions of amino acid deprivation [4], as previously reported
[20]. Additionally, this assay was able to demonstrate that Mex67
and Crm1 are required for the re-export of tRNAPheGAA, whereas
the canonical tRNA exporters, Los1 and Msn5, are not [4]. Overall,
use of the HCl/aniline assay will lead to the identification and
characterization of additional gene products involved in the retro-
grade tRNA nuclear import and re-export processes and deepen
our understanding of the role of tRNA trafficking in cell biology.

2 Materials

All solutions should be prepared in filter sterilized double distilled

water (ddH2O). Reagents should be stored at room temperature,
unless otherwise indicated.

2.1 HCl/Aniline 1. Stock HCl solution: Dilute 37% (w/w) hydrochloric acid
Assay 100 fold in water.
2. Working HCl solution: In a 1.5 mL microcentrifuge tube, mix
975 μL water and 25 μL of the stock HCl solution.
3. Working aniline solution (0.5 M aniline, pH 4.5): Add 8.2 mL
water to a 15 mL tube. Then add 910 μL aniline (ACS grade; ≥
99.5%) and 795 μL glacial acetic acid. Cap and flip the tube
several times to mix. Wrap the tube in foil to protect from light.
This solution should be prepared just before use (see Notes 1
and 2).
4. 5 mM potassium hydroxide (KOH).
5. 100% ethanol: Store at 4 °C.
6. 3 M sodium acetate, pH 5.2.
7. GlycoBlue coprecipitant (15 mg/mL; Invitrogen): This
reagent consists of a blue dye covalently linked to glycogen,
which coprecipitates with RNA. GlycoBlue enhances precipita-
tion of low quantities of RNA and also increases the visibility of
the RNA pellet during RNA precipitation.
 HCl/Aniline Assay for tRNAPheGAA Nuclear Import and Re-export 7

2.2 Gel 1. 10% polyacrylamide: In a 1 L beaker, add 424.2 g urea to

Electrophoresis 300 mL water and stir on a heating plate until dissolved, but
be careful not to bring to a boil. Remove from heat and allow to
cool at room temperature for 5–10 min. After the solution is
slightly cooled but still warm, add 250 mL 40% acrylamide/bis
solution (19:1), 100 mL 10× TBE (see #5 below for recipe) and
bring to 1 L with water. Filter sterilize, cover with foil and store
at 4 °C.
2. Ammonium persulfate (APS): 10% solution in water (see
Note 3).
3. N,N,N,N′-Tetramethyl-ethylenediamine (TEMED): Store at
4 °C.
4. 10% polyacrylamide, 8 M urea gel: In a 50 mL conical tube,
combine 35 mL 10% polyacrylamide, 300 μL 10% APS and
22.5 μL TEMED. Prepare just before casting gel.
5. 10× TBE Running buffer: In a 1 L beaker, dissolve 108 g Tris
base, 55 g boric acid and 7.5 g EDTA, disodium salt in 800 mL
water. Stir with heat until dissolved. Bring to 1 L with water.
When ready to use, prepare 1× TBE by adding 100 mL 10×
TBE to 900 mL water (see Note 4).
6. 2× Loading Dye: Combine 24 g urea, 2 mL 0.5 M EDTA
(pH 8.0) and 0.1 mL 1 M Tris buffer (pH 7.5). Adjust the
volume to 50 mL with water. Then add 0.05 g of xylene cyanol
and 0.05 g bromophenol blue. Store at 4 °C.
7. Gel electrophoresis apparatus.

2.3 Transfer and UV 1. 50× TAE transfer buffer: In a 1 L beaker, dissolve 242 g Tris
Cross Linking base and 18.61 g EDTA, disodium salt in 700 mL water and
stir until dissolved. Add 57.1 mL glacial acetic acid, and adjust
the volume to 1 L. When ready to use, prepare 1× TAE by
adding 20 mL 50× TAE to 980 mL water (see Note 4).
2. Hybond N+ nylon membrane.
3. Spectrolinker XL-100 UV cross-linker: RNAs can be fixed to
the Hybond N+ nylon membrane by UV cross-linking at an
energy dosage of 120 millijoules/cm2.
4. Transfer apparatus.

2.4 DIG-Labeled 1. tRNAPheGAA 5′/3′ exon probe: 5’ CGAACACAGGACCTC

Probes CAGATCTTCAGTCTGGCGCTCTCCC 3’
This 40 nt oligo is complementary to 20 nts of the 3′ end
of the 5′ exon and 20 nts of the 5′ end of the 3′ exon of
tRNAPheGAA in Saccharomyces cerevisiae.
8 Regina T. Nostramo and Anita K. Hopper

2. tRNAPheGAA 5′ exon probe: 5’ CAACTGAGCTAAGTCCGC 3’

This oligo is complementary to the 18 nts at the 5′ end of
the 5′ exon of tRNAPheGAA in Saccharomyces cerevisiae.
3. tRNAPheGAA 3′ exon probe: 5’ TGCGAACTCTGTGGATC-
GAACACAGGACCT 3’
This oligo is complementary to the 30 nts at the 3′ end of
the 3′ exon of tRNAPheGAA in Saccharomyces cerevisiae.
4. 5S rRNA probe: 5’ GCACCTGAGTTTCGCGTATGGT 3’
This oligo is complementary to 5S rRNA in Saccharomyces
cerevisiae and can be used as a control for equal gel loading (see
Note 5).
5. DIG Oligonucleotide Tailing Kit, second generation (Roche):
DIG-label the 3′ end of the probes listed above using
DIG-dUTP/dATP by first adding 1 μL of 100 μM oligo to
9 μL dH2O, and then adding reagents #1–5, according to the
manufacturer’s protocol. After incubation at 37 °C for 15 min,
stop the reaction by adding 0.8 μL of 0.5 M EDTA, pH 8.0.
One labeling reaction yields a total volume of 20.8 μL
DIG-labeled probe, which is enough probe for about
20 blots (1 μL DIG-labeled probe per blot). DIG-labeled
probes should be stored at -20 °C.

2.5 Northern Blot 1. Hybridization tubes.

Hybridization and 2. Hybridization oven (rotisserie).
Detection of DIG-
3. 20× saline-sodium citrate (SSC) buffer: In a 1 L beaker, com-
labeled Probes
bine 175.3 g NaCl and 77.4 g sodium citrate. Add 800 mL
water and pH with 12 N HCl to 7.0. Bring to 1 L.
4. Prehybridization buffer: 5× saline-sodium citrate (SSC), 0.1%
(w/v) N-lauroylsarcosine, 0.02% (w/v) sodium dodecyl sulfate
(SDS), 1% (w/v) Roche Blocking Reagent (see Note 6).
5. Wash buffer 1: 2× SSC containing 0.1% SDS.
6. Wash buffer 2: 0.1 M Maleic acid, 0.15 M NaCl, pH 7.5, 0.3%
Tween 20.
7. Blocking buffer: 1% (w/v) Roche Blocking Reagent, 0.1 M
Maleic acid, 0.15 M NaCl, pH 7.5.
8. Anti-DIG antibody conjugated with alkaline phospha-
tase (Roche, Cat. #11093274910).
9. 1× Detection buffer: 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5.
10. Hybridization bags.
11. CDP-STAR: Disodium 2-chloro-5-(4-methoxyspiro
(1,2-dioxetane-3,2′-(5′-chloro) tricyclo [3.3.1.13,7]decan)-4-
yl)-1-phenyl phosphate (Roche, Cat. #12041677001).
 HCl/Aniline Assay for tRNAPheGAA Nuclear Import and Re-export 9

3 Methods

3.1 HCl/Aniline 1. Prepare one 1.5 mL microcentrifuge tube for each sample. On
Assay ice, mix 10 μg of RNA with water to a volume of 30 μL.
2. Add 20 μL of working HCl solution to each tube, bringing the
final volume to 50 μL.
3. To induce wybutosine base excision, incubate tubes at 37 °C
for 3 h (see Note 7).
4. Neutralize the HCl-treated RNA solutions by adding 11.38 μL
of 5 mM KOH and keep on ice (see Note 8).
5. Prepare minus (-) HCl controls for each sample by mixing
1955.04 ng RNA and water to a volume of 12 μL in a 2 mL
microcentrifuge tube. Keep on ice (see Notes 9 and 10).
6. Preheat a heating block to 60 °C.
7. Prepare 0.5 M aniline, pH 4.5 (see Note 11).
8. Label a 2 mL microcentrifuge tube for each HCl-treated sample
(see Note 10). To each tube, add 12 μL 0.5 M aniline, pH 4.5,
and 12 μL neutralized, HCl-treated RNA. For -HCl samples, add
12 μL aniline directly to the RNA prepared in step 5.
9. Incubate samples at 60 °C for 20 min to induce chain scission
at abasic sites.
10. Briefly centrifuge samples, then add the following in order:
451 μL water, 47.5 μL 3 M sodium acetate pH 5.2, 1410 μL
ice cold 100% ethanol, and 1 μL of 15 mg/mL GlycoBlue
coprecipitant (see Note 12). Flip the tubes several times to
mix and store at -80 °C for at least 1 h or overnight to
precipitate the RNA.
11. Centrifuge samples for 20 min at 4 °C at 15,000 rpm
(microcentrifuge).
12. Preheat a water bath to 55 °C.
13. After centrifugation, a small blue pellet should be visible.
Remove as much supernatant as possible. Wash pellet in 1 mL
cold 70% ethanol. Centrifuge for 5 min at 4 °C at 15,000 rpm.
14. Remove 70% ethanol. Quick centrifuge the samples and
remove all remaining ethanol with gentle suction using a
10 μL pipette tip, being careful not to aspirate the pellet (see
Note 13).
15. Allow samples to sit at room temperature with the caps open to
air dry for 10–15 min.
16. Add 20 μL water to each tube. Quick spin the samples to
ensure the pellet is in the water. Incubate at 55 °C for
10 min. Quick spin again.
17. Add 20 μL of 2× northern loading dye to each tube of 20 μL of
RNA. At this point, the samples can be stored at -80 °C.
10 Regina T. Nostramo and Anita K. Hopper

3.2 Northern Blot 1. Prepare a 10% polyacrylamide gel by combining 35 mL 10%

polyacrylamide solution, 300 μL 10% ammonium persulfate,
and 22.5 μL TEMED in a 50 mL conical centrifuge tube. Flip
upside down a few times to mix and cast gel within a
16 cm × 18 cm × 1.5 mm gel cassette with 15-well or 24-well
comb. Allow approximately 45 min for the gel to solidify.
2. Add 1× TBE to the gel apparatus and clean out the wells to
remove any unpolymerized polyacrylamide using a needle and
syringe.
3. Heat the samples at 85 °C for 5 min. Load 20 μL per lane.
Electrophorese at 4 °C at 50 V for approximately 18 h, until the
dye front is about 1 inch from the bottom of the gel.
4. Following electrophoresis, gently pry the gel plates open,
allowing the gel to remain on the bottom plate. Cut off the
wells of the gel and just below the dye front. Gently rinse the
gel with water to remove any gel fragments that could interfere
with the transfer step.
5. Cut a piece of Hybond N+ nylon membrane to the size of the
gel. Cut a small piece off the top left corner of the membrane.
Assemble the transfer cassette in a plastic container containing
1× TAE buffer. In order, add one foam pad, three sheets of
filter paper, the membrane (with the cut corner oriented at the
top left), the gel (with lane 1 on the left), three more sheets of
filter paper, and another foam pad. Seal the cassette and insert
into a transfer apparatus containing 1× TAE buffer. Transfer at
15 V for 15 min, then 0.6 A for 2 h at 4 °C.
6. After the transfer, disassemble the cassette and dry the mem-
brane on a piece of filter paper for a few minutes. Orient the
membrane with the RNA side facing up (the cut corner should
be at the top left). Then UV cross-link the front of the mem-
brane at an energy dosage of 120 millijoules/cm2. Flip the
membrane over and UV cross-link the back of the membrane.
At this point, the membrane can be stored dry in plastic wrap at
-20 °C.
7. Place the membrane in a hybridization tube with 10 mL pre-
hybridization buffer, with the RNA side facing the center of the
hybridization tube. Assure that the membrane is not overlap-
ping itself. Place the tube in a rotisserie hybridization oven and
incubate at 37 °C for 30 min with rotation. (see Notes 14
and 15).
8. Replace the pre-hybridization buffer with hybridization buffer,
which is 10 mL prehybridization buffer containing 1 μL
tRNAPheGAA DIG-labeled oligo. Any of the tRNAPheGAA oligos
HCl/Aniline Assay for tRNAPheGAA Nuclear Import and Re-export 11

listed in Subheading 2.4 can be used in this step. Each of these

tRNAPheGAA oligos hybridizes to different parts of tRNAPhe-
GAA (5′ exon, 3′ exon or 5′/3′ exon junction) yielding slightly
different detection patterns. See Supplemental Fig. 1C in Nos-
tramo and Hopper (2020) for a comparison of the detection
patterns that are observed using each of these probes. Since the
5′/3′ exon junction probe will detect both halves of the cleaved
tRNAPheGAA, quantitation is easier when using either the 5′ or
3′ exon only probes. Incubate the membrane at 37 °C over-
night with rotation.
9. Remove hybridization buffer (see Note 16). Wash the mem-
brane four times with 15 mL Wash Buffer 1 for 10 min each.
Perform the first three washes at 37 °C with rotation. During
the fourth wash, turn the temperature of the hybridization
oven down to room temperature. The remaining steps should
all be performed at room temperature.
10. Discard Wash Buffer 1 and equilibrate the membrane in 10 mL
Wash Buffer 2 for 3 min with rotation.
11. Discard Wash Buffer 2 and incubate the membrane in 10 mL
Blocking Buffer for 30 min with rotation.
12. Discard the Blocking Buffer, and add 10 mL fresh Blocking
Buffer containing 1:10,000 dilution of anti-DIG antibody
conjugated to alkaline phosphatase. Incubate with rotation
for 30 min.
13. Discard the antibody solution, and wash the membrane twice
with 15 mL Wash Buffer 2 for 15 min each with rotation.
14. Discard Wash Buffer 2, and equilibrate the membrane in
10 mL Detection Buffer for 3 min with rotation.
15. Discard the Detection Buffer, and place the membrane on the
bottom layer of a hybridization bag. Add 1 mL of CDP-STAR
to the membrane and cover with the top layer of the bag. Seal
using a heat sealer, avoiding air bubbles. Store the membrane
in the dark for 5–10 min.
16. Image the membrane in a Chemiluminescence imager (see
Note 17).
17. After imaging, place the membrane back into a hybridization
tube and wash twice with 15 mL Wash Buffer 1 at 37 °C for
15 min each with rotation (see Note 18).
18. To measure 5S rRNA levels, repeat steps 8–16 using 1 nM
DIG-labeled oligo for 5S rRNA (see Note 19).
12 Regina T. Nostramo and Anita K. Hopper

4 Notes

1. Aniline is very hygroscopic. After opening, the aniline should

be tightly sealed, wrapped in Parafilm, and stored at 4 °C.
When stored in this manner, the aniline is good for at least
6 months.
2. The pH of the solution will be 4.5 and does not need to be
adjusted.
3. APS should be made fresh just before use, or it can be prepared
and frozen at -20 °C in aliquots. Do not reuse after thawing.
4. After use, this buffer can be stored at 4 °C and used for two
additional times.
5. Detection of 5S rRNA as a loading control should be per-
formed using hybridization of the indicated probe to the mem-
brane, rather than by standard ethidium bromide staining of
the gel. Staining of the gel with ethidium bromide following
HCl and aniline treatment leads to a smear in the gel, making
the 5S rRNA band difficult to see.
6. For blocking, it is important to use the Roche Blocking
Reagent supplied in the Roche DIG Block and Wash Buffer
Set, which is supplied as a 10× solution. In our hands, the
Roche Blocking Reagent that can be purchased in powdered
form and reconstituted to form a 10× solution did not work
well, yielding high background levels.
7. Incubation at 37 °C for 3 h is sufficient for near complete
removal of the wybutosine base.
8. At this point, RNA can be stored at -80 °C for later treatment
with aniline.
9. The -HCl controls are prepared in this way in order to conserve
RNA, since in the next step only a portion of the HCl-treated
RNA will be used. Alternatively, -HCl samples can be prepared
exactly as above for +HCl samples.
10. In this step, a 2 mL microcentrifuge tube and not a 1.5 mL
microcentrifuge tube is required. This is to ensure that the
aniline can be adequately diluted and removed following
RNA precipitation.
11. The working aniline solution should be prepared just prior
to use.
12. GlycoBlue coprecipitant is needed to help precipitate the low
quantities of RNA and to visualize the RNA pellet after precip-
itation in ethanol.
13. It is important to remove as much liquid as possible since any
residual aniline will not easily dry.
 HCl/Aniline Assay for tRNAPheGAA Nuclear Import and Re-export 13

14. During the prehybridization, hybridization, and detection

steps, assure that the membrane is kept wet in order to avoid
high nonspecific background signals.
15. If a rotisserie hybridization oven is not available, the membrane
can be placed in a glass dish with the RNA side facing down and
incubated on an orbital platform shaker at the appropriate
temperature.
16. Hybridization buffer can be stored at -20 °C for up to 1 year
and reused once.
17. Typically, an exposure time of 2–3 min is sufficient to detect a
strong signal.
18. Alternatively, the membrane can be stored in the heat-sealed
hybridization bag with the CDP-Star reagent at -20 °C for at
least a year. For all steps after prehybridization, the membrane
should be kept wet. Do not let the membrane dry or store dry.
19. For 5S rRNA, typically an exposure time of 30–60 s is sufficient
to detect a strong signal.

Acknowledgments

This work was supported by funding from the National Institutes

of Health [grant number GM122884 to A.K.H.].

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 Chapter 2

Analysis of Gene Expression Patterns and RNA Localization

by Fluorescence in Situ Hybridization in Whole Mount
Drosophila Testes
Jaclyn M. Fingerhut and Yukiko M. Yamashita

Abstract
Researchers have used RNA in situ hybridization to detect the presence of RNA in cells and tissues for
approximately 50 years. The recent development of a method capable of visualizing a single RNA molecule
by utilizing tiled fluorescently labeled oligonucleotide probes that together produce a diffraction-limited
spot has greatly increased the resolution of this technique, allowing for the precise determination of
subcellular RNA localization and relative abundance. Here, we present our method for single molecule
RNA fluorescence in situ hybridization (smFISH) in whole mount Drosophila testes and discuss how we
have utilized this method to better understand the expression pattern of the highly unusual Y-linked genes.

Key words RNA fluorescence in situ hybridization, RNA localization, Single molecule RNA fluores-
cence in situ hybridization, Gene expression, RNA quantification

1 Introduction

Cells can be largely defined by their transcriptomes, but knowing

what RNAs are expressed in a certain cell type is just the tip of the
iceberg. For example, just because an RNA is present in a cell does
not mean that it is being actively translated or otherwise utilized.
The ability to visualize a single RNA species within a cell can reveal a
multitude of information, including whether it is strongly or weakly
expressed and whether it has a distinct subcellular localization, such
as to a specific ribonucleoprotein (RNP) granule. Visualizing a
single RNA species within a tissue can tell RNA’s story over devel-
opmental time from initiation of transcription to eventual transla-
tion and degradation. RNA fluorescence in situ hybridization
(RNA FISH) allows us to explore these types of questions.
Since it was first utilized, RNA in situ hybridization has become
increasingly sensitive and specific. Initially, such experiments relied
on radiolabeled cDNA probes [1, 2]; however, this method was

Ren-Jang Lin (ed.), RNA-Protein Complexes and Interactions: Methods and Protocols,
Methods in Molecular Biology, vol. 2666, https://doi.org/10.1007/978-1-0716-3191-1_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

15
16 Jaclyn M. Fingerhut and Yukiko M. Yamashita

plagued by low spatial resolution and the difficulties associated with

working with radioactive materials. Fluorescence-based methodol-
ogies were later applied, which relied on the conjugation of hep-
tanes (e.g., biotin) or fluorophores to modified cDNA or RNA
probes [3]. These methods also lacked sensitivity, and low abun-
dance RNAs could easily be missed. Recently, methodologies were
developed that have allowed researchers to visualize a single RNA
molecule (referred to as single molecule RNA FISH or smFISH)
and precisely localize that molecule within a cell [4–6]. smFISH
methods generally rely on either signal amplification or direct
detection. Signal amplification is largely an extension of previous
fluorescence-based methods with improved specificity and sensitiv-
ity [7–9]. Compared to direct detection methods, signal amplifica-
tion is a better choice for short transcripts and for utilizing
polymorphisms to differentiate between alleles [8, 10]. In our
work on RNA expression and localization in the Drosophila testis
[11, 12], we have made use of the direct detection method, in
which a pool of oligonucleotide probes, each conjugated with a
dye or fluorophore, creates a diffraction limited spot when hybri-
dized to the target RNA that is easily detectable above background
(Fig. 1) [5, 6]. While off-target binding of probes is minimized
though the use of probe design software, the off-target binding of
one probe within the pool is not sufficient to produce detectable
signal, reducing the number of false positives [5, 6]. Additionally,
the number of probes used is high enough such that even if not all
bind to every target, the signal will be sufficient for detection,

Fig. 1 Direct detection single molecule RNA fluorescence in situ hybridization

(smFISH). The direct detection method for smFISH relies on the use of many
fluorescently conjugated short oligonucleotides (black line with blue star) that all
bind to the target RNA (gray curved line). In the cell, signal from these
oligonucleotides will appear as diffraction limited spots
 RNA FISH in Drosophila Testes 17

reducing false negatives [5, 6]. The drawbacks to the direct detec-
tion methods are that (1) the target RNA must be long enough to
bind a sufficient number of fluorescent probes and (2) the signal
produced can be weak, and sensitive imaging systems may be
required. For visualizing multiple target RNAs simultaneously,
the direct detection method is ideal as each pool of oligonucleo-
tides can be conjugated with a different dye/fluorophore.
RNAs are not always evenly distributed within the cytoplasm.
smFISH allows researchers to determine whether an RNA of inter-
est is polarized within a cell or whether it localizes to a specific
subcellular compartment. These asymmetries are of great biological
importance [13]. For example, asymmetric RNA localization in
oocytes and embryos is critical for proper development of the
organism [14]. Additionally, during cell division, asymmetric
RNA localization can help specify daughter cell fate, such as in
the division of Drosophila neuroblasts [15, 16]. Moreover, actin
mRNAs localize to the leading edge to help facilitate cell migration
[17, 18]. RNAs can be enriched in RNP granules for storage via
translational repression or for processing, among other purposes.
The sensitivity of smFISH is sufficient to detect whether a particular
RNA is located within a specific sub-compartment of an RNP
granule, which could have important implications for its eventual
fate [12, 19–22]. These specific localizations can be confirmed by
combining smFISH with immunofluorescence staining using anti-
bodies to mark these cellular compartments [6, 12, 23]. The colo-
calization of an RNA with its RNA-binding protein(s) could also be
shown in this manner.
Some RNAs are robustly expressed while others may only have
a few transcripts present per cell. Using smFISH, the number of
transcripts per cell can be easily quantified, and the effect of various
perturbations on gene expression levels can be assessed [24]. This
data could nicely complement large-scale RNA sequencing meth-
ods. Within a tissue, the expression level of a single RNA in differ-
ent cell types could also be assessed.
We have applied the power of smFISH to the study of a set of
highly unusual genes in whole mount Drosophila testes, which
allowed us to gain tremendous insight into the expression of
these genes over developmental time [11, 12]. The Drosophila testis
is an excellent model for gene expression studies because, within a
single tissue, every stage of germ cell development from germline
stem cells to mature sperm can be seen simultaneously. Addition-
ally, the testis is spatiotemporally organized with the earliest germ
cells at one end and mature sperm at the other [25], making it easy
to follow the expression of a single gene during germ cell differen-
tiation (Fig. 2a). The genes we have focused on are the Y-linked
fertility genes. They encode axonemal dynein motor proteins essen-
tial for sperm motility [26–28], and they are highly unusual as they
are among the largest genes known, spanning over 4 Mb [29–
31]. This is due to the presence of satellite DNA (short tandem
18 Jaclyn M. Fingerhut and Yukiko M. Yamashita

Fig. 2 Single molecule RNA fluorescence in situ hybridization (smFISH) to follow gene expression over
developmental time. (a) Top: Diagram of Drosophila spermatogenesis: Early germ cells (blue) reside at one
end of the testis and undergo several founds of mitotic division before becoming spermatocytes (green).
Spermatocytes develop over 80–90 h (depicted by darkening of the green color) before initiating the meiotic
divisions. Middle: RNA FISH visualizing expression of the large Y-linked gene kl-3 in single spermatocyte
nuclei (yellow dashed line) at different stages of spermatocyte development. Early exon (blue), intron (green),
late exon (red), DAPI (white), nuclei of neighboring cells (white dashed line), and cytoplasmic mRNA granules
(yellow arrows). Bar: 10 μm. Bottom: Diagram of the Y-linked gene kl-3. Exons (vertical rectangles), introns
(black line), intronic satellite DNA repeats (dashed line), and regions of kl-3 targeted by RNA FISH probes
(colored bars). (This figure is partially reproduced from Fingerhut et al., 2019 under a Creative Commons
License (CC BY 4.0) [11]). (b) Diagram of a spermatocyte (nucleus, cytoplasm, and DNA in shades of gray)
showing the organization of early exon (blue), intron (green), and late exon (red) transcripts in the nucleus and
mRNA granules in the cytoplasm

repeats arrayed in vast tracks of 100’s of kilobases to several mega-

bases) within the introns as the coding regions are only around
14 kb [32–35]. We designed pools of smFISH probes targeting
different regions of these genes (one set targeting an early exon and
another targeting a late exon) [11]. We also used a single fluores-
cently labeled oligonucleotide to target transcript from the satellite
DNA in the introns (Fig. 2a). Using these probe sets, we could
follow the expression of these genes over time and have elucidated
 RNA FISH in Drosophila Testes 19

the “life story” of these transcripts [11]. This story begins in early
spermatocytes (cells in meiosis I prophase) where we detect tran-
script from only the early exon probes. Spermatocyte development
takes 80–90 h [36], and as spermatocytes mature, we start to detect
the intron transcript and finally the late exon transcript. In late
spermatocytes, we detect mRNA (both exon probe sets without
signal from the intron probe) in RNP granules in the cytoplasm
(Fig. 2). There are three large Y-linked axonemal dynein genes, and
we detect mRNAs from all three in these granules and can also
detect sub-compartmentalization of these mRNAs within the gran-
ules [12] (Fig. 3a, b). We identified a protein marker for these
granules (Pontin, which is necessary for the assembly of these
RNP granules [12]) by combining smFISH with antibody staining
(Fig. 4).

Fig. 3 Single molecule RNA fluorescence in situ hybridization (smFISH) to analyze the subcellular localization
of RNAs. (a) smFISH against transcripts from the Y-linked genes kl-3, kl-5, and kl-2 in spermatocytes. Each
transcript has distinct nuclear localization and all three colocalize in ribonucleoprotein (RNP) granules in the
cytoplasm. kl-3 (blue), kl-2 (green), kl-5 (red), DAPI (white), RNP granules (yellow arrows), spermatocyte
nuclei (yellow dashed line), and nuclei of neighboring cells (white dashed line). Bar: 10 μm. (b) An enlarged
RNP granule. smFISH allows for analysis of sub-compartmentalized mRNA localization within RNP granules.
kl-3 (blue), kl-2 (green) and kl-5 (red). Bar: 1 μm. (c) smFISH shows the polarized localization of RNP granules
within an elongating spermatid cyst. kl-3 (blue), kl-2 (green), kl-5 (red), DAPI (white) and spermatid cyst (cyan
dashed line). Bar: 25 μm. (This figure is reproduced from Fingerhut and Yamashita, 2020 under a Creative
Commons License (CC BY-NC-SA 4.0) [12])
20 Jaclyn M. Fingerhut and Yukiko M. Yamashita

Fig. 4 Analysis of RNA/protein colocalization by single molecule RNA fluores-

cence in situ hybridization (smFISH) combined with immunofluorescent staining.
(a, b) Immunofluorescent staining for Pontin protein and smFISH for kl-3 and kl-5
mRNAs in a spermatocyte (a) and within an ribonucleoprotein (RNP) granule (b).
Pontin (green), kl-3 (blue), kl-5 (red), DAPI (white), spermatocyte nuclei (yellow
dashed line), nuclei of neighboring cells (white dashed line), and RNP granules
(yellow arrows). Bar: 10 μm (a) or 1 μm (b)

As germ cells enter into the meiotic divisions, we observe these

RNP granules seemingly segregating such that each haploid sper-
matid receives a RNP granule. Drosophila sperm are among the
longest in the animal kingdom, reaching 1.9 mm in length
[37, 38]. smFISH allowed us to see that these RNP granules
become highly polarized as spermatids elongate, staying near the
very distal end of the cell (Fig. 3c), which we showed is important
for the axonemal dynein motor proteins to properly incorporate
into the axoneme [12]. The story of these mRNAs ends with the
dissociation of these RNP granules, which correlates with the accu-
mulation of axonemal dynein protein and the presumed degrada-
tion of the mRNAs.
Here, we present our method for RNA FISH in whole mount
Drosophila testes and also extend this method to the simultaneous
detection of RNA and protein.

2 Materials

Prepare all solutions using RNase free reagents. This includes all
starting solutions as well as pipette tips and collection tubes. As
much as possible, keep all materials (e.g., tube racks, collection
tubes, and pipette tips) used for RNA FISH separate from materials
used in other procedures, and only access these materials during the
RNA FISH procedure to prevent accidental contamination with
RNases.
 RNA FISH in Drosophila Testes 21

1. Fixative: 4% formaldehyde in 1× phosphate-buffered saline

(PBS). It is made from 16%, methanol-free, ultrapure EM
grade formaldehyde and 10× PBS (1.37 M NaCl, 0.027 M
KCl, 0.08 M Na2HPO4, 0.02 M KH2PO4, pH 7.4) diluted
in ultrapure distilled water. Store 1 mL aliquots at -20 °C.
2. 1× PBS: 10× PBS diluted in ultrapure distilled water. Store at
room temperature.
3. Seventy percent ethanol: We dilute 200 proof ethanol in ultra-
pure distilled water. Store at room temperature.
4. Oligonucleotide probes: We use fluorescently conjugated short
oligonucleotide probes to visualize RNAs (see Subheading 3.2
for information on probe design). We are primarily interested
in two types of RNAs: (1) RNAs derived from protein coding
genes and (2) RNAs derived from highly repetitive regions of
the genome, such as satellite DNAs. In order to visualize RNAs
from protein coding genes, we utilize direct detection smFISH
technologies, such as the Stellaris RNA FISH method by Bio-
search Technologies, Inc. By this method, we obtain pools
of fluorescently conjugated oligonucleotides for each transcript
of interest. These probes sets are kept at stock concentrations of
100 μM in 1 M Tris–HCl pH 7.4, diluted to working concen-
trations of 10 μM in ultrapure distilled water, and used at a final
concentration of 100 nM in hybridization buffer. To visualize
satellite DNA transcripts, we use a single fluorescently conju-
gated oligonucleotide at stock concentrations of 10 μM in 1 M
Tris–HCl pH 7.4, diluted to working concentrations of 5 μM
in ultrapure distilled water, and used at a final concentration of
50 nM in hybridization buffer.
5. Hybridization buffer: 2× saline-sodium citrate (SSC, 20 stock
solution contains 3 M NaCl, 0.3 M Na3C6H5O7), 10% dextran
sulfate, 1 mg/mL Escherichia coli tRNA, 2 mM ribonucleoside
vanadyl complex, 0.5% UltraPure bovine serum albumin
(BSA), and 10% formamide in ultrapure distilled water. Store
at -20 °C. (see Note 1).
6. Wash buffer: 2× SSC and 10% formamide in ultrapure distilled
water. Store at room temperature or 4 °C.
7. Blocking buffer: 1× PBS, 0.05% UltraPure BSA, 50 μg/mL
E. coli tRNA, 10 mM ribonucleoside vanadyl complex, 0.2%
Tween-20 in ultrapure distilled water. Make fresh (see Note 2).
8. 1× PBST: 1× PBS with 0.2% Tween-20 in ultrapure distilled
water. Store at room temperature.
9. Desired primary antibodies and appropriate AlexaFluor-
conjugated secondary antibodies (see Note 3).
10. VECTASHIELD with 4′,6-diamidino-2-phenylindole
(DAPI).
22 Jaclyn M. Fingerhut and Yukiko M. Yamashita

11. Stereomicroscope equipped for fly sorting and dissection.

12. Dissection dishes and forceps.
13. 1.5 mL tubes and 50 mL conical tubes.
14. Nutating 3D platform mixer.
15. Water bath.
16. Microscope slides and cover slips.
17. Widefield fluorescent or confocal microscope with the
appropriate filters/lasers to match the dyes used, a sensitive
camera (e.g., a cooled CCD), a high numerical aperture
(NA) objective, and appropriate image capture and analysis
software.

3 Methods

3.1 Fly Husbandry Flies are raised on standard Bloomington medium at 25 °C and
assayed at the desired age. For standard RNA FISH in the Drosoph-
ila testis, we typically dissect 1- to 5 day-old adults. However, we
have applied this method to a variety of larval and adult tissues
[39, 40], and fly age should be determined by experimental
question.

3.2 RNA FISH Probe As mentioned above, we primarily use RNA FISH to visualize
Design transcripts from either single copy genes or highly repetitive regions
of the genome. For single-copy genes, we have utilized the Stellaris
system by Biosearch Technologies, Inc., which makes use of a pool
of up to 48 short (18–22 bp) fluorescently tagged oligonucleotides
that bind along the transcript of interest to produce a diffraction
limited spot easily seen by conventional microscopy methods. Bio-
search Technologies, Inc.’s Stellaris® RNA FISH Probe Designer,
which is available online at www.biosearchtech.com/
stellarisdesigner, lets the user specify the target sequence, allowing
you to design probes against specific exons (to address questions
surrounding transcriptional timing or to target specific splice iso-
forms) and/or introns, or avoid designing a probe in a certain
region (such as an exon–exon junction). The designer software
screens for common sequences (across all organisms and within
your organism of choice) and avoids designing in those regions.
It also contains a masking level feature that allows the user to
control the stringency to help avoid off-target background noise
(see Notes 4 and 5). The designer also lets you control the probe
length (18–22 bp) and the spacing between probes. An ideal probe
set will contain the maximum number of probes (48, see Note 6).
Biosearch Technologies, Inc. also offers a wide variety of dyes and
modifications to allow for multiplexing or additional downstream
applications (see Note 7). When designing your probes, also
 RNA FISH in Drosophila Testes 23

consider what controls are necessary for your experiments—they

provide predesigned ship-ready sets for common positive controls,
such as housekeeping genes or RNA pol II, and you can design/
order a set against a target not present in your sample as a negative
control if an efficient RNAi line or mutant strain is not available.
To visualize transcripts originating from repetitive regions of
the genome, we utilize a single ~30 bp oligonucleotide. The repet-
itive nature of the target sequence allows for sufficient signal ampli-
fication. Many of the repeats of interest to us are 5 bp satellite DNA
repeats. For these repeats, our oligo is a 6 mer of the repeat
conjugated with a dye or fluorophore, such as Alexa-488, Cy3, or
Cy5 (see Note 8). These oligonucleotides can also be easily multi-
plexed. We order these probes HPLC purified.

3.3 RNA FISH All solutions listed are RNase free (see Subheading 2). Before start-
ing, clean your workspace and all materials, such as pipettes, tube
racks, and nutators, with either 70% ethanol or a commercial
reagent (such as RNase Zap) to remove RNase contamination.
1. Dissect testes (or other tissue of interest) in 1× PBS, and collect
them in a 1.5 mL tube containing 1 mL of 1× PBS. Dissections
should be completed within 30 min.
2. Aspirate off the 1× PBS and add 1 mL of fixative. Place the tube
on a nutator, and incubate for 30 min at room temperature.
3. Remove the fixative and wash the testes twice, 5 min per wash,
with 1 mL of 1× PBS on a nutator.
4. Remove the 1× PBS and add 1 mL of 70% ethanol. Place the
tubes in an RNase free container and incubate overnight at 4 °
C on a nutator (see Notes 9 and 10).
5. Add the appropriate volume of probe solution to hybridization
buffer to achieve the desired final probe concentration and
pipette to mix (see Subheading 2, step 4 for final probe con-
centration recommendations based on probe type and Note
11). Prepare 100 uL of this mixture per sample.
6. Aspirate the ethanol and add 1 mL of wash buffer. Let this
nutate for 3 min and sit for 2 min at room temperature.
7. Aspirate the wash buffer and add 100 μL of hybridization
solution containing the probe(s) (see Notes 11 and 12).
8. Seal the tube with parafilm and incubate overnight in a 37 °C
water bath. Ensure the sample is protected from light.
9. Add 1 mL of wash buffer without first removing the hybridiza-
tion solution. Incubate at 37 °C for 30 min. Continue to
protect from light (see Note 13).
10. Aspirate off the wash buffer + hybridization solution and wash
one more time with 1 mL of wash buffer at 37 °C for 30 min in
the dark (see Note 14).
24 Jaclyn M. Fingerhut and Yukiko M. Yamashita

11. Remove the wash buffer and add VECTASHIELD mounting

media with DAPI. Samples can be stored at 4 °C or imaged
immediately (see Notes 15 and 16).

3.4 Combining RNA Protein markers are often needed to better characterize the location
FISH with Protein of an RNA within a cell (e.g., to discern whether the RNA coloca-
Localization lizes with a specific type of RNP granule) or to assess translational
timing (e.g., RNA synthesis and translation could occur at different
developmental times, indicating the presence of additional layers of
gene expression regulation), among other applications. If the fly
strain dissected contains a fluorescently tagged transgene, we typi-
cally skip the overnight ethanol permeabilization step (Subheading
3.3, step 4 above) and proceed directly to the hybridization as
ethanol can denature fluorophores (see Note 10). If instead you
wish to use antibody staining to visualize your protein(s) of interest
alongside your RNA(s) of interest, an immunofluorescent staining
procedure can be included between steps 4 and 5 in the above
protocol.
1. Remove the ethanol and rinse with 100 μL of 1× PBS three
times.
2. Wash with 1 mL of 1× PBST three times for 5 min each on a
nutator.
3. Aspirate off the 1× PBST and add 100 μL of blocking buffer.
Incubate at 37 °C for 30 min (see Note 17).
4. Remove the blocking buffer and add 100 μL of primary anti-
body diluted in blocking buffer. Incubate at 37 °C for 1 h (see
Note 18).
5. Remove the primary antibody and wash three times with 1 mL
of 1× PBST for 5 min each on a nutator.
6. Aspirate the 1× PBST and incubate in 100 μL of blocking
buffer for 5 min at 37 °C.
7. Remove the blocking buffer and incubate in 100 μL of second-
ary antibody diluted in blocking buffer for 30 min at 37 °C (see
Note 18).
8. Wash three times with 1 mL of 1× PBST for 5 min each on a
nutator.
9. Remove the wash buffer and refix the tissue by adding 500 μL
of fixative and incubating for 10 min at room temperature
while rocking.
10. Aspirate the fixative and wash twice with 1 mL 1× PBS for
5 min each on a nutator.
11. Continue with the RNA FISH procedure starting from Sub-
heading 3.3, step 5 above.
 RNA FISH in Drosophila Testes 25

4 Notes

1. The dextran sulfate takes time to dissolve. Mixing by pipetting

or vortexing is messy and results in bubbles and loss of buffer.
We have found that it is best to leave the solution on a nutator
at room temperature until completely dissolved or at 4 °C
overnight.
2. Blocking buffer could be stored at -20 °C; however, we have
found that making this fresh before each experiment greatly
improves the staining quality of both the immunofluorescence
and the subsequent RNA FISH when some antibodies, poten-
tially dirtier ones, are used.
3. Not all antibodies work well in combination with RNA FISH.
This may be partially dependent on how the antibody is
prepared (how pure the serum is and whether it contains
abundant RNases). If possible, use purified antibodies, but if
this is not possible, such as for custom-made antibodies, using
fresh blocking buffer, which contains a high concentration of
ribonucleoside vanadyl complex to inhibit ribonucleases, and
minimizing antibody incubation times have typically yielded
sufficient quality results.
4. A masking level of 5 is the most stringent. We always start here
and only lower the masking level if the designer is unable to
generate a sufficient number of probes.
5. If your gene of interest has a paralog, it is recommended that
you align the generated probes against the paralogous
sequence to ensure specificity.
6. Biosearch Technologies, Inc., recommends at least 25 probes
for each set, although in our experience the necessary mini-
mum number of oligos depends on transcript abundance. We
have had success with sets containing as few as eight oligos for
highly expressed transcripts. If the default parameters do not
yield the recommended maximum of 48 oligos, it is best to try
different oligo lengths and spacing to increase the probe num-
ber first, then try lowering the masking level while being mind-
ful that this could increase off target binding (and therefore
background noise). The more probes you can get, the better
the sensitivity and specificity.
7. We have had very good results with the Quasar dyes, which are
similar to Cy3 and Cy5. In our experience, the Fluorescein dye
works in cases where the RNAs are enriched within a specific
location within the cell, such as within an RNP granule, but the
autofluorescence of the testis limits the detection of diffuse
single transcripts for probes sets conjugated with this dye. We
frequently multiplex Fluorescein with Quasar 570 and
Quasar 670.
26 Jaclyn M. Fingerhut and Yukiko M. Yamashita

8. These oligos work very well for both DNA and RNA FISH
applications.
9. The ethanol serves to weakly permeabilize the tissue, which is
all the permeabilization needed for the short oligo probes to
enter the tissue.
10. Ethanol can denature fluorophores. If your tissue contains a
fluorescently tagged transgene, this step can be omitted—we
have not had any issue with the probes being unable to diffuse
into the tissue. We have noticed that the expression pattern of
the RNA within a nucleus can look slightly different when this
step is omitted, but only for repetitive transcripts. It is possible
that these highly repetitive RNAs adopt complex secondary
structures that in turn interact with the ethanol during per-
meabilization. Be sure to know what your RNA expression
pattern looks like under standard RNA FISH conditions before
omitting this step. We have not noticed any differences in
expression pattern for our smFISH probes with and without
ethanol permeabilization.
11. When pipetting the hybridization solution, cut the end off of
your pipette tip as the hybridization buffer is viscous. When
adding the hybridization solution and probe mixture to your
tissue, pipette to mix, again with a cut pipette tip, which also
prevents damage to the tissue. You should be able to see the
tissue suspended in the hybridization solution. Failure to mix
the tissue with the hybridization solution could result in poor
or no hybridization.
12. Testes often stick to the sides and inside the cap of the 1.5 mL
tubes. Use some of the wash buffer and a pipette to wash them
down to the bottom of the tube. If sticking becomes a large
issue, 0.1% Tween-20 can be added to the wash buffer.
13. The hybridization solution is too viscous to remove without
also removing the testes. Diluting it with wash buffer for
30 min both washes the sample and allows the hybridization
solution to be removed.
14. Additional washes can be added if there is high background
present during imaging.
15. We have kept samples for several months at 4 °C with no
decrease in signal quality.
16. We typically image using a Leica STELLARIS 8 or SP8 confo-
cal microscope with a 63× oil immersion objective lens
(NA = 1.4), but it is important to note that this is not the
recommended method for imaging smFISH samples. As con-
focal microscopy relies on point illumination, which limits the
focal plane and restricts out of focus light, it can reduce the
sensitivity of low-light imaging, which can include smFISH
 RNA FISH in Drosophila Testes 27

depending on expression level of the target gene. A traditional

widefield fluorescence microscope could yield brighter signal at
the sacrifice of some resolution.
17. We seal the tubes with parafilm prior to all incubations in a
water bath to prevent any accidental contamination.
18. Antibody incubation times will need to be optimized for each
antibody. We have used anywhere from 30 min at 37 °C to
overnight at 4 °C. Keep in mind see Note 3 when deciding
what incubation times to try.

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 Chapter 3

Electrophoretic Mobility Shift Assay (EMSA) and Microscale

Thermophoresis (MST) Methods to Measure Interactions
Between tRNAs and Their Modifying Enzymes
Andrzej Chramiec-Gła˛bik, Michał Rawski, Sebastian Glatt,
and Ting-Yu Lin

Abstract
The Elongator complex is a unique tRNA acetyltransferase; it was initially annotated as a protein acetyl-
transferase, but in-depth biochemical analyses revealed its genuine function as a tRNA modifier. The
substrate recognition and binding of the Elongator is mainly mediated by its catalytic Elp3 subunit. In
this chapter, we describe protocols to generate fluorescently labeled RNAs and outline the principles
underlying electrophoretic mobility shift assays (EMSA) and microscale thermophoresis (MST). These
two methods allow qualitative and quantitative examinations of the binding affinity of various tRNAs
toward the homologs of Elp3 from various organisms. The rather qualitative results from EMSA analyses
can be nicely complemented by MST measurements allowing precise determination of the dissociation
constant (KD). We also provide detailed notes for users to mitigate potential ambiguities and technical
pitfalls during the procedures.

Key words Biophysical measurements, In vitro transcription, Fluorescent, Nonradioactive isotope

labeling, tRNA, Elongator, EMSA, MST

1 Introduction

Currently, more than 170 modifications of RNA nucleotides have

been discovered, and they greatly expand the capacity of nucleic
acids by a variety of chemically added functional moieties [1]. Mod-
ifications occur on various RNAs, such as tRNAs, rRNAs, or
mRNAs. They affect RNA stability, secondary structures, base pair-
ing, and contribute to the biological functions of individual RNAs
[2]. For instance, the modifications on the 34th or 37th position in
the tRNA anticodon stem loop are crucial for fine-tuning transla-
tion [3]. In detail, these modifications provide additional bonds
between the tRNA and its cognate or near-cognate codon in the
ribosome during the decoding process.

Ren-Jang Lin (ed.), RNA-Protein Complexes and Interactions: Methods and Protocols,
Methods in Molecular Biology, vol. 2666, https://doi.org/10.1007/978-1-0716-3191-1_3,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

29
30 Andrzej Chramiec-Gła˛bik et al.

Although the Elongator complex was originally annotated as a

protein acetyltransferase [4], it is now recognized as the enzyme
responsible for the 5-carboxy-methyl-uridine (cm5U34) modifica-
tion of “wobble uridines” [5, 6]. The evolutionarily conserved
Elongator complex exists in all eukaryotes, harboring two copies
of each of its six subunits (Elp1–6). Elp3, the catalytic center,
accommodates the tRNA substrates and catalyzes the reaction
with the support of other requisite subunits [7, 8]. Interestingly,
Elp3 homologs are also present in some bacteria, viruses, and
archaea, while the other Elongator subunits are absent from their
genomes [6]. Purification of the eukaryotic Elongator is difficult,
and it is hard to obtain sufficient amounts of purified complex for
biochemical analyses. On the other hand, archaeal and bacterial
Elp3 proteins show much better solubility and can be efficiently
produced in heterologous organisms, which facilitates biochemical
studies of the underlying tRNA modification reaction [6, 9, 10].
To understand RNA substrate selectivity and specificity of the
protein, several standard methods for interaction studies can be
performed, such as filter binding analysis [11], fluorescent polari-
zation [12], Biacore/surface plasmon resonance [13], isothermal
titration calorimetry (ITC) [14, 15], electrophoretic mobility shift
assay (EMSA) [7, 16], and ultraviolet (UV) cross-linking and
immunoprecipitation (CLIP) [17]. ITC is a biophysical method
that measures the exchange of heat (e.g., heat generation or absorp-
tion), when a molecule comes in contact with its binding partner
[14]. However, ITC requires large quantities (i.e., several milli-
grams of purified protein and the RNA of interest) and is thus a
considerable material-consuming method. On the other hand,
EMSA requires relatively small reaction volume compared to ITC.
In EMSA, the reaction usually is prepared in less than 20 μL reac-
tion volumes and requires less sample [16]. The principle of EMSA
is based on the separation of the samples via a polymerized gel
under native non-denaturing conditions. The free nucleic acid
and the nucleic acid-protein complex show different mobilities as
the former runs faster and the latter migrates slower. The mobility
retardation of the nucleic acid-protein complex is contributed by
many factors, such as an increase in mass or a decrease in charge
density. EMSA can determine affinities and binding stoichiometries
that involve multiple binding sites or cooperative binding events.
Furthermore, to enhance the detection sensitivity and quantitative
analysis, the nucleic acid molecule is predominantly radioisotope
labeled with 32P using T4 polynucleotide kinase. Recently, the use
of multifluorescence-labeled DNA in EMSA has been widely
applied [9, 18]. Additionally, the high sensitivity of fluorescence
labeling can be easily measured using an imaging system without
safety requirements related to radioactive substances. CLIP is a
relatively recent technique that combines the in vivo UV
cross-linking and immunoprecipitation and followed by
Biophysical Methods to Measure tRNA and Modifying Enzyme Interaction 31

high-throughput sequencing. Recently, several approaches have

been implemented to improve the resolution and provide kinetic
parameters for RNA-protein interaction, such as enhanced CLIP
[19] and kinetic KIN-CLIP [20], respectively. The methods enable
the transcriptome-wide analysis of RNA-protein interactions with
high positional resolution and specificity. While ITC and EMSA can
reveal in vitro interactions, CLIP globally maps the in vivo protein-
RNA interaction sites [21]. For instance, a recent study employed
CLIP to identify the RNA binding sites of YTHDC1, a N6-methy-
ladenosine (m6A) binder, and used ITC to reveal the sequence
selectivity of YTHDC1 [22].
Apart from these gold standard methods mentioned above, we
use a relatively newly developed method called microscale thermo-
phoresis (MST) [23]. This method can be employed to measure
molecular interactions as a function of both temperature and bind-
ing partner concentration. In detail, an infrared source is used to
locally increase the temperature of the sample and create a thermo-
optical gradient. This induces two parallel effects, namely
(a) temperature-related intensity change of fluorescence (TRIC)
and (b) thermophoresis as the molecules move through the tem-
perature gradient [24]. The technique allows to monitor and ana-
lyze the state of molecules, including unbound and RNA-bound
state. The principle is based on the temperature-dependent fluores-
cence changes due to molecular size, charge, and conformation of
biomolecules. These changes in turn enable a quantitative measure-
ment of binding affinity. Furthermore, MST measurements are
performed in aqueous solutions, which can match “native”
biological conditions. By using fluorescently labeled samples, the
required sample quantity for the MST measurements is further
reduced. In addition, MST has no restrictions on molecular mass
and is, therefore, suited even for large molecular assemblies, like
ribosomes [25].
A bacteriophage RNA polymerase-driven in vitro transcription
reaction is a standard method [26, 27] to produce the RNA of
interest for these kinds of interaction assays. The T7, T3, and SP6
phage RNA polymerases are commonly used for this purpose.
Because each RNA polymerase has its own specific promoter
sequence, the DNA template should consist of the enzyme-specific
promoter sequence followed by the DNA-encoded sequence of
interest. The template can be further cloned into a vector backbone
(e.g., pUC plasmids) to preserve the gene and allow large-scale
template amplification in bacteria. When the DNA template-
containing plasmid is used for the in vitro transcription, the precise
length of the RNA product needs to be defined by DNA lineariza-
tion. The principle of in vitro transcription is to synthesize RNA in
the 5′!3′ direction while incorporating unlabeled, radiolabeled, or
other modified ribonucleotides depending on the downstream
applications. During a “run-off reaction,” the T7 RNA polymerase
Another Random Scribd Document
with Unrelated Content
und werden vollkommener; aber wann? Wenn wir das Weib tiefer
und gründlicher verstehen lernen. Denk an die üppigen Perser: sie
haben ihre Frauen zu Sklavinnen gemacht, und was ist das Ergebnis?
Sie haben kein Verständnis für das Gefühl des Schönen — dieses
unendliche Meer geistiger Genüsse. Kein Funke schlägt aus ihrem
Herzen empor beim Anblick der Göttin des Praxiteles; ihre Seele
spricht nicht begeisterungsvoll mit der unsterblichen Seele des
Marmors, und kein verständnisvoller Laut tönt ihr aus ihm entgegen.
Was ist das Weib? — Die Sprache der Götter. Wir wundern uns über
das milde heitere Haupt des Mannes; aber wir glauben nicht das
Ebenbild der Götter in ihm zu sehen; das sehen wir im Weibe und
bewundern es im Weibe, und in ihm erst bewundern wir die Götter.
Sie ist die Poesie! sie ist der Gedanke, wir dagegen sind bloß seine
Verkörperung in der Wirklichkeit. Der Eindruck von ihr glüht in
unserer Seele, und je stärker und je umfassender und größer die
Wirkung ist, die er auf uns ausübt, um so edler und schöner werden
wir. Solange das Bild noch im Kopfe des Künstlers weilt, sich
unkörperlich in ihm formt und gestaltet, ist es — ein Weib; sobald es
sich materialisiert und greifbare Gestalt annimmt, wird es zum —
Manne. Warum strebt aber dann der Künstler mit so unersättlicher
Begierde danach, seine unsterbliche Idee in grobe Materie zu
verwandeln und sie unseren gemeinen Sinneswerkzeugen zu
unterwerfen? Weil er von den hohen Gefühlen geleitet wird — von
dem Wunsche, die Gottheit der Materie einzuverleiben und den
Menschen wenigstens einen Teil von der unendlichen Welt seines
Inneren zugänglich zu machen, d. h. das Weib im Manne zu
verkörpern. Und wenn das Auge eines Jünglings, dessen Herz
glühend und verständnisvoll für die Kunst schlägt, zufällig auf das
unsterbliche Bild des Künstlers fällt, — was sucht es, was ergreift es
in ihm? Sieht es etwa die Materie in ihm? Nein, sie verschwindet,
und er erblickt die grenzenlose, unendliche, unkörperliche Idee des
Künstlers vor sich. Wie erklingen da die Saiten seiner Seele, welch
lebendige Lieder ertönen in seinem Inneren! Wie deutlich und
lebendig spricht, wie auf den Ruf der Heimat, das Vergangene, das
unwiederbringlich dahin ist, und die unabwendliche Zukunft in ihm!
Wie unkörperlich umarmt seine Seele die göttliche Seele des
Künstlers! Wie verschmelzen ihre Geister in einem unaussprechlichen
Kusse der Seelen! Was wären die hohen Tugenden des Mannes,
wenn sie nicht geschmückt und nicht geformt würden durch die
milden sanften Tugenden des Weibes? Sein Mut, seine Festigkeit,
seine stolze Verachtung des Lasters würden sich in Barbarei
verwandeln. Raube der Welt das Licht — und die bunte Vielfältigkeit
der Farben fällt dahin; Himmel und Erde verschwimmen und gehen
in der Finsternis unter, die noch dunkler ist als die Gestade des
Hades. Was ist die Liebe? — Die Heimat der Seele, die hehre
Sehnsucht des Menschen nach der Vergangenheit, in der der reine
Ursprung seines Lebens verborgen liegt, wo alles noch den
unaussprechlichen, unverwischbaren Stempel kindlicher Unschuld
trägt und wo uns alles heimatlich berührt. Und wenn die Seele
versinkt im ätherischen Schoße der weiblichen Seele, wenn sie in ihr
ihren Vater — den ewigen Gott — und ihre Brüder, d. h. Gefühle und
Erscheinungen, die keines irdischen Ausdruckes fähig sind, findet —
was geschieht dann mit ihr? Dann tönen in ihr die alten Klänge
wider, dann gedenkt sie des früheren paradiesischen Lebens am
Busen Gottes, und sie setzt es fort bis in die Unendlichkeit.“
Das begeisterte Auge des Weisen blickte starr und unbeweglich
vor sich hin: vor ihnen stand Alkinoe, die während ihres Gespräches
unbemerkt eingetreten war. Auf ein Götterbild gestützt, schien sie
völlig in stumme Aufmerksamkeit versunken, und ihr herrliches
Gesicht belebte häufig ganz plötzlich der Ausdruck einer göttlichen
Seele. Die marmorweiße Hand, durch die die blauen, von
himmlischer Ambrosia durchfluteten Adern hindurchschienen,
schwebte frei in der Luft; der schlanke, von den purpurroten
Bändern des Beinharnischs umschlungene Fuß, den sie einen Schritt
vorgesetzt hatte, hatte die neidische Hülle abgestreift und schien
kaum die niedrige Erde zu berühren; der hohe göttliche Busen
wogte, gespannt von unruhigen Seufzern, auf und ab, und das
Gewand, das die beiden durchsichtigen Wolken des Busens nur halb
verdeckte, bebte und fiel in herrlichen malerischen Linien auf den
Fußboden herab. Es schien, als ob der dünne lichte Äther, in dem
sich die Himmelsbewohner baden, durchflutet von einer rosigen und
bläulichen Flamme, die sich in unendlichen, in tausend Farben
spielenden Strahlen zerstreut, für die es auf Erden keine Namen gibt,
und in denen ein duftenden Meer eines unbegreiflichen Wohllautes
wogt — es schien, als ob dieser Äther sichtbare Form angenommen
hätte und, indem er nun vor ihnen schwebte, die herrliche Gestalt
des Menschen noch verklärte und vergöttlichte. Die nachlässig
zurückgeworfenen Locken umdrängten schwarz wie die dunkle
beseelte Nacht ihre lilienreine Stirn und fielen in dunklen Kaskaden
auf die leuchtenden Schultern herab. Die Blitze, die ihren Augen
entsprühten, schienen ihre ganze Seele zu offenbaren. Nein, selbst
die Königin der Liebe war nie so schön, nicht einmal in dem
Augenblick, als sie so wunderbar dem Schaum der jungfräulichen
Wellen entstieg.
Erstaunt und in ehrfurchtsvoller Andacht warf sich der Jüngling
der stolzen Schönen zu Füßen, und eine heiße Träne, die dem Auge
der sich über ihn beugenden Halbgöttin entstieg, tropfte auf seine
brennenden Wangen.
Fragmente
Gedichte und poetische Versuche
Sturm
„ W arum so trüb?“ — „Einst war ich heiter,“
Sag’ ich zu meiner Lust Genossen.
„Ich hab’ mein Herz dem Schmerz erschlossen;
Die Freude starb: ich lebe weiter.
Jung war ich, und mein heller Blick
hat Trauer nicht und Mißgeschick
Gekannt; jetzt welkt die Jugend hin,
Stirbt wie der Herbst, und ich verblute
Gleich ihm. Nie wird mir froh zumute.
Die Freude lockt nicht meinen Sinn.“
Die Freunde lachen: „Was du nur
Zu weinen hast! Das Wetter ist
So heiter klar, und die Natur
Nicht halb so trüb, wie du es bist.“
Und ich: „Mir gilt das alles nichts.
Ob Tag zu Tag und Jahr sich türmt,
Ob’s hell, ob’s dunkel ist, was ficht’s
Mich an, wenn mir’s im Herzen stürmt.“ —
 Albumblatt

D as Licht verliert im Auge des Träumers schnell seine Wärme. Er

findet die Hoffnungen, die ihn belebten, unerfüllt, seine
Erwartungen unbefriedigt, und die Glut des Genießens verraucht
in seinem Herzen ... Er befindet sich in einem Zustande der Starrheit
und Leblosigkeit. Wie glücklich ist er, wenn er den Wert der
Erinnerungen vergangener Tage erkennt: der Tage einer glücklichen
Kindheit, da er die keimenden Zukunftsträume von sich warf und
seine Freunde verließ, die ihm von ganzem Herzen ergeben waren.
Hans Küchelgarten

Eine Idylle
in ** Bildern
von
W. Alow
1827

Deutsch von Ulrich Steindorff

D as vorliegende Werk hätte nie das Licht der Welt erblickt, wenn
nicht besondere Umstände, die nur für den Verfasser von
Bedeutung sind, die Veranlassung dazu gegeben hätten. Dies
Werk ist eine Frucht seiner achtzehnjährigen Jugend. Wir haben
nicht die Absicht, hier ein Urteil über die Vorzüge oder Mängel dieser
Dichtung abzugeben — das überlassen wir dem Publikum — wir
wollen nur bemerken, daß viele von den Bildern dieser Idylle leider
verloren gegangen sind; sie haben wahrscheinlich das Band
zwischen den nun unverbunden dastehenden Teilen gebildet und die
Zeichnung des im Mittelpunkt stehenden Charakters vollendet. Wir
rechnen es uns indessen zum Verdienst an, daß wir dem Publikum,
soweit dies möglich war, Gelegenheit gaben, das Werk eines jungen
Talentes kennen zu lernen.
Erstes Bild
E s tagt. Das Dorf taucht aus dem Dämmerdunst
Mit seinen Häusern, seinen Gärten. Alles liegt
In hellem Licht. Der Glockenturm erglänzt
Wie lauter Gold, und auf dem alten Zaun
Tanzt froh ein Sonnenstrahl. Die Silberflut
Gleicht einem Zauberspiegel, der getreu
Das Konterfei von Zaun und Gärtchen gibt.
Und nichts hält Ruhe in dem Silberspiegel.
Blau wölbt der Himmel sich; die Wolken ziehn
Wie Wellen hin, und flüsternd rauscht der Wald.
Dort, wo das Ufer weit ins Meer sich wagt,
Da steht behaglich unter Lindenschatten
Ein Pfarrhaus, schon jahrzehntelang bewohnt
Von seinem greisen Herrn und arg verfallen.
Das Dach geworfen und der Schornstein schwarz,
Von blüh’ndem Moos bedeckt das Mauerwerk;
Die Fenster windschief. Aber immer ist
Das Häuschen traulich nett. Um keinen Preis
Der Welt wär’ es dem Alten feil. — Dort steht
Die Linde, sein geliebter Ruheplatz.
Auch sie ist alt. Doch Jugendfrische weht
Rings von den Rosenbäumen. Vögel nisten
In ihrem Dunkel und erfüllen Garten
Und Haus mit ihrer Lieder frohem Schall.
— Weil ihn der Schlaf die ganze Nacht gemieden,
Ging schon vorm Morgengraun der Pfarrer, hier
Ein wenig in der Frische noch zu schlummern.
Im alten Lehnstuhl unterm Lindendach
Schläft er. Der sanfte Wind kühlt sein Gesicht
Und spielt voll Keckheit mit den grauen Haaren.

Wer ist die Schöne, die mit Blicken

Ihm naht, in denen alle Glut,
Des Morgens ganze Frische ruht,
Und vor ihn tritt? Welch ein Entzücken,
Wi i it lili iß H d
Wie sie mit lilienweißer Hand
Ihn sanft berührt, um ihn zu wecken,
Bemüht, ihn ja nicht zu erschrecken.
Doch eh’ er aus dem Schlaf sich fand
Zur Welt, sprach er, die Lider kaum
Geöffnet, leise wie im Traum:

„Du wunder-, wunderbarer Gast,

Der du mein Heim besuchet hast,
Warum füllt Kummer mich und schwillt
Durch meine Seele. Was bewegt
Mich Greisen denn dein Engelsbild
So tief, so seltsam tief, und regt
Den Sinn mir auf? Sieh mich und schilt,
Schilt nicht: mein Leib ist schwach und alt
Und allem, was da lebt, längst kalt.

Seit ich mich tot in mir verscharrte,

Ist’s Ruhe nur, auf die ich warte,
Die ich begehre immerfort.
Ihr gilt mein Denken, gilt mein Wort.
Und nun kommst du, du Junge, mir
Zu Gaste, lockst mich heiß zu dir?
Ach nein, aus deinem lichten Munde
Flammt einer neuen Hoffnung Kunde.
Rufst du zum Himmel mich? Zur Stunde
Bin ich bereit. Allein mir fehlt
Die Würde. Meine Sündenlast
Ist groß. Ich war in dieser Welt
Ein arger Streiter und gehaßt
Von Hirt und Herde. Grausamkeit
War mir nicht fremd. Allein ich schwor
Den Teufel ab, und ich verlor
Zur Buße keinen Tag, allzeit
Entsühnend die Vergangenheit.“

Voll schwerer Sorge und verwirrt

 o sc e e So ge u d e t
Fragt sie sich bang: „Soll ich’s ihm sagen, —
Wer weiß, wohin die Träume ihn verschlagen, —
Sag’ ich ihm, daß er phantasiert?“
Doch Nebel des Vergessens hängt
Um ihn, den neuer Schlaf umfängt.
Sie neigt sich über ihn, verstohlen.
Wie sanft er schläft, wie still er ruht!
Kaum merklich hebt beim Atemholen
Die Brust sich. Licht in Ätherflut
Hält ihn ein Engel in der Hut,
Und paradiesisch Lächeln flicht
Sich leuchtend um sein Angesicht.

Nun öffnet er die Augen: „Wer,

Wer ist’s? — Luise? — Seltsam, ach;
Mir träumte — —, du, wo kommst du her?
Bist, Wildfang, du so früh schon wach?
Noch liegt der Tau. — Es nebelt schwer.“ —

„Großvater, nein, ’s ist hell und klar.

Im Walde blitzt das Sonnenlicht.
Und schon am frühsten Morgen war
Es heiß wie jetzt. Es regt sich nicht
Ein Blatt. — Weißt du, warum ich kam?
Es gibt ein Fest. Wir feiern heut.
Der alte Geiger Lodelham
Und auch der Fritz sind längst bereit.
Erst kommt die Kahnfahrt bis zur Mittagszeit
Und dann — —; ach, wenn nur Hans — —!“ Den Greis
Umspielt ein weises Lächeln. Still
Hört er, was sie erzählen will,
Das sorglos junge Blut. „Ich weiß,
Großväterchen, nur du hast Macht,
Ein bitter großes Weh zu bannen.
Mein Hans ist krank. Bald in der Nacht
Und bald am Tag schleicht er von dannen
Zum dunklen Meer. Nichts ist ihm recht,
Nichts freut ihn mehr. Wenn man ihn fragt,
Dann hört er gar nicht, was man sagt.
Er spricht nur mit sich selbst. So schlecht,
So elend sieht er aus. Wenn ihn sein Schmerz
Noch lange quält, geht er zugrund.
‚Zugrund‘, wie zittert, wenn mein Mund
Das harte Wort gebraucht, mein Herz.
Meinst du, daß er vielleicht mit mir
Nicht mehr zufrieden ist, daß er
Mich nicht mehr liebt? Das träfe schwer
Und hart wie Stahl mein Herz. Sag’s mir,
Du Engelsguter!“ — Und sie schlang
Die Arme fest um ihn. Kaum ging
Ihr Atem, als sie an ihm hing
In ihrer Liebe so verwirrt und bang.
Als sich die Träne ihr ins Auge stahl,
Wie war sie schön in ihrer Qual.

„Gib Ruh’, mein Kind, nicht weinen, nein.

Schämst du dich nicht?“ Der Pfarrer mühte
Sich tröstend um sie. „Gottes Güte
Wird dir Geduld und Kraft verleihn.
Wenn du ihn innig bittest, wirst
Du auch bei ihm Erhörung finden.
Hans lebt ja nur für dich. Du irrst.
Du mußt die Zweifel überwinden.
Du darfst dir nicht mit solchen leeren
Gedanken deine Ruhe stören.“ — —

Und als er noch der weinenden Luise

Zuspricht, die an die welke Brust sich lehnt,
Da bringt die alte Gertrud schon den Kaffee,
Den heißen, bernsteinklaren, den der Greis
So gern im Freien nahm. Er liebte es,
Die Weichselpfeife dann dabei zu rauchen.
So stieg denn bald der Rauch in klaren Ringen
So stieg denn bald der Rauch in klaren Ringen.
Luise fütterte gedankenschwer
Den Kater, der mit lautem Schnurren,
Vom süßen Duft gelockt, sie lang umstrichen.
Der Greis erhob sich vom geblümten Sessel
Aus Väterzeit, sprach sein Gebet und drückte
Der Enkelin die Hand. Dann zog er sich
Den sonntäglichen, taftnen Schlafrock an,
Den silberschimmernden, und nahm das Käppchen,
Das Hans ihm kürzlich aus der Stadt gebracht
Und ihm geschenkt. So ging er denn gemächlich,
Sich auf Luisens weiße Schulter stützend, —
Hell schlug der Sang der Lerchen himmelwärts —
Ins Feld hinaus. — Wie herrlich war der Tag!
Es ließ ein Wind das Gold der Felder wogen,
Das, überragt von dichten, früchteprangenden
Laubkronen, in der Sonne flimmerte.
Fern dunkelten die grünen Wälder.
Dem regenbogenfarbnen Sommerdunst
Entströmten Fluten wundersamster Düfte.
Die Bienen waren fleißig unterwegs
Und sogen Honig aus den jungen Blüten.
Die Grillen zirpten froh. Und aus der Weite
Klang laut und lauter kräft’ger Rudersang.
Und lichter ward der Wald. Das Tal erschien.
Das frohe Schrein der Herden scholl herauf.
Tief in der Ferne sah man schon das Dach
Vom Haus Luisens winken, sah das Rot
Der Ziegel schimmern, wenn die Sonnenstrahlen
In keckem Tanzspiel blitzend es umhuschten. — —
Zweites Bild
Noch ungeklärt sind die Gedanken,
Die Hans bewegen, und sein Blick
Sieht wirr die Welt des Lebens wanken
Und sucht sein künftiges Geschick. —
In stillem Frieden war die Zeit
Dem Tändelnden vorbeigeflossen;
Noch hatte keine Bitterkeit
Sich in der Seele Unschuld ihm gegossen.
Kind dieser Erdenwelt war er.
Doch ihrer Leidenschaften Brand
War seinem Herzen unbekannt.
Ganz sorglos war und leicht bisher
In Heiterkeit und Glück und Lust
Das Kind beim Spiel der Kinderschar.
Das Böse war noch seiner Brust
Ganz fremd. Ihm blühte wunderbar
Die Welt. — Schon in der frühsten Zeit
Der Kindheit war sein Kamerad
Luise, deren Heiterkeit
Und Milde seinen Lebenspfad
Erhellt. Wenn sie im grünen Kleid
Zu tanzen anfing oder sang,
Dann schoß durchs blonde Ringelhaar
Manch Blitz, der zündend weitersprang.
Ihr rosa Miedertüchlein glitt
Herab. Man sah bei jedem Schritt
Das feine, zarte Füßchenpaar.
Sie war ein Kind, und kindlich war
Ihr Tun. — Im Walde spielte sie
Mit ihm. Sie fingen sich. Dann lief
Sie fort, versteckte sich und schrie
Ihm plötzlich zu, daß er erschreckte.
Sie schwärzte heimlich, wenn er schlief,
Ihm sein Gesicht, und lachend weckte
Sie ihn dann aus dem süßen Schlafe.
Und er, er küßte sie zur Strafe. —

Und Lenz auf Lenz zog hin ins Land.

Die Spiele wollten nicht mehr taugen.
Die gegenseit’ge Keckheit schwand.
Es schwand das Feuer seiner Augen.
Und sie hält Traurigkeit gebannt
Und Schüchternheit. — Ihr, junger Herzen
Verliebte, erste Worte, wart
Gekommen, und es blieben nicht erspart
Die Tage voller süßer Schmerzen.
Was blieb ihm denn zu wünschen weiter,
Wo er Luise bis zur Nacht,
Gefesselt wie von Zaubermacht,
Nicht ließ, ihr treuester Begleiter,
Ihr Schatten, wo sie ging und stand.
Mit innig tiefer Freude sahen
Die Eltern, wie das Glück sich fand,
Und sahen sich nicht satt. Die nahen,
Leidvollen, zweifelvollen Zeiten
hielt noch ein Engel sanft verhüllt den beiden. —

Doch allzubald befiel ein Schmerz,

Ein tiefer, ihn. Matt ward vor Gram
Sein Blick; er starrte himmelwärts
Und war ganz unstet, ach, und wundersam.
Es schien, als suchte stets sein Geist,
Als hegte er geheimen Groll.
Die Seele sehnte sich zumeist
Gedankenschwer und kummervoll. —
Er sitzt und schaut hinab vom Strand
Hinaus aufs Meer wie festgebannt.
Und wenn im Takt die Wellen rauschen,
Scheint einer Stimme er zu lauschen.
———————————
Bald geht er grübelnd durch das Tal,
Die Augen feierlich voll Glanz
Die Augen feierlich voll Glanz,
Wenn bei der Wolken Wirbeltanz
Der Donner grollt, ein Feuerstrahl
Durchs Dunkel zuckt und wilder Regen
Heiß prasselt und mit einemmal
In Strömen rauscht auf allen Wegen.
Bald sitzt er in der Mitternacht
Vor alten Sagen auf und wacht
Und hofft, daß sich die Lettern regen
In ihrer Stummheit, wenn die Seiten
Er wendet, die so tiefe Kunde
Ihm bringen von den grauen Zeiten.
Ins Buch versunken manche Stunde,
Sitzt er und wendet kaum das Haupt.
Wer ihn in dieser schweren Not
Gesehn, der hätte fest geglaubt,
Die Zeit, da er gelebt, sei tot.
Gedanken, wunderbare, hatten
Mit ihrem Zauber ihn gebannt.
Er suchte dunkler Eichen Schatten
Auf seinem Weg durchs Sommerland.
Aus diesen tiefen Schatten sprach
Manch Rätsel, das er nicht verstand,
Und träumend streckte er die Hand
Liebkosend aus und griff darnach. —

Luise ist die ganze Zeit

Allein in ihrem tiefen Kummer.
Ihr Herz ist einzig ihm geweiht.
Sie findet nächtens keinen Schlummer
Und bringt die gleiche Zärtlichkeit
Ihm dennoch stets entgegen, hält
Die zarten Arme um ihn, küßt
Ihn sanft, daß er den Schmerz vergißt,
Bis er der Schwermut neu verfällt.

Schön sind die Stunden, wunderbar,

Sc ö s d d e Stu de , u de ba ,
Wenn ferne Träume ihn umschweben
Und der Gesichte lichte Schar
Ihn fortträgt in ein andres Leben.
Doch, wenn der Seele Land zerstört,
Der stille Erdenfleck vergessen,
Der Scholle nicht sein Herz gehört,
Die schlichten Menschen er vermessen
Nicht achtet, werden Traumgestalten
Auch dann noch froh im Herzen walten? — —

Indessen laßt sein unstet Wesen

Belauschen uns. Macht euch bereit,
Die Rätsel seines Geists zu lösen
In ihrer Mannigfaltigkeit. —
Drittes Bild
Du klassisch schöner Werke klassisch schönes Land!
Des Ruhmes und der Freiheit Land, Athen!
An dich, in wundersamer Gluten Wehn,
Ist meine Seele festgebannt.

Vom Tempel hoch bis hin zu des Piräus Mauern

Ergießen sich und wogen feierliche Massen.
Äschines’ Worte blitzen, donnern und durchschauern,
Der Iliß Wassern gleich, und fassen

Gebietrisch alle wie der laute Sturm der Welle.

Gewaltig ragt empor die Marmorherrlichkeit
Der Parthenon, wo Säule sich an Säule reiht;
Empor Minerva, von des Phidias Stahl geweiht.
Und Zeuxis’ wie Parrhasios’ Pinsel strahlen Helle.

Im Portikus steht göttergleich ein Greis

Und redet weise von der andern Welt;
Sagt, wer für Tugend einst Unsterblichkeit erhält,
Wen Schande trifft und wen der Preis.

Horch! Rohes Tosen mischt sich in das Springbrunnrauschen.

Der Tag ist wach, und dem Theater voll Verlangen
Zu strömt das Volk. Wie Persiens Farben prangen!
Sieh, wie die Tuniken sich bauschen!

Noch eh’ die Leidenschaft des Sophokles verklungen,

Schwirrt Kranz auf Kranz, von den Begeisterten geschwungen.
Von Epikurens Honigmund, dem liebgewohnten,
Enteilt sind Amors Diener, Krieger und Archonten,
Daß ihnen sich die hohe Wissenschaft enthülle,
Wie man Genüsse schlürft und trinkt des Lebens Fülle.
Aspasia kommt! Ihr Blick, vom Wimpernschwarz verbrämt,
Trifft einen Jüngling, und sein Atem stockt verschämt.
Wie heiß die Lippen sind! Wie loht der Rede Glut!
Die schwarzen, losen Locken fallen wie die Nacht
Auf ihrer Schultern Marmorpracht,
Auf ihre Brüste wie die Flut. —

Und jetzt? — Tympane tosen und die Becher klirren.

Bacchantinnen in wilder Raserei, geschmückt
Mit Efeu, stürmen durch den heil’gen Hain in wirren,
Gehetzten Haufen. — Wo? Wohin? — Entrückt, entrückt.

Allein! — Verschwunden ist der Chor.

Und Gram befällt mich neu und Wehe.
Stieg’ doch vom Tal ein Faun empor;
Dräng’ aus des Gartens dunkler Nähe
Mir einer Nymphe Sang ans Ohr!

Ihr Griechen, wunderbarlich habt

Die Welt mit Träumen ihr erfüllt,
In Zauber alles eingehüllt!
Heut ist sie ärmlich, grau, verschabt
Und wohl quadriert, mit Nichts begabt. — —

Doch neue Träume kommen und heben

Und ziehen ihn lockend himmelan
Empor aus der Sorgen Ozean,
hinweg von allem kleinlichen Leben. —
Viertes Bild
Im Land, wo des Lebens Wunderquellen
Entspringen und strahlend rings alles erhellen;
Wo schwer die Nächte vom Ambraduft,
Von Lotossüße geschwängert die Luft;
Wo Räucherwerkwolken die Bläue durchfluten
Und Mangostans Früchte golden gluten;
Wo Kandahars Wiesengrund samten sich breitet;
Wo kühn sich ob allem der Himmel weitet
Und Blüten regnet in üppigem Glanz;
Wo Schwärme von Faltern auffunkeln im Tanz:
Dort sieht mein Blick eine Peri: versunken,
Nichts sehend, nichts hörend; traumestrunken.
Gleich Sonnen leuchtet ihr Augenpaar,
Wie Hemasagara funkelt ihr Haar.
Ihr Atem gleicht dem, den die Lilie haucht,
Wenn die Nacht den Garten in Schlummer taucht
Und im Wind ihre Seufzer von dannen schwingen;
Ihre Stimme den nächtlichen Ton von Syringen,
Dem silbernen Tone, wenn Israfil
Die Flügel schlägt in mutwilligem Spiel;
Dem heimlichen Plätschern des Tschindara-Fluß.
Und ihr Lächeln erst! Und erst ihr Kuß!
Was ist? — Sie hebt sich, ein Hauch, und entschwindet
In Himmeln, wo sie Verwandte findet.
Bleib! Blicke dich um! Bleib! — Taub meinem Schrei,
Verrinnt sie im Regenbogen. — Vorbei!
Erinnrung an sie bleibt und hält
Sich fest; und Duft erfüllt die Welt. —

Bunt war sein Träumen überstrahlt;

Vom Drang der Jugend heiß durchflossen.
Die Hoheit, die sein Herz genossen,
Hat herrlich oft sich abgemalt
Auf seinem Angesicht. Allein,
Was ihn in seinen T ä me ein
Was ihn in seinen Träumerein,
Was die erregte Seele quälte,
Wonach er schrie, wonach er bangte,
In wilder Leidenschaft verlangte,
Als gält’ es, daß er sich vermählte
Der ganzen Welt mit ganzer Lust,
Verstand er nicht. — Voll Staub und Dust,
Von Dumpfheit voll und Schwere fand
Er diese Welt und wirr. Es flog
Sein Herz und schlug und schlug und zog
Ihn hin nach fernem, fernem Land.
Wer sah ihn so? Sein Atem ächzte.
Die Brust ging keuchend auf und nieder.
Stolz funkelte durch seine Lider.
Ach, wie die Seele darnach lechzte,
Am flücht’gen Traum sich festzusaugen.
Ach, welche Feuer in ihm brannten,
Wie ihn die Tränen übermannten,
Das Leben schürend in den heißen Augen. —
Sechstes Bild
Zwei Meilen nur von Wismar liegt das Dorf,
Wo unserer Geschichte Welt, die Welt,
Wo ihre Menschen leben, Grenzen findet.
Das heitre Lünensdorf, so hieß es einst;
Doch weiß ich nicht, ob es noch heut so ist. —
Weit schimmerte dem Wanderer entgegen
Das kleine, weiße Häuschen Wilhelm Bauchs,
Des Musikers, das er vor langer Zeit,
Als er des Pastors Kind zum Weibe nahm,
Erbaut. Es war ein liebes, heitres Haus;
Grün war’s gestrichen; rote Ziegelplatten
Erklirrten hell im Wind. Kastanienbäume
Umstanden es und drängten in die Fenster.
Durch ihre Stämme sah ein Weidenzaun,
Den Wilhelm selbst aus Ruten sich geflochten.
Jetzt rankte sich der Hopfen an ihm hoch.
Vom Fenster zu dem Zaun lief eine Stange,
Behangen mit der Wäsche, die im Glanz
Der heißen Mittagssonne lustig blinkte.
Durch eine Speicherluke drängte sich
Laut girrend eine Taubenschar; es schrien
Die Puter, und mit seinen Flügeln schlagend
Entbot der Hofhahn seinen Morgengruß
Dem Tag und pickte den behäbig bunten Hennen
Die Körner fort. Zwei fromme Ziegen rupften
Das junge Gras. Schon lange stieg der Rauch
In krausen Wolken aus dem Schornstein auf
Zum Himmel, um den Morgendunst zu mehren.
Dort auf der Seite, wo der Mauerputz
Ein wenig abgebröckelt von den grauen Ziegeln,
Dort, wo die alten Bäume Schatten geben,
Stand schon seit frühstem Morgen säuberlich
Gedeckt ein Eichentisch voll guter Dinge:
Radieschen, gelber Käse, eine Dose
In Entenform mit Butter; Wein und Bier,
Der süße Bischof, Zucker, Waffelkuchen
Und dann ein Korb mit leuchtend reifen Früchten:
Himbeeren voller Duft, glashelle Trauben
Und bernsteinfarbne Birnen, blaue Pflaumen
Und rote Pfirsiche in buntem Durcheinander. —
Es war so festlich, denn Herr Wilhelm wollte
Der lieben Frau Geburtstag in dem Kreise
Der Töchter und des alten Pfarrherrn feiern.
Luise kam, doch ihre Schwester Fanny,
Die Jüngere, war fortgeeilt, um Hans
Zu holen, und war noch nicht zurück.
Vermutlich irrte er verträumt umher.
Luise blickte unverwandt zum dunklen Fenster
Im Nachbarhaus empor; lag es doch nur
Zwei Schritt von ihr. — Sie war nicht selbst gegangen,
Damit er nicht den Gram von ihrer Stirn,
Aus ihren Augen keinen Vorwurf läse.
Da wandte Wilhelm sich, Luisens Vater,
Zu ihr und sprach: „Du mußt den Hans mal schelten,
Daß er so lange nicht mehr bei uns war.
Pass’ auf, du hast ihn dir zu sehr verwöhnt.“
Doch sie war um die Antwort nicht verlegen:
„Mir fehlt der Mut, den braven Hans zu tadeln.
Er ist schon ohnedies so bleich und elend.“
„Was, krank, sagst du?“ fiel Mutter Berta ein.
„Es ist nicht Krankheit, nur Melancholie,
Die ihn jetzt plagt, und die wird sehr bald weichen,
Seid ihr einmal vermählt. Ein junger Sproß,
Den halbverdorrt ein Sommerregen trifft,
Fängt plötzlich an zu blühn. — Ist denn die Frau
Nicht Lichtflut für den Mann?“ — „Ein kluges Wort,“
Warf da der Pfarrer ein. „Wenn Gott es will,
Glaubt mir, wird alles noch vorübergehn!“
Er klopfte wieder seine Pfeife aus.
Dann fing er an, mit Wilhelm sich zu streiten;
Sie sprachen von den Tagesneuigkeiten,