Table Of ContentMethods in
Molecular Biology 1095
Christoph Arenz Editor
miRNA
Maturation
Methods and Protocols
M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651
miRNA Maturation
Methods and Protocols
Edited by
Christoph Arenz
Institute for Chemistry, Humboldt-Universität zu Berlin, Berlin, Germany
Editor
Christoph Arenz
Institute for Chemistry
Humboldt-Universität zu Berlin
Berlin, Germany
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-62703-702-0 ISBN 978-1-62703-703-7 (eBook)
DOI 10.1007/978-1-62703-703-7
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Pref ace
The last decade has seen a dramatic development in the fi eld of micro RNAs (miRNAs).
Starting with a small set of small noncoding regulatory RNAs in D. melanogaster and C.
elegans miRNAs are now regarded as important regulatory components in many eukaryotic
species including humans. The number of reported miRNAs exceeds 1,000 and many of
these miRNAs have been implicated in important biological processes and human diseases.
In this volume of “Methods in Molecular Biology” we concentrate on the pathway of
microRNA maturation. After its synthesis, the primary miRNA transcript (pri-miRNA) is
cleaved by an endonuclease called Drosha to yield the precursor miRNA (pre-miRNA).
After being transported to the cytoplasm, the latter is further cleaved by another nuclease
called Dicer to yield the mature double-stranded miRNA. This mature miRNA consists of
the active guide strand which remains bound to the miRNA effector complex, whereas the
passenger strand is degraded. These few relatively simple steps (although the cellular
machinery promoting this pathway is rather complex) are common to most miRNAs and
thus are highly important to study. In this book we concentrate on three important aspects
of this maturation pathway: First of all, we give an overview over the current knowledge of
the pathway of miRNA maturation (Chapter 1 ) and how this pathway relates to human
disease (Chapter 2 ). In the third review, established and novel approaches to manipulate
miRNA maturation and activity are described.
During the last 5 years many correlations between the levels of certain miRNAs and
various human malignancies have been identifi ed, and in some cases the causative role of
certain miRNAs in disease formation has been shown. Specifi c miRNAs can regulate certain
proteins causing or preventing disease formation. These miRNA–disease correlations call
for easy and reliable methods for quantifying specifi c miRNA species from biological sam-
ples, a topic which is only partially covered in this book. While miRNAs today have some-
how lost their “exotic” touch with many standard methods established in laboratories
around the world, novel methods with the potential to expand the view on miRNAs are
currently being developed. Such innovation can arise from improving existing methods or
by the development of completely new methods allowing for addressing questions other
than previous ones.
In this book, established methods (qRT-PCR of miRNA maturation components,
qRT-PCR of miRNAs) are completed with fl uorescent and nonfl uorescent methods for
homogenous assays of Dicer-mediated miRNA maturation or an in vivo assay for Drosha
activity. Since miRNAs appear to be emerging drug targets, anti-miRs are already commer-
cially available. Less common is the use of biologically stable PNA as anti-miRs or even
miRNA maturation inhibitors, doubtlessly adding to the arsenal of current oligonucleotide
approaches to manipulate miRNA activity. Apart from oligonucleotides affecting miRNA
activity, which are already under clinical investigation, there is a huge interest in fi nding
v
vi Preface
small molecules with equivalent effects. Specially adapted luciferase assays have proven as
powerful tools for identifying candidate small molecule miRNA effectors.
I am convinced that the collection of methods of this book is suitable to widen the view
on miRNA as biological mediators and potential drug targets and thus stimulate future
research in this highly dynamic and thrilling topic.
Berlin, Germany Christoph Arenz
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I REVIEWS
1 The Pathway of miRNA Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Michael Sand
2 MicroRNA Maturation and Human Disease . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Marlen Hesse and Christoph Arenz
3 Approaches to the Modulation of miRNA Maturation. . . . . . . . . . . . . . . . . . . 27
Valerie T. Tripp, Jaclyn R. McKenna, and Douglas D. Young
PART II METHODS
4 Expression Profiling of Components of the miRNA
Maturation Machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Michael Sand and Marina Skrygan
5 Primary MicroRNA Processing Assay Reconstituted
Using Recombinant Drosha and DGCR8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Ian Barr and Feng Guo
6 In Vivo Processing Assay Based on a Dual-Luciferase
Reporter System to Evaluate DROSHA Enzymatic Activity. . . . . . . . . . . . . . . 87
Vera Bilan, Danilo Allegra, Florian Kuchenbauer, and Daniel Mertens
7 Assaying Dicer-Mediated miRNA Maturation
by Means of Fluorescent Substrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Marlen Hesse, Brian P. Davies, and Christoph Arenz
8 A Fluorescence Correlation Spectroscopy-Based Enzyme Assay
for Human Dicer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Eileen Magbanua and Ulrich Hahn
9 Detection of microRNA Maturation Using Unmodified
pre-microRNA and Branched Rolling Circle Amplification . . . . . . . . . . . . . . . 109
Saskia Neubacher and Christoph Arenz
10 Quantitative RT-PCR Specific for Precursor
and Mature miRNAs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Hannah Zöllner, Stephan A. Hahn, and Abdelouahid Maghnouj
vii
viii Contents
11 Cellular MicroRNA Sensors Based on Luciferase Reporters. . . . . . . . . . . . . . . 135
Colleen M. Connelly and Alexander Deiters
12 Identification of Inhibitors of MicroRNA Function
from Small Molecule Screens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Colleen M. Connelly and Alexander Deiters
13 Inhibition of miRNA Maturation by Peptide Nucleic Acids. . . . . . . . . . . . . . . 157
Concetta Avitabile, Enrica Fabbri, Nicoletta Bianchi, Roberto Gambari,
and Alessandra Romanelli
14 Molecular Methods for Validation of the Biological Activity
of Peptide Nucleic Acids Targeting MicroRNAs . . . . . . . . . . . . . . . . . . . . . . . 165
Eleonora Brognara, Enrica Fabbri, Nicoletta Bianchi, Alessia Finotti,
Roberto Corradini, and Roberto Gambari
15 Lentiviral Overexpression of miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Hannah Zöllner, Stephan A. Hahn, and Abdelouahid Maghnouj
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Contributors
DANILO ALLEGRA • Internal Medicine III, University of Ulm, Ulm, Germany; German
Cancer Research Center (DKFZ), Heidelberg, Germany
CHRISTOPH ARENZ • Institute for Chemistry, Humboldt-Universität zu Berlin ,
Berlin , Germany
CONCETTA AVITABILE • Dipartimento delle Scienze Biologiche, Università di Napoli
“Federico II” , Napoli , Italy
IAN BARR • Department of Biological Chemistry, University of California Los Angeles,
Los Angeles, CA, USA
NICOLETTA BIANCHI • Department of Life Sciences and Biotechnology , Ferrara University ,
Ferrara , Italy ; Laboratory for the Development of Pharmacological and
Pharmacogenomic Therapy of Thalassaemia, Biotechnology Center , Ferrara University ,
Ferrara , Italy
VERA BILAN • Internal Medicine III, University of Ulm , Ulm , Germany
ELEONORA BROGNARA • Department of Life Sciences and Biotechnology , Ferrara University ,
Ferrara , Italy
COLLEEN M. CONNELLY • Department of Chemistry , North Carolina State University ,
Raleigh , NC , USA
ROBERTO CORRADINI • Department of Organic Chemistry , Parma University , Parma , Italy
BRIAN P. DAVIES • Institute for Chemistry, Humboldt Universität zu Berlin, Germany
ALEXANDER DEITERS • Department of Chemistry , North Carolina State University , Raleigh ,
NC , USA
ENRICA FABBRI • Department of Life Sciences and Biotechnology , Ferrara University ,
Ferrara , Italy
ALESSIA FINOTTI • Laboratory for the Development of Pharmacological and
Pharmacogenomic Therapy of Thalassaemia, Biotechnology Center , Ferrara University ,
Ferrara , Italy
ROBERTO GAMBARI • Department of Life Sciences and Biotechnology , Ferrara University ,
Ferrara , Italy ; Laboratory for the Development of Pharmacological and
Pharmacogenomic Therapy of Thalassaemia, Biotechnology Center , Ferrara University ,
Ferrara , Italy
FENG GUO • Department of Biological Chemistry , University of California Los Angeles ,
Los Angeles, CA , USA
STEPHAN A. HAHN • Labor für Molekulare Gastroenterologische Onkologie (MGO),
Zentrum für Klinische Forschung (ZKF), Ruhr Universität Bochum , Bochum , Germany
ULRICH HAHN • Institute for Biochemistry and Molecularbiology, University of Hamburg ,
Hamburg, Germany
MARLEN HESSE • Institute for Chemistry, Humboldt Universität zu Berlin, Germany
FLORIAN KUCHENBAUER • Internal Medicine III, University of Ulm, Ulm, Germany
EILEEN MAGBANUA • Institute for Biochemistry and Molecularbiology, University of
Hamburg, Hamburg, Germany
ix
