Table Of ContentMethods in
Molecular Biology 1724
Christoph Dieterich
Argyris Papantonis Editors
Circular
RNAs
Methods and Protocols
M M B
ethods in olecular iology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
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Circular RNAs
Methods and Protocols
Edited by
Christoph Dieterich
Department of Internal Medicine III, Klaus Tschira Institute for Integrative Computational Cardiology,
University Hospital Heidelberg, Heidelberg, Germany
Argyris Papantonis
Center for Molecular Medicine Cologne (CMMC),
University of Cologne, Cologne, Germany
Editors
Christoph Dieterich Argyris Papantonis
Department of Internal Medicine III Center for Molecular Medicine Cologne
Klaus Tschira Institute for Integrative (CMMC)
Computational Cardiology University of Cologne
University Hospital Heidelberg Cologne, Germany
Heidelberg, Germany
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-7561-7 ISBN 978-1-4939-7562-4 (eBook)
https://doi.org/10.1007/978-1-4939-7562-4
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Preface
Although isolated examples of circularized RNA molecules were already described more
than 20 years ago [1, 2], it was not until quite recently that the prevalence of circular RNAs
(circRNAs) was revealed. The advent of massively parallel sequencing technology allowed
researchers to catalogue circRNAs across eukaryotic and prokaryotic systems (e.g., [3–7]).
Their surprisingly high titers within cells are a result of (1) their circular form that renders
them far less susceptible to degradation by exoribonucleases, and (2) their multiple regions
of origin. circRNAs originate from thousands of different genes active per cell type (both
protein-coding and noncoding), and the final circular RNA molecule may include exonic,
intronic, or exonic and intronic sequences [8].
Despite their abundance, the functions served by circRNAs remain largely enigmatic,
and it is has been suggested that many of them might rather be long-lived transcriptional
by-products [9]. To date, mainly three types of functions have been described. First, their
role as potent miRNA sponges: ciRS-7 constitutes a prime example, carrying >70 r ecognition
sites for human miR-7, thus sequestering this miRNA from intracellular circulation [10, 11],
while circHIPK3 acts as a sponge of multiple miRNAs [12]. Nonetheless, only very few
circRNAs are predicted to be able to carry out such a “sponging” activity. Second, their role
as (post-) transcriptional regulators: For example, particular exon-intron circRNAs have
been described that can regulate the expression levels of their parental genes via RNA-RNA
interactions (which may also involve the U1 snRNA; [13]), via direct competition with pre-
mRNA splicing [9, 14], or even by modulation of t ranscription factor activity [15, 16].
Finally, there exist circRNAs with a role in translation: circRNAs were shown to be transla-
tionally competent and to carry short open reading frames [17, 18]; hence, three recent
studies exemplifying their translatability greatly expand the coding and r egulatory eukary-
otic landscape [15, 16, 19, 20]. Although not a functional aspect per se, one should also note
here that circRNAs show significant potential as biomarkers, for e xample in tissue aging
[21, 22] and in cancer [13, 23].
CircRNA expression is cell type- and tissue-specific and can be largely independent
of the expression level of the linear host gene. Thus, regulation of expression might be
an important aspect with regard to control of circular RNA function. Initial evidence
suggests that circular RNA biogenesis proceeds through RNA hairpin intermediates,
which are modulated by RNA modifications (e.g., A->I editing at flanking inverted
repeats; [24]) and/or RNA-binding proteins (e.g., QKI; [25]). Conceptually, RNA
structure shapes in such a way that the downstream 5’ splice site is close to a 3’ upstream
splice site [26]. This facilitates a back-splicing event leading to a circularized RNA mol-
ecule with different covalent configurations: 3′–5′ linkages, containing only exonic
sequence; 2′–5′ linkages (intronic lariats); or 3′–5′ linkages that contain retained intronic
sequences [27]. There is an active discussion whether circular RNAs predominantly
emerge from direct back-splicing or exon skipping events [9].
Taken together, circRNAs still comprise unexplored territory as regards many of their
basic biogenesis mechanisms and functional implications. The molecular and b ioinformatics
toolkit for studying circRNAs is continuously expanding, and the present volume aims at
v
vi Preface
providing access to well-established approaches for identifying, characterizing, and
manipulating circRNAs in vitro, in vivo, and in silico—and in doing so this compilation of
17 chapters also highlights the breakthroughs and the challenges in this new field of research.
Heidelberg, Germany Christoph Dieterich
Cologne, Germany Argyris Papantonis
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Preface vii
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Contents
Preface............................................................ v
Contributors........................................................ xi
1 Detection and Reconstruction of Circular RNAs from Transcriptomic Data. . . . 1
Yi Zheng and Fangqing Zhao
2 Deep Computational Circular RNA Analytics from RNA-seq Data . . . . . . . . . . 9
Tobias Jakobi and Christoph Dieterich
3 Genome-Wide circRNA Profiling from RNA-seq Data . . . . . . . . . . . . . . . . . . . 27
Daphne A. Cooper, Mariela Cortés-López, and Pedro Miura
4 Analysis of Circular RNAs Using the Web Tool CircInteractome. . . . . . . . . . . . 43
Amaresh C. Panda, Dawood B. Dudekula, Kotb Abdelmohsen,
and Myriam Gorospe
5 C haracterization and Validation of Circular RNA
and Their Host Gene mRNA Expression Using PCR . . . . . . . . . . . . . . . . . . . . 57
Andreas W. Heumüller and Jes-Niels Boeckel
6 Detecting Circular RNAs by RNA Fluorescence In Situ Hybridization . . . . . . . 69
Anne Zirkel and Argyris Papantonis
7 S ingle-Molecule Fluorescence In Situ Hybridization (FISH)
of Circular RNA CDR1as . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Christine Kocks, Anastasiya Boltengagen, Monika Piwecka,
Agnieszka Rybak-Wolf, and Nikolaus Rajewsky
8 A Highly Efficient Strategy for Overexpressing circRNAs . . . . . . . . . . . . . . . . . 97
Dawei Liu, Vanessa Conn, Gregory J. Goodall, and Simon J. Conn
9 C onstructing GFP-Based Reporter to Study Back Splicing
and Translation of Circular RNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Yun Yang and Zefeng Wang
10 Northern Blot Analysis of Circular RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Tim Schneider, Silke Schreiner, Christian Preußer,
Albrecht Bindereif, and Oliver Rossbach
11 Nonradioactive Northern Blot of circRNAs. . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Xiaolin Wang and Ge Shan
12 Characterization of Circular RNA Concatemers. . . . . . . . . . . . . . . . . . . . . . . . . 143
Thomas B. Hansen
13 Characterization of Circular RNAs (circRNA) Associated
with the Translation Machinery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Deniz Bartsch, Anne Zirkel, and Leo Kurian
14 Synthesis and Engineering of Circular RNAs. . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Sonja Petkovic and Sabine Müller
ix
x Contents
15 Preparation of Circular RNA In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Naoko Abe, Ayumi Kodama, and Hiroshi Abe
16 Discovering circRNA-microRNA Interactions from CLIP-Seq Data . . . . . . . . . 193
Xiao-Qin Zhang and Jian-Hua Yang
17 Identification of circRNAs for miRNA Targets by Argonaute2 RNA
Immunoprecipitation and Luciferase Screening Assays. . . . . . . . . . . . . . . . . . . . 209
Yan Li, Bing Chen, and Shenglin Huang
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Contributors
Kotb Abdelmohsen • Laboratory of Genetics and Genomics, National Institute on
Aging-Intramural Research Program, National Institutes of Health, Baltimore, MD,
USA
hiroshi Abe • Department of Chemistry, Graduate School of Science, Nagoya University,
Nagoya, Japan
nAoKo Abe • Department of Chemistry, Graduate School of Science, Nagoya University,
Nagoya, Japan
deniz bArtsch • Cologne Excellence Cluster on Cellular Stress Responses in Aging-
Associated Diseases, University of Cologne, Cologne, Germany; Institute for
Neurophysiology, University of Cologne, Cologne, Germany; Laboratory for Developmental
and Regenerative RNA Biology, Center for Molecular Medicine (CMMC), University of
Cologne, Cologne, Germany
Albrecht bindereif • Institute of Biochemistry, Department of Biology and Chemistry,
Justus Liebig University Giessen, Giessen, Germany
Jes-niels boecKel • Institute for Cardiomyopathies, Division of Cardiology, Department
of Internal Medicine III, University Clinic Heidelberg, Heidelberg, Germany
AnAstAsiyA boltengAgen • Systems Biology of Gene-Regulatory Elements, Berlin Institute
for Medical Systems Biology (BIMSB), Max Delbrück Center (MDC) for Molecular
Medicine in the Helmholtz Association, Berlin, Germany
bing chen • Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences
and Shanghai Medical College, Fudan University, Shanghai, China
simon J. conn • Centre for Cancer Biology, An Alliance Between University of South
Australia and SA Health, Adelaide, SA, Australia
VAnessA conn • Centre for Cancer Biology, An Alliance Between University of South
Australia and SA Health, Adelaide, SA, Australia
dAphne A. cooper • Department of Biology, University of Nevada, Reno, Reno, NV,
USA
mArielA cortés-lópez • Department of Biology, University of Nevada, Reno, Reno, NV,
USA
christoph dieterich • Section of Bioinformatics and Systems Cardiology, Department of
Internal Medicine III, Klaus Tschira Institute for Integrative Computational
Cardiology, University Hospital Heidelberg, Heidelberg, Germany; German Center for
Cardiovascular Research (DZHK)—Partner site Heidelberg/Mannheim, Heidelberg,
Germany
dAwood b. dudeKulA • Laboratory of Genetics and Genomics, National Institute on
Aging-Intramural Research Program, National Institutes of Health, Baltimore, MD,
USA
gregory J. goodAll • Centre for Cancer Biology, An Alliance Between University of
South Australia and SA Health, Adelaide, SA, Australia
myriAm gorospe • Laboratory of Genetics and Genomics, National Institute on Aging-
Intramural Research Program, National Institutes of Health, Baltimore, MD, USA
xi
