MicroRNAs FROM BASIC SCIENCE TO DISEASE BIOLOGY docx

Brock Ordway Research Institute 150 New Scotland Avenue Albany, NY 12208, USA George Adrian Calin Center for Human Cancer Genetics The Ohio State University Comprehensive Cancer Center 3

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MicroRNAs (miRNAs) are evolutionary conserved RNA molecules that ulate gene expression, and their recent discovery is currently revolutionizingboth basic biomedical research and drug discovery Expression levels ofmiRNAs have been found to vary between tissues and with developmentalstages and hence evaluation of the global expression of miRNAs potentiallyprovides opportunities to identify regulatory points for many different bio-logical processes This wide-ranging reference work, written by leadingexperts from both academia and industry, will be an invaluable resourcefor all those wishing to use miRNA techniques in their own research, fromgraduate students, post-docs and researchers in academia to those working

reg-in research and development reg-in biotechnology and pharmaceuticalcompanies who need to understand this emerging technology From thediscovery of miRNAs and their functions to their detection and role indisease biology, this volume uniquely integrates the basic science withindustry application towards drug validation, diagnostic and therapeuticdevelopment

KR I S H N A R A OAP P A S A N Iis the Founder and Chief Executive Officer of GeneExpression Systems, a gene discovery company, focusing on functionalgenomics in cancer research He is the Editor of RNA Interference: From BasicScience to Drug Development (2005)

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Cambridge University Press

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Introduction

I Discovery of microRNAs in various organisms 5

1 The microRNAs of C elegans

Ines Alvarez-Garcia and Eric A Miska 7

2 Non-coding RNAs – development of man-made vector-based

intronic microRNAs (miRNAs)

Shao-Yao Ying, Joseph D Miller and Shi-Lung Lin 22

3 Seeing is believing: strategies for studying microRNA expression

Joshua W Hagen and Eric C Lai 42

4 MicroRNAs in limb development

Danielle M Maatouk, Jason R Rock and Brian D Harfe 58

5 Identification of miRNAs in the plant Oryza sativa

Hui Zhou, Yue-Qin Chen, Yu-Chun Luo, Jia-Fu Wang and Liang-Hu Qu 70

II MicroRNA functions and RNAi-mediated pathways 83

6 Inhibition of translation initiation by a microRNA

David T Humphreys, Belinda J Westman, David I K Martin

7 In situ analysis of microRNA expression during vertebrate

development

Diana K Darnell, Stacey Stanislaw, Simran Kaur, Tatiana A Yatskievych,

Sean Davey, Jay H Konieczka and Parker B Antin 102

8 MicroRNA function in the nervous system

Gerhard Schratt and Michael Greenberg 115

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9 MicroRNA expression that controls the amount of branched chain

a-ketoacid dehydrogenase in mitochondria of human cells

Dean J Danner, John Barnes and Benjamin Mersey 129

10 MicroRNAs and the regulation of leaf shape

Ramiro E Rodriguez, Carla Schommer and Javier F Palatnik 137

11 miRBase: a database of microRNA sequences, targets and

nomenclature

Anton J Enright and Sam Griffiths-Jones 157

12 Computational prediction of microRNA targets in vertebrates,

fruitflies and nematodes

Dominic Gru ¨ n and Nikolaus Rajewsky 172

13 Computational approaches to elucidate miRNA biology

Praveen Sethupathy, Molly Megraw and Artemis G Hatzigeorgiou 187

14 The RNAhybrid approach to microRNA target prediction

15 Machine learning predicts microRNA target sites

Pa˚l Sætrom and Ola Snøve Jr. 210

16 Models of microRNA–target coordination

Neil R Smalheiser and Vetle I Torvik 221

IV Detection and quantitation of microRNAs 227

17 Detection and analysis of microRNAs using LNA (locked nucleic

Hatim T Allawi and Victor I Lyamichev 242

19 A single molecule method to quantify miRNA gene expression

Sonal Patel, Joanne Garver, Michael Gallo, Maria Hackett, Stephen McLaughlin, Steven R Gullans, Mark Nadel, John Harris, Duncan Whitney and Lori A Neely 255

20 Real-time quantification of microRNAs by TaqMan1

assays

Yu Liang, Linda Wong, Ruoying Tan and Caifu Chen 269

21 Real-time quantification of miRNAs and mRNAs employing universalreverse transcription

Gregory J Hurteau, Simon D Spivack and Graham J Brock 283

Colour-plate section

22 Dysregulation of microRNAs in human malignancy

Kathryn A O’Donnell and Joshua T Mendell 295

23 High throughput microRNAs profiling in cancers

Muller Fabbri, Ramiro Garzon, Amelia Cimmino, George Adrian Calin

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24 Roles of microRNAs in cancer and development

Andrea Ventura, Madhu S Kumar and Tyler Jacks 322

25 miR-122 in mammalian liver

Jinhong Chang and John M Taylor 338

26 MiRNAs in glioblastoma

27 Role of microRNA pathway in Fragile X mental retardation

28 Insertion of miRNA125b-1 into immunoglobulin heavy chain gene

locus mediated by V(D)J recombination in precursor B cell acute

lymphoblastic leukemia

Takashi Sonoki and Norio Asou 372

29 miRNAs in TPA-induced differentiation of HL-60 cells

30 MiRNAs in skeletal muscle differentiation

Irina Naguibneva, Anna Polesskaya and Annick Harel-Bellan 392

31 Identification and potential function of viral microRNAs

Finn Grey, Alec J Hirsch and Jay A Nelson 405

32 Lost in translation: regulation of HIV-1 by microRNAs and a key

enzyme of RNA-directed RNA polymerase

33 MicroRNAs in the stem cells of the mouse blastocyst

34 The role of miRNA in hematopoiesis

Michaela Scherr and Matthias Eder 467

35 MicroRNAs in embryonic stem cell differentiation and prediction

of their targets

Yvonne Tay, Andrew M Thomson and Bing Lim 476

36 Generation of single cell microRNA expression profile

Fuchou Tang, Kaiqin Lao and M Azim Surani 489

37 Piwi-interacting RNAs (piRNAs)

38 MicroRNAs in immunology, cardiology, diabetes, and

unicellular organisms

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The Wellcome Trust/Cancer Research UK

Gurdon Institute and Department of Biochemistry

The Henry Wellcome Building of Cancer and Developmental BiologyUniversity of Cambridge

Tennis Court Road

Cambridge CB2 1QN, United Kingdom

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Department of Cell Biology and Yale Stem Cell Center

Yale University School of Medicine

333 Cedar Street, SHM I-213

New Haven, CT 06511, USA

Graham J Brock

Ordway Research Institute

150 New Scotland Avenue

Albany, NY 12208, USA

George Adrian Calin

Center for Human Cancer Genetics

The Ohio State University Comprehensive Cancer Center

385L Comprehensive Cancer Center

Biotechnology Research Center

Key Laboratory of Gene Engineering of the Ministry of Education

Zhongshan University

Guangzhou, 510275, People’s Republic of China

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Caifu Chen

Research & Development

Applied Biosystems

850 Lincoln Centre Drive

Foster City, CA 94404, USA

Silvia Anna Ciafre`

Experimental Medicine and Biochemical SciencesUniversity of Rome

Via Montpellier 1, Rome 00133, Italy

Amelia Cimmino

The Ohio State University Comprehensive Cancer Center385L Comprehensive Cancer Center

410 West 10th Avenue

Columbus, OH 43210, USA

Carlo Maria Croce

Dean J Danner (deceased)

Department of Human Genetics

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Hanover Medical School

Center for Internal Medicine

Department of Hematology and Oncology

Carl-Neuberg Strasse 1

D-30623 Hanover, Germany

Anton J Enright

Computational and Functional Genomics

The Wellcome Trust Sanger Institute

Wellcome Trust Genome Campus

Hinxton, Cambridge CB10 1SA, United Kingdom

Muller Fabbri

The Ohio State University Comprehensive Cancer Center

385L Comprehensive Cancer Center

Yoichi R Fujii

Molecular Biology and Retroviral Genetics Group

Division of Nutritional Sciences

Graduate School of Pharmaceutical Sciences

Nagoya City University

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Ramiro Garzon

Michael Greenberg

Department of Neurobiology

Children’s Hospital, 300 Longwood Avenue

Harvard Medical School

Boston, MA 02115, USA

Finn Grey

Vaccine & Gene Therapy Institute

Oregon Health & Sciences University

Portland, OR 97201, USA

Dominic Gru¨n

Center for Functional Comparative Genomics

Department of Biology, NYU, 1009 Main Building

100 Washington Square East

New York, NY 10003-6688, USA

Sam Griffiths-Jones

Computational and Functional Genomics

The Wellcome Trust Sanger Institute

Wellcome Trust Genome Campus

Hinxton, Cambridge CB10 1SA, United Kingdom

Memorial Sloan-Kettering Institute

Department of Developmental Biology

521 Rockefeller Research Labs

1275 York Avenue, Box 252

New York, NY 10021, USA

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Annick Harel-Bellan

Laboratoire Oncogenese, Differenciation et Transduction du Signal

CNRS UPR 9079

Institut Andre Lwoff,

Batiment B, 1er Etage

7 rue Guy Moquet

94800 Villejuif, France

Brian D Harfe

Department of Molecular Genetics & Microbiology

University of Florida College of Medicine

Hristo B Houbaviy

Department of Cell Biology

University of Medicine & Dentistry of New Jersey

Two Medical Center Drive

Stratford, NJ 08084-1489, USA

David T Humphreys

Molecular Genetics Program

Victor Chang Cardiac Research Institute

384 Victoria Street, Darlinghurst

Sidney, NSW 2010 Australia

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Gregory J Hurteau

Ordway Research Institute

150 New Scotland Avenue

Albany, NY 12208, USA

Tyler Jacks

Massachusetts Institute of Technology

Department of Biology and Center for Cancer Research

40 Ames Street, E17-518

Cambridge, 02139 MA, USA

Peng Jin

Emory University School of Medicine

615 Michael Street, Room 325.1

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Tomoko Kozu

Division of Cancer Treatment

Research Institute for Clinical Oncology

Saitama Cancer Center

818 Komuro, Ina, Saitama 362-0806, Japan

Madhu S Kumar

Eric C Lai

Memorial Sloan-Kettering Institute

Department of Developmental Biology

521 Rockefeller Research Labs

1275 York Avenue, Box 252

New York, NY 10021, USA

Kaiqin Lao

Advanced Research Technology

Applied Biosystems

850 Lincoln Center Drive

Yu Liang

Applied Biosystems

Bing Lim

Stem Cell and Developmental Biology

Genome Institute of Singapore

60 Biopolis Street, #02-01, Genome

Singapore 138672

Haifan Lin

Department of Cell Biology and Yale Stem Cell Center

Yale University School of Medicine

333 Cedar Street, SHM I-213

New Haven, CT 06511, USA

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Shi-Lung Lin

Department of Cell and Neurobiology

Keck School of Medicine

University of Southern California

1333 San Pablo Street, BMT-403

Los Angeles, CA 90033-9112, USA

Yu-Chun Luo

Key Laboratory of Gene Engineering of the Ministry of EducationZhongshan University

1600 SW Archer Road

Gainesville, FL 32610-0266, USA

David I K Martin

Program in Human Genetics and Molecular Biology

Institute of Genetic Medicine

Johns Hopkins University School of Medicine

Baltimore, MD 21205, USA

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Eric A Miska

Gurdon Institute and Department of Biochemistry

The Henry Wellcome Building of Cancer and Developmental Biology

University of Cambridge

Tennis Court Road

Cambridge CB2 1QN, United Kingdom

7 rue Guy Moquet

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Jay A Nelson

Kathryn A O’Donnell

Program in Human Genetics and Molecular Biology

Institute of Genetic Medicine

Johns Hopkins University School of Medicine

Baltimore, MD 21205, USA

Javier F Palatnik

Molecular Biology Division

IBR – Institute of Molecular and Cellular Biology of RosarioSuipacha 531

7 rue Guy Moquet

94800 Villejuif, France

Thomas Preiss

Sidney, NSW 2010, Australia

Liang-Hu Qu

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Nikolaus Rajewsky

Assistant Prof of Biology and Mathematics

New York University

Center for Functional Comparative Genomics

Department of Biology, NYU, 1009 Main Building

100 Washington Square East

New York, NY 10003-6688, USA

and

MDC/Charite Medical University

Max Delbruck Centrum for Molecular Medicine

1600 SW Archer Road

Gainesville, FL 32610-0266, USA

Ramiro E Rodriguez

Molecular Biology Division

IBR – Institute of Molecular and Cellular Biology of Rosario

Medisinsk Teknisk Senter

NO-7489 Trondheim, Norway

Michaela Scherr

Hanover Medical School

Center for Internal Medicine

Department of Hematology and Oncology

Carl-Neuberg Strasse 1

D-30623, Hanover, Germany

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Carla Schommer

Department of Molecular Biology

Max Plank Institute for Developmental BiologyTubingen 72076, Germany

Gerhard Schratt

University of Heidelberg

Interdisciplinary Center for Neurosciences

Im Neuenheimer Feld 345, Room 160

Medisinsk Teknisk Senter

NO-7489 Trondheim, Norway

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Gurdon Institute of Cancer and Developmental Biology

Tennis Court Road

Cambridge CB2 1QR, United Kingdom

Keith Szulwach

Emory University School of Medicine

615 Michael Street, Room 325.1

Atlanta, GA 30322, USA

Ruoying Tan

Applied Biosystems

Fuchou Tang

Gurdon Institute of Cancer and Developmental Biology

Tennis Court Road

Cambridge CB2 1QR, United Kingdom

Yvonne Tay

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Andrew M Thomson

Jia-Fu Wang

Belinda J Westman

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Hui Zhou

Key Laboratory of Gene Engineering of the Ministry of Education

Zhongshan University

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Sidney Altman

Twenty-five years ago it was possible to predict that many more RNAs that wereknown at that time would be found: Some of them would be catalytic and some ofthem would not be but would serve other functions The prediction turned out to

be spectacularly true and this book deals with one class of novel RNAs, themicroRNAs (miRNAs) The task now is to elucidate the nature and function ofall these new RNAs

This book is a compendium of experimental methods, in silico and the tional kind, of analyzing the miRNAs There are many chapters on miRNAs onintra-cellular functions, in developmental events and in disease A newcomerwould do well to have this volume handy for purposes of reference and for itseducational value per se There are not many books on miRNAs that have such anextensive and complete view of the field as it now exists My hope is that theproblems presented here will be worked on assiduously to pave the way for a newsynthesis of various RNAs as important regulatory genes in eukaryotes

tradi-Yale UniversityNew Haven, CT, USA

xxv

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Victor R Ambros

MicroRNA research is enjoying an inflationary period of astonishingly rapidprogress that began in 2001 after a relatively long gestation The idea that smallRNAs could regulate gene expression by base-pairing to messenger RNAs can betraced back more than 45 years In a 1961 Journal of Molecular Biology paper, Jacoband Monod proposed an antisense RNA base-pairing model for the lac repressor/operator interaction that they had defined genetically The lac repressor turnedout to be a protein, but the field of antisense RNA mediated gene regulation grewsteadily through studies of authentic antisense regulatory RNAs in bacteria Theidentification in 1993 of the lin-4 microRNA and its antisense regulation of lin-14did not trigger a surge of interest in antisense gene regulation in eukaryotes,primarily because lin-4 did not exhibit any evident conservation outside nema-todes (although we now know that lin-4 is related to vertebrate mir-125, but is notclose enough to be detected by the methods available in the era before theavailability of genome sequences) Even the identification in 2000 of let-7, asecond microRNA in C elegans, did not immediately stimulate a sense amongbiologists that these exceptionally small RNAs could represent a general phenom-enon I must admit that I was among the skeptical majority

The modern era of microRNA biology began with the finding from the Ruvkunlaboratory, reported in a 2000 Nature paper, that the let-7 microRNA has beenconserved almost precisely in all its 21 nt for more than 400 million years: sincethe common ancestor of all bilaterally symmetric animals Note that this report

of the conservation of let-7 closely followed the identification in 1999 by theBaulcombe laboratory of similar c.22 nt RNAs associated with gene silencing inplants These convergent results from the Ruvkun and Baulcombe labs really rangthe bells around our ears, announcing undeniably that the lin-4 and let-7 RNAsmust be part of a widespread and ancient suite of genetic regulatory phenomenainvolving small RNAs The realization that there must be more small RNAs likelin-4 and let-7, spurred several labs to immediately try to identify other microRNAsencoded in eukaryotic genomes

Within a year or two of the discovery of C elegans let-7, numerous novelmicroRNAs were identified in flies, worms, vertebrates and plants, including

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many others that, like let-7, are deeply conserved The subsequent years havebrought remarkable progress towards understanding the diversity of microRNAs,their evolution, biogenesis, expression patterns, biological functions, and mole-cular mechanisms MicroRNAs continue to surprise and delight us in unexpectedways These little regulators seem to be engaged in practically every aspect ofmetazoan biology, regulating diverse processes including viral replication, cellfate, morphogenesis, differentiation, physiology, and disease This versatility ofmicroRNAs is reflected by the diverse array of topics included in this volume,which capture the status of understanding of microRNA biology in 2008 Thesechapters cover the identification and detection of microRNAs in vivo, the com-putational analysis of mRNA microRNA-target interactions, and the molecularmechanisms of gene regulation by microRNAs Further and reflecting an area ofparticular excitement currently, a large portion of the volume describes geneticand molecular studies of the roles of microRNAs in development and humandisease The full scope of the involvement of microRNAs in human disease isdifficult to envision at this early stage, but it is already clear from the workreported here that microRNAs will figure significantly in the molecular pathology

of many diseases

This volume marks the state of the field at this moment, circa 2008, but we can

be assured that there are still major surprises and important principles to emergefrom future research on microRNAs This is apparent from the many questionsthat arise from the work reported here Each chapter addresses important funda-mental questions, but in so doing also leaves us with an enhanced appreciation ofthe unanswered questions and technical challenges before us Much more work isrequired to fully understand the developmental and physiological roles of parti-cular microRNAs How well do genetic studies using model organisms informabout the functions in humans of the conserved microRNAs? Indeed, we have noidea how is it that the entire 21–22 nucleotides of a microRNA such as let-7 can beprecisely conserved over vast evolutionary time; what selective forces could haveconstrained every single nucleotide of an RNA for so long? MicroRNA genes canalso evolve rapidly, either de novo or by gene duplication; so how do the evolution

of microRNAs and their targets affect human population diversity and diseasesusceptibility? Finally, improvements in microRNA detection technology andcomputational methods will certainly lead to the discovery of important newmicroRNA genes and microRNA–target interactions, particularly for rare cell typesand stem cell niches Progress in microRNA research has been astonishingly rapidover the past six years or so, and we can be sure that the future will see anacceleration of this pace of discovery, yielding a wealth of exciting new biologicalinsights and human health applications

Dartmouth Medical School

Hanover, NH, USA

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Krishnarao Appasani

It is now clear an extensive miRNA world was flying almost unseen by genetic radar.

Gary Ruvkun, Professor, Harvard Medical School; Cell, S116, S95, 2004

RNomics is a newly emerging sub-discipline of genomics that categoricallystudies the structure, function and processes of non-coding ribo nucleic acids in

a cell MicroRNomics is a sub-field of RNomics that describes the biogenesis andmechanisms of tiny RNA regulators, and their involvement in the processes ofdevelopment, differentiation, cell proliferation, cell death, chromosomal segrega-tion and metabolism The discovery of microRNAs in species ranging fromCaenorhabditis elegans (C elegans) to humans, and their regulatory functions,opened a new tier of understanding of gene expression In addition, the study ofRNA interference (RNAi) and its mechanisms propelled the elucidation of variousfunctions of microRNAs The development and brisk progress in microRNAsresearch over the past few years allowed us to comprehend the genomics at anewer scale Therefore, it is now an ideal time to examine the available evidence in

a systematic way in order to know where this impressive field is going

MicroRNAs: From Basics to Disease Biology is mainly intended for readers in thefields of molecular cell biology, genomics, biotechnology and molecular medi-cine This book may be useful in more advanced graduate level courses This book,which focuses on the concepts of microRNA biology, key implications in variousforms of diseases, and applications in diagnostic and drug development, consists

of thirty-eight chapters, grouped into six sections Most of the chapters are written

by the original discoverers or their associated scientists from academia, biotechand pharma, exclusively from the microRNomics field

Although several reviews have been published covering these tiny molecules,there is no single volume currently available in the marketplace This is the firstbook that integrates the academic science with industrial applications from thediverse role of microRNAs development to disease biology, covering bioinfor-matics, quantitation, prognosis and diagnostics as well drug development andvalidation This book will serve as a reference for graduate students, post-doctoralresearchers, and professors from academic research institutions who wish to initi-ate microRNA research in their own laboratories Additionally, this book will serve

MicroRNAs: From Basic Science to Disease Biology, ed Krishnarao Appasani Published by Cambridge University Press # Cambridge University Press 2008.

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as a descriptive and in-depth scientific explanatory analysis for executives andscientists in research and development from biotech and pharmaceutical compa-nies It will provide a valuable executive scientific summary for investors andthose responsible for business development in the life sciences, who need to keepabreast with the microRevolution Most importantly, my hope is that this volumewill serve both as a prologue to the field for newcomers, as well as an update tothose already active in the field.

Sydney Brenner from the MRC Molecular Biology Laboratory in Cambridgeopened the field of C elegans biology (mid 1970s), when the lin-4 mutant wasoriginally discovered Later (his post-doctoral fellow) Howard Robert Horvitz fromMassachusetts Institute of Technology isolated lin-14 Although both these geneswere discovered in the study of developmental timing defects of the worm theirnature and functions were not explored until Horovitz’s post doctoral fellows,Ambros and Ruvkun, studied them Fourteen years ago, Victor Ambros and hiscolleagues from Dartmouth Medical School identified for the first time that lin-4was the first short non-coding microRNA that regulates gene expression in C.elegans At the same time Gary Ruvkun and his colleagues from the HarvardMedical School demonstrated lin-14 to be the first microRNA target gene.Together, these two seminal discoveries identified a novel mechanism of post-transcriptional gene regulation A successful initial collaboration and subsequentindependent contributions by Ambros and Ruvkun paved the way to our currentunderstanding of microRNA biology The microRNAs world did not take off untilthe discovery of RNAi, and let-7, a second microRNA discovered by Ruvkun andhis colleagues in 2000 The highly conserved nature of let-7 attracted a great deal

of attention to microRNAs research and since then many groups have identifiedmicroRNAs in various organisms, from protozoans to humans The paradigmfrom the heterochromic lin-4/lin-14 genes remains the model; miRNAs havenow been shown to control mRNA abundance in plants, and are thought toregulate many more steps beyond translation Despite considerable attentionhaving been paid to microRNAs over the past five years, a lot of questions remainunanswered The mechanism of action of microRNAs is still controversial andonly a few in vivo functions of microRNAs have been demonstrated in anyorganism to date In this book I intend to bring the various aspects of microRNAresearch together, including the biology, applications, their role in stem cell anddisease biology As the field of microRNomics is rapidly growing, any volume thatdescribes the material might be little outdated The role of microRNAs in immu-nology, cardiology, diabetes, and unicellular organisms has been recentlyreported, after I communicated all the original chapters to the press Therefore Ihave added two new chapters reviewing these topics at the end of this book

As Ambros mentioned in an earlier paper, the lin-4 story is one of persistentcuriosity, luck, timing, and the generosity of colleagues Indeed, it is a key break-through in the modern molecular biology In the coming years, microRNAresearch will necessitate the rewriting of our textbooks and alter the basic concept

of the central dogma In the near future we will be able to use microRNA-basedmolecular signatures or panels of multiplexed microRNA biomarkers to identify

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whether a tumor is malignant or benign, its site of origin, its prognostic subtype,

and even predict its response to therapy Clearly, high-throughput approaches

and bioinformatics will have a primary role in the future of clinical oncology and

pathology In addition, microRNAs will be used in targeted cardiovascular therapy

and they will be a central tenet in ‘translational medicine’ To fully realize the

potential of microRNAs as biomarkers to aid in drug development, industry must

implement best practices for biomarker development, and promote translational

research strategies The biggest obstacles to turning discovery from bench to

bed-side are not only, in the advancement of technology, but also the necessary

regulatory approvals In a nutshell, microRNA array technology has the potential

to accommodate all required assay formats on one testing platform, and to

provide better reagents for pathological diagnosis in the future It is hoped that

by fully understanding the functions of microRNAs in various forms of human

diseases and their subsequent development as theranostics, so we will be the new

era of ‘translational medicine’

Many people have contributed to making my involvement in this project

possible I thank my teachers throughout my life for their excellent teaching,

guidance and mentorship, which caused me to become a scientist I am thankful

to all of the contributors to this book Without their commitment this book

would not have emerged for the reader Many people have had a hand in the

launch of this book Each chapter has been passed back and forth between the

authors for criticism and revision, so that each chapter represents a joint

compo-sition Thanks to you, the reader, who makes the hours I have spent putting

together this volume worthwhile, if you find value in the hours you spend with

this book I am indebted to the staff of Cambridge University Press, and in

particular Katrina Halliday for her generosity and efficiency throughout the

edit-ing of this book; she truly understands the urgency and need of this volume I

would specially like to thank Nobel Laureate Sidney Altman and Victor Ambros

for their kindness and support in writing the Forewords to this book Last, but not

least, I thank my two wonderful sons, Raakish and Raghu, for their understanding

and cooperation during the development of this book

A portion of the royalties will be contributed to the Dr Appasani Foundation, a

non-profit organization devoted to bring social change through education of

youth in developing nations

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Discovery of

microRNAs in

various organisms

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1 The microRNAs of C elegans

Ines Alvarez-Garcia and Eric A Miska*

Discovery

MicroRNA (miRNA) is a class of short non-coding RNA that regulates gene sion in many eukaryotes MicroRNA was first discovered in Caenorhabditis elegans byVictor Ambros’ laboratory in 1993 (Lee et al.,1993) At the same time Gary Ruvkun’slaboratory identified the first microRNA target gene (Wightman et al., 1993).Together, these two seminal discoveries identified a novel mechanism of post-transcriptional gene regulation The history of these events has been reported recently,

expres-as remembered by the main researchers involved (Lee et al.,2004; Ruvkun et al.,

2004) The realization of the importance of the discovery of microRNA, however,took about seven years and was preceded by the identification of a second microRNA

in C elegans by the Ruvkun and Horvitz laboratories and also by the rise in interest

in another class of short RNA, siRNA, involved in the process of RNAi and relatedphenomena in plants and animals (Hamilton and Baulcombe,1999; Zamore et al.,

2000) Although the discovery of microRNA was unexpected, theoretical work onthe mechanism of the lac repressor by Jacob and Monod had postulated a microRNA-like mechanism forty years earlier, see Figure1.1(Jacob and Monod,1961)

Biogenesis and mechanism of action

From studies in C elegans, Drosophila melanogaster and mammalian cell culture

a model for microRNA biogenesis in animals is emerging (see Figure 1.2a andFigure1.2b): microRNAs are transcribed by RNA polymerase II as long RNA precur-sors (pri-miRNAs) (Kim,2005), which are processed in the nucleus by the RNase IIIenzyme Drosha and DGCR8/Pasha, to form the approximately 70 base pre-miRNA(Lee et al.,2003; Denli et al.,2004; Gregory et al.,2004; Han et al.,2004; Landthaler

et al.,2004) Pre-miRNAs are exported from the nucleus by Exportin-5 (Lund et al.,

2004) and processed by the RNase III enzyme, Dicer, and incorporated into anArgonaute containing silencing complex (RISC) (Du and Zamore,2005) From this

* Author to whom correspondence should be addressed.

MicroRNAs: From Basic Science to Disease Biology, ed Krishnarao Appasani Published by Cambridge University Press # Cambridge University Press 2008.

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general model a number of factors have been directly implicated in microRNAfunction in C elegans Drsh-1 (Drosha) and Pash-1 (DGCR8/Pasha) are required forpri-miRNA processing (Denli et al.,2004) Dcr-1 (Dicer) is required for pre-miRNAprocessing (Grishok et al., 2001; Ketting et al., 2001; Knight and Bass, 2001).Interestingly, two Argonaute family members, Alg-1 and Alg-2 are also required forthe generation of the mature microRNA from pre-miRNA, suggesting that at least in

C elegans Argonaute proteins not only play roles in microRNA mechanism of actionbut also microRNA biogenesis (Grishok et al.,2001) In animals microRNAs arethought to function through the inhibition of effective mRNA translation of targetgenes through imperfect base-pairing with the 30-untranslated region (30UTR) oftarget mRNAs (see Figure1.2c) (Bartel,2004; Pillai et al.,2005) However, alternativemechanisms of action include direct mRNA cleavage (Mansfield et al.,2004; Yekta

et al.,2004) and mRNA stability (Bagga et al.,2005; Jing et al.,2005; Giraldez et al.,

2006) In vivo microRNA targets are largely unknown, but estimates range from one

to hundreds of target genes for a given microRNA, based on microRNA targetpredictions using a variety of bioinformatics approaches (Lewis et al.,2003; Stark

et al.,2003; John et al.,2004; Rajewsky and Socci,2004; Brennecke et al.,2005; Farh

et al.,2005; Lewis et al.,2005; Stark et al.,2005; Xie et al.,2005; Lall et al.,2006)

ProteinsMessengersGenes

Structuralgenes

Oper atorgene

Structuralgenes

Oper atorgene

Figure 1.1 Models for the regulation of protein synthesis Reproduced from Jacob and Monod ( 1961 ) Model II depicts a microRNA-like mechanism Both models were proposed for the lac repressor and turned out to be false, as the lac repressor is a protein transcription factor However, model I and II may underlie short RNA mediated gene regulation at the transcriptional and post- transcriptional level respectively.

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pre-miRNA (~70 bases)

Dicer/A go

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MicroRNA gene identification and expression analysis

The first microRNAs were identified through forward genetic screens in C elegans,

as is described below However, the majority of microRNAs in C elegans and otherorganisms were identified using molecular biology techniques and bioinformatics(Lau et al.,2001; Lee and Ambros,2001) Strict criteria for the identification andnaming of microRNAs have been agreed upon (Ambros et al.,2003a) Importantly,microRNAs need to be derived from stem-loop precursors and verified experimen-tally by either northern blotting or polymerase chain reaction using specificprimers, unless they represent clear orthologs of microRNAs in related species(Lau et al.,2001; Lee and Ambros,2001; Grad et al.,2003; Lim et al.,2003; Ohler

et al., 2004) The public database that has been established for microRNAs,miRBase, currently lists 115 C elegans microRNAs (release 8.0)(Griffiths-Jones,

2004; Griffiths-Jones et al.,2006) However, of these only 98 microRNAs are likely

to be real (Lim et al.,2003; Ohler et al.,2004) Of these, at least 40% are conserved

in humans (Lim et al.,2003) In addition, other classes of short RNAs, distinctfrom microRNAs have been identified in C elegans, endogenous siRNAs and tinynon-coding RNAs (tncRNAs) (Ambros et al.,2003b; Lee et al.,2006) It is likely thatthe number of short RNAs identified in C elegans will continue to grow and itwould not be surprising if additional microRNAs were identified

To aid the study of microRNA function, analysis of microRNA expression mayprovide useful information The temporal expression of many microRNAs in

C elegans has been determined previously using northern blotting (see Table1.1)(Lee et al., 1993; Reinhart et al.,2000; Lau et al.,2001; Lee and Ambros, 2001;Lim et al.,2003; Grad et al.,2003; Ohler et al.,2004) These studies revealed thatpossibly one third of C elegans microRNAs are differentially expressed during larvaldevelopment However, a large number of microRNAs appear to be expressedthroughout embryonic and larval stages and adulthood It would be interesting toextend these studies to include embryonic stages and a collection of mutant back-grounds The availability of microRNA microarray technology should aid futuremicroRNA expression studies (Krichevsky et al.,2003; Miska et al.,2004) In addi-tion to information regarding temporal expression of microRNAs, spatial informa-tion would be very informative Spatial expression of microRNAs in C elegans so farhas been restricted to indirect methods such as promoter GFP fusion transgenes(Johnson et al.,2003) It remains an open question if in situ approaches to directlydetect microRNAs using oligonucleotide probes that have been successful in plants,flies and fish will also be applicable to C elegans (Kidner and Martienssen,2004;Sokol and Ambros,2005; Wienholds et al.,2005)

MicroRNA target predictions

The first microRNA targets, lin-14, lin-28 and lin-41 mRNAs were identified throughgenetic interactions with the lin-4 and let-7 microRNAs respectively (Ambros,1989;Ruvkun and Giusto,1989) However, a number of microRNA target predictionalgorithms have been described (Lewis et al.,2003; Stark et al.,2003; John et al.,

Tiêu đề	MicroRNAs from Basic Science to Disease Biology
Tác giả	Krishnarao Appasani
Người hướng dẫn	Victor R. Ambros Dartmouth Medical School, Sidney Altman Yale University
Trường học	Gene Expression Systems, Inc.
Chuyên ngành	Biology
Thể loại	Sách tham khảo
Năm xuất bản	2005

Định dạng
Số trang	580
Dung lượng	8,99 MB