Regulation of microRNAs - 1st Edition

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PREFACE

When in 2001 the first draft versions of the human genome revealed that there were no
more than 25,000 human genes, much soul searching resulted. How could a complicated
human being develop and function with a gene set not much bigger than that of a worm,
Caenorhabditis elegans?
Although the meaning of ‘gene counts’ is debatable when a single gene can give rise to
a multitude of different gene products (and when in fact much of the ‘inter-genic’ genome
appears to be transcribed), the apparent conundrum highlighted the importance of gene
regulation in making complex organisms. It thus appears particularly appropriate that it
was also in 2001 that microRNAs (miRNAs) were finding their way into the limelight.
These regulatory RNAs, named for their small size of some 22 nucleotides, had been
discovered in 1993 in C. elegans, but were initially considered a worm oddity and largely
ignored. It was only when small RNA cloning efforts started to reveal hundreds of different
miRNAs in a typical animal or plant genome that they were widely noticed. Today, it
appears that hardly any cellular or developmental pathway has escaped the control that
miRNAs exert by silencing target mRNAs through an antisense mechanism. Accordingly,
miRNAs dysregulation contributes to numerous diseases, most notably diverse cancers.
Given this pervasiveness and importance of miRNA-mediated gene regulation,
it should come as little surprise that miRNAs themselves are also highly regulated.
However, the recent explosion of knowledge on this topic has been remarkable, providing
a primary motivation for publication of this book. As miRNAs are transcribed by RNA
polymerase II, the enzyme that also generates mRNAs, it was perhaps not unexpected
that miRNA transcription would be subject to regulation, and we have willfully omitted
this aspect from this monograph. However, what has been unexpected is the extent of
post-transcriptional regulation of miRNAs that is illustrated in this book.
In the first chapter, René Ketting provides the background against which all of the
regulatory processes occur by revealing the complex biogenesis and function of miRNAs
and the related siRNAs. Akiko Hata and Brandi Davis then describe how SMAD proteins,
generally known for their function in controlling transcription, reveal another side in
regulating the processing of certain primary miRNA (pri-miRNA) transcripts by the
RNase Drosha. Drosha-mediated processing of pri-miRNAs into the short precursor

v
vi PREFACE

miRNAs (pre-miRNAs) is also modulated by the RNA-binding proteins hnRNP A1, as

discussed by Javier Caceres and colleagues and KSRP (Michele Trabucchi et al). Whereas
SMADs, hnRNP A1 and KSRP promote processing of specific pri-miRNAs, estrogen
receptor alpha represses this biogenesis step as related by Shigeaki Kato and colleagues.
Robinson Triboulet and Richard Gregory further reveal that the pri-miRNA processing
complex undergoes autoregulation.
KSRP not only promotes processing of pri-miRNAs, but also the subsequent
cleavage of pre-miRNAs by the RNase Dicer. Conversely, Lin28 was found to repress
pri-miRNA as well pre-miRNA processing as discussed by Nicolas Lehrbach and
Eric Miska. This inhibition involves 3’ end uridylation of the pre-miRNA. Another
RNA modification that occurs on miRNAs is adenosine-to-inosine editing, and Mary
O’Connell and colleagues critically evaluate its incidence and how editing affects
processing and functionality of miRNAs.
Gregory Wulczyn and colleagues discuss the Trim-NHL protein family whose
members utilize diverse mechanisms to regulate miRNA levels and activity both positively
and negatively. Nicole Meisner and Witold Filipowicz review HuR, an RNA-binding
protein that regulates mRNAs through a number of mechanisms, including at least one
instance in which HuR reverses miRNA-mediated mRNA silencing.
Finally, although mature miRNAs have long been viewed as highly stable molecules,
miRNA degradation pathways have now been identified in plants and algae, as revealed
by Heriberto Cerutti and Fadia Ibrahim, and in animals, as discussed by us.
Even if this monograph cannot strive to be comprehensive in a field developing at
such an amazing pace, I hope that the examples provided here will serve to illustrate the
diversity of mechanisms regulating miRNAs, as well as highlight some unifying themes,
particularly among the mechanisms regulating miRNA biogenesis. Undoubtedly, many
more examples of regulation of miRNAs remain to be discovered and mechanistic details
on known pathways to be revealed, promising an exciting future to this field of research.

Helge Großhans, PhD

Friedrich Miescher Institute for Biomedical Research
Basel, Switzerland
ABOUT THE EDITOR...

HELGE GROßHANS, PhD is a research group leader at the Friedrich Miescher

Institute for Biomedical Research (FMI), which is part of the Novartis Research
Foundation in Basel, Switzerland. His main research interests are in the developmental
function, regulation, and mechanism of action of microRNAs. He received his PhD
from the University of Heidelberg, Germany and did his postdoctoral training at Yale
University, USA. He is the winner of a 2009 ERC Award.

vii
PARTICIPANTS

Paola Briata Brandi N. Davis

Istituto Nazionale per la Ricerca Department of Biochemistry
sul Cancro (IST) Tufts University School of Medicine
Genova and
Italy Molecular Cardiology Research Institute
Tufts Medical Center
Javier F. Cáceres Boston, Massachusetts
Medical Research Council USA
Human Genetics Unit
Institute of Genetics Witold Filipowicz
and Molecular Medicine Friedrich Miescher Institute
Western General Hospital for Biomedical Research
Edinburgh Basel
UK Switzerland

Heriberto Cerutti Eleonora Franzoni

School of Biological Sciences Center for Anatomy
Center for Plant Science Innovation Institute of Cell Biology and Neurobiology
University of Nebraska Charité-Universitätsmedizin Berlin
Lincoln, Nebraska Berlin
USA Germany

Saibal Chatterjee Sally Fujiyama-Nakamura

Friedrich Miescher Institute Institute of Molecular and Cellular
for Biomedical Research Biosciences
Basel University of Tokyo
Switzerland Tokyo
Japan
Elisa Cuevas
Center for Anatomy
Institute of Cell Biology and Neurobiology
Charité-Universitätsmedizin Berlin
Berlin
Germany

ix
x PARTICIPANTS

Roberto Gherzi Fadia Ibrahim

Istituto Nazionale per la Ricerca School of Biological Sciences
sul Cancro (IST) Center for Plant Science Innovation
Genova University of Nebraska
Italy Lincoln, Nebraska
USA
Richard I. Gregory
Stem Cell Program Shigeaki Kato
Children’s Hospital Boston Institute of Molecular and Cellular
and Biosciences
Department of Biological Chemistry University of Tokyo
and Molecular Pharmacology Tokyo
Harvard Medical School Japan
Harvard Stem Cell Institute
Boston, Massachusetts Liam P. Keegan
USA Medial Research Council
Human Genetics Unit
Helge Großhans Institute of Genetics and Molecular
Friedrich Miescher Institute Medicine
for Biomedical Research Western General Hospital
Basel Edinburgh
Switzerland UK

Sonia Guil René F. Ketting

Cancer Epigenetics and Biology Program Hubrecht Institute-KNAW
(PEBC) University Medical Centre Utrecht
Catalan Institute of Oncology Utrecht
(ICO-IDIBELL) The Netherlands
L’Hospitalet Barcelona
Catalonia Nicolas J. Lehrbach
Spain Wellcome Trust Cancer Research
Gurdon Institute and Department
Akiko Hata of Biochemistry
Department of Biochemistry University of Cambridge
Tufts University School of Medicine Cambridge
and UK
Molecular Cardiology Research Institute
Tufts Medical Center Nicole-Claudia Meisner
Boston, Massachusetts Novartis Institutes for Biomedical Research
USA Novartis Campus
Basel
Bret S.E. Heale Switzerland
Medial Research Council
Human Genetics Unit Eric A. Miska
Institute of Genetics and Molecular Wellcome Trust Cancer Research
Medicine Gurdon Institute and Department
Western General Hospital of Biochemistry
Edinburgh University of Cambridge
UK Cambridge
UK
PARTICIPANTS xi

Gracjan Michlewski Michele Trabucchi

Medical Research Council Howard Hughes Medical Institute
Human Genetics Unit Department and School of Medicine
Institute of Genetics and Molecular University of California, San Diego
Medicine La Jolla, California
Western General Hospital USA
Edinburgh
UK Robinson Triboulet
Stem Cell Program
Mary A. O’Connell Children’s Hospital Boston
Medial Research Council and
Human Genetics Unit Department of Biological Chemistry
Institute of Genetics and Molecular and Molecular Pharmacology
Medicine Harvard Medical School
Western General Hospital Harvard Stem Cell Institute
Edinburgh Boston, Massachusetts
UK USA

Andres Ramos F. Gregory Wulczyn

Division of Molecular Structure Center for Anatomy
National Institute for Medical Research Institute of Cell Biology and Neurobiology
The Ridgeway, London Charité-Universitätsmedizin Berlin
UK Berlin
Germany
Michael G. Rosenfeld
Howard Hughes Medical Institute Kaoru Yamagata
Department and School of Medicine Institute of Molecular and Cellular
University of California, San Diego Biosciences
La Jolla, California University of Tokyo
USA Tokyo
Japan
Agnieszka Rybak
Max Delbrück Center for Molecular
Medicine
Berlin
Germany
 CONTENTS

1. microRNA BIOGENESIS AND FUNCTION:

AN OVERVIEW ...............................................................................................1
René F. Ketting

Abstract......................................................................................................................................... 1
Introduction: PTGS in Plants and Small RNAs........................................................................ 1
RNAi .............................................................................................................................................. 2
Dicer .............................................................................................................................................. 2
Argonaute ..................................................................................................................................... 4
The First microRNAs and Links to RNAi ................................................................................. 4
miRNAs: Ancient Mediators of Gene Regulation ..................................................................... 5
Primary miRNA Processing by Drosha ..................................................................................... 6
Small RNA Selectivity of Argonaute Proteins ........................................................................... 7
Target Recognition ....................................................................................................................... 7
Mechanisms of miRNA-Mediated Silencing.............................................................................. 8
Regulating miRNAs ................................................................................................................... 10
Conclusion .................................................................................................................................. 10

2. REGULATION OF pri-miRNA PROCESSING THROUGH Smads................15

Akiko Hata and Brandi N. Davis

Abstract....................................................................................................................................... 15
Introduction: Basic TGF` Signaling ........................................................................................ 15
miRNA Biogenesis ...................................................................................................................... 17
The First Processing Step by Drosha ....................................................................................... 19
R-Smads Regulate miRNA Maturation ................................................................................... 20
0HFKDQLVPRI5HJXODWLRQRI6SHFL¿FSULPL51$VE\6PDG ................................................. 22
Transcriptional Regulation of miRNA Genes by Smads ........................................................ 24
Conclusion and Future Prospects ............................................................................................. 24

xiii
xiv CONTENTS

3. STIMULATION OF pri-miR-18a PROCESSING BY hnRNP A1 .....................28

Gracjan Michlewski, Sonia Guil and Javier F. Cáceres

Abstract....................................................................................................................................... 28
Introduction ................................................................................................................................ 28
hnRNP A1 Binds to The pri-miR-18a Stem-Loop Structure ................................................. 29
hnRNP A1 Promotes The Drosha-Mediated Processing of pri-miR-18a .............................. 30
Role of The Terminal Loops in miRNA Processing ................................................................ 31
Conclusion and Future Prospects ............................................................................................. 33

4. KSRP PROMOTES THE MATURATION OF A GROUP OF miRNA

PRECURSORS ...............................................................................................36
Michele Trabucchi, Paola Briata, Witold Filipowicz, Andres Ramos,
Roberto Gherzi and Michael G. Rosenfeld

Abstract....................................................................................................................................... 36
Introduction ................................................................................................................................ 36
Co-Activators and Co-Repressors of miRNA Precursor Maturation ................................... 37
Impact of KSRP and Other Co-Activators and Co-Repressors of miRNA
Precursor Maturation on Cell Proliferation, Differentiation and Cancer.................... 39
Conclusion .................................................................................................................................. 41

5. HORMONAL REPRESSION OF miRNA BIOSYNTHESIS THROUGH

A NUCLEAR STEROID HORMONE RECEPTOR ..................................43
Sally Fujiyama-Nakamura, Kaoru Yamagata and Shigeaki Kato

Abstract....................................................................................................................................... 43
Introduction ................................................................................................................................ 43
p68/p72 DEAD-Box RNA Helicases Serve as RNA-Binding Components
in the Drosha Complex ...................................................................................................... 44
Gene Regulation by Nuclear Estrogen Receptors ................................................................... 46
Estrogen-Induced mRNA Stability is Mediated through Hormonally Regulated
miRNA Biosynthesis........................................................................................................... 49
Conclusion .................................................................................................................................. 52

6. AUTOREGULATORY MECHANISMS CONTROLLING

THE MICROPROCESSOR ..........................................................................56
Robinson Triboulet and Richard I. Gregory

Abstract....................................................................................................................................... 56
Introduction ................................................................................................................................ 56
Posttranscriptional Regulation of DGCR8 by the Microprocessor....................................... 59
Stabilization of Drosha Protein by DGCR8 ............................................................................. 61
Conclusion .................................................................................................................................. 61
CONTENTS xv

7. REGULATION OF pre-miRNA PROCESSING ................................................67

Nicolas J. Lehrbach and Eric A. Miska

Abstract....................................................................................................................................... 67
Introduction ................................................................................................................................ 67
miRNAs and Developmental Timing in C. elegans ................................................................. 68
The Heterochronic Gene lin-28 encodes a Regulator of let-7 microRNA Processing .......... 69
Lin28/LIN-28 Promotes Uridylation and Degradation of Pre-let-7 ...................................... 71
Heterochronic Gene Orthologues: Ancient Stem Cell Regulators? ...................................... 71
Conclusion .................................................................................................................................. 73

8. THE EFFECT OF RNA EDITING AND ADARs ON miRNA

BIOGENESIS AND FUNCTION ..................................................................76
Bret S.E. Heale, Liam P. Keegan and Mary A. O’Connell

Abstract....................................................................................................................................... 76
Introduction ................................................................................................................................ 76
Prevalence of Edited miRNAs................................................................................................... 78
Effects of Editing of pri-miRNAs and pre-miRNAs on Biogenesis ....................................... 80
The Effect of Editing on miRNA Function .............................................................................. 81
ADARs as Competing dsRNA-Binding Proteins .................................................................... 82
(GLWLQJRI6HHG7DUJHW6HTXHQFHVZLWKLQƍ875V ................................................................... 82
Conclusion .................................................................................................................................. 83

9. miRNAs NEED A TRIM: REGULATION OF miRNA ACTIVITY

BY Trim-NHL PROTEINS ............................................................................85
F. Gregory Wulczyn, Elisa Cuevas, Eleonora Franzoni and Agnieszka Rybak

Abstract....................................................................................................................................... 85
Introduction ................................................................................................................................ 86
The Trim-NHL Family of Developmental Regulators ............................................................ 87
The Trim Domain as E3 Ubiquitin Ligase ............................................................................... 89
Functional Analysis of Individual Trim-NHL Family Members ........................................... 90
Trim-NHL Proteins as Regulators of the miRNA Pathway ................................................... 95
Conclusion .................................................................................................................................. 99

10. PROPERTIES OF THE REGULATORY RNA-BINDING PROTEIN

HuR AND ITS ROLE IN CONTROLLING miRNA REPRESSION ......106
Nicole-Claudia Meisner and Witold Filipowicz

Abstract..................................................................................................................................... 106
Introduction to HuR and ARE Elements ............................................................................... 106
Regulation of the Regulator .................................................................................................... 108
Molecular Mechanisms of Posttranscriptional Control by HuR ......................................... 112
Function of HuR in the Relief of miRNA-Mediated Repression ......................................... 114
Synergism between HuR and let-7 in Translational Repression of c-Myc mRNA............. 117
Conclusion ................................................................................................................................ 118
xvi CONTENTS

11. TURNOVER OF MATURE miRNAs AND siRNAs IN PLANTS

AND ALGAE .................................................................................................124
Heriberto Cerutti and Fadia Ibrahim

Abtract ...................................................................................................................................... 124

Introduction .............................................................................................................................. 125
Small RNA Processing ............................................................................................................. 126
6PDOO51$0RGL¿FDWLRQE\v-O-Methylation ...................................................................... 126
Small RNA Loading and Activation of the RNA-Induced Silencing Complex................... 128
Mature Small RNA Degradation by Ribonucleases .............................................................. 130
Quality Control of Mature Small RNAs ................................................................................ 133
Conclusion ................................................................................................................................ 134

12. MicroRNases AND THE REGULATED DEGRADATION

OF MATURE ANIMAL miRNAs ................................................................140
Helge Großhans and Saibal Chatterjee

Abstract..................................................................................................................................... 140
Introduction .............................................................................................................................. 140
microRNA Biogenesis and Function....................................................................................... 141
XRN-2, a Multifunctional Exoribonuclease .......................................................................... 143
Degradation of C. elegans miRNAs by XRN-2 ...................................................................... 144
,VPL51$7XUQRYHUD6XEVWUDWH6SHFL¿F(YHQW" .................................................................. 148
A Function of XRN-2 in miRNA Turnover beyond C. elegans? .......................................... 148
Half-Lives of miRNAs—Not all miRNAs are Equal ............................................................. 149
Target Availability Affects Release of miRNAs
from AGO and their Subsequent Degradation.............................................................. 151
Conclusion ................................................................................................................................ 152

INDEX........................................................................................................................157
 CHAPTER 1

microRNA BIOGENESIS AND FUNCTION

An Overview

René F. Ketting*

Abstract: During the last decade of the 20th century a totally novel way of gene regulation
ZDVUHYHDOHG)LQGLQJVWKDWDW¿UVWJODQFHDSSHDUHGIUHDNIHDWXUHVRISODQWVRUC.
elegans turned out to be mechanistically related and deeply conserved throughout
evolution. This important insight was primed by the landmark discovery of
51$LQWHUIHUHQFHRU51$LLQ7KLVZRUNVWDUWHGDQHQWLUHQRYHO¿HOGRI
research, now usually referred to as RNA silencing. The common denominator
RIWKHSKHQRPHQDJURXSHGLQWKLV¿HOGDUHVPDOO51$PROHFXOHVRIWHQGHULYHG
from double stranded RNA precursors, that in association with proteins of the
so-called Argonaute family, are capable of directing a variety of effector complexes
to cognate RNA and/or DNA molecules. One of these processes is now widely
known as microRNA-mediated gene silencing and I will provide a partially
historical framework of the many steps that have led to our current understanding
of microRNA biogenesis and function. This chapter is meant to provide a general
overview of the various processes involved. For a comprehensive description of
current models, I refer interested readers to the reviews and primary literature
references provided in this chapter and to the further contents of this book.

INTRODUCTION: PTGS IN PLANTS AND SMALL RNAs

In the early 90s a number of papers were published that revealed an activity in Tobacco
and Petunia plants that was triggered by repetitive transgenic DNA and that resulted in
WKHVLOHQFLQJRIWKDW'1$DQGDQ\RWKHU'1$EHDULQJVLJQL¿FDQWKRPRORJ\WRWKHWULJJHU
(cosuppression).1,2 At least part of these phenomena acted downstream of transcription,
through destabilization of mRNA and hence was named “Post-Transcriptional Gene
Silencing” (PTGS). The molecular trigger of this phenomenon was not clear, although

*René F. Ketting—Hubrecht Institute-KNAW and University Medical Centre Utrecht, Uppsalalaan 8,

3584 CT Utrecht, The Netherlands. Email: [Link]@[Link]
Regulation of microRNAs, edited by Helge Großhans.
©2010 Landes Bioscience and Springer Science+Business Media.

1
2 REGULATION OF microRNAs

it was speculated that “aberrant” RNA or double stranded RNA (dsRNA) were good
candidates for priming PTGS. Although aberrant RNAs still play an important role in
many models on RNA-mediated silencing events in plants, for example as templates
on which dsRNA is synthesized, we now know that dsRNA is indeed in most cases the
primary trigger. Furthermore, in 1999 a landmark paper from David Baulcombe and
FROOHDJXHV LGHQWL¿HG VPDOO 51$ PROHFXOHV DV SRWHQWLDO ³VSHFL¿FLW\ GHWHUPLQDQWV´ LQ
PTGS.37KLVK\SRWKHVLVKDVWXUQHGRXWWREHDEVROXWHO\FRUUHFWDQGWKHLGHQWL¿FDWLRQRI
this type of small RNA species helped to lay the basis for an outburst of research activity
on RNA-based silencing processes in the years that followed.

RNAi

,QGRXEOHVWUDQGHG51$ GV51$ ZDV¿UVWGHVFULEHGDVDYHU\SRWHQWDQG

VSHFL¿FDJHQWIRUJHQHVLOHQFLQJLQC. elegans. The term RNA interference was coined
to refer to the described silencing effects, a term that is now usually abbreviated to
RNAi.4 This ground-breaking work, published by Craig Mello, Andrew Fire and their
colleagues, was awarded the Nobel Prize in 2006. Mello and Fire noted that RNAi
targets exonic regions in RNA and leads to decreased RNA levels, consistent with a
PRGHOLQZKLFK51$LOHDGVWRVHTXHQFHVSHFL¿FP51$GHVWDELOL]DWLRQDVKDGEHHQ
found for cosuppression in plants. It was also noted that dsRNA could very well be a
trigger in plant cosuppression, since inverted repeat sequences had been described as
YHU\SRWHQWWULJJHUVRI37*66RRQDIWHUWKLVSDSHU51$LOLNHSURFHVVHVZHUHLGHQWL¿HG
in numerous other systems.5 Biochemical experiments in Drosophila started to reveal
a mechanistic framework of RNAi,6,7 while genetics in C. elegans was revealing
endogenous functions for RNAi and genes required for it.8,9 It appeared that RNAi could
mechanistically be roughly divided into two steps: an initiation step and an effector
step (Fig. 1).10 In the initiation step small RNAs are generated from the dsRNA trigger;
in the effector step these small RNAs guide an Argonaute protein-containing complex
named RNA-Induced Silencing Complex (RISC) to cognate mRNAs. The realization
that small RNA molecules (then named siRNAs, for short interfering RNAs), like those
described by Baulcombe in Tobacco plants undergoing PTGS, rather than long dsRNA
PROHFXOHVSURYLGHGWKHVHTXHQFHVSHFL¿FLW\RIWKHZKROHSURFHVV7,11HQDEOHGHI¿FLHQW
RNAi also in mammalian cells.12 7KLV SURYLGHG D KLJKO\ HI¿FLHQW ZD\ WR SHUIRUP
UHYHUVHJHQHWLFH[SHULPHQWVLQFHOOFXOWXUHV\VWHPVD¿QGLQJWKDWKDVUHYROXWLRQL]HG
research on mammalian cells.

DICER

'LFHUWKHHQ]\PHWKDWJHQHUDWHVVL51$VIURPGV51$ZDVLGHQWL¿HGLQ13
This enzyme contains two RNase III active sites, a so-called PAZ domain (named after
WKUHHSURWHLQVLQZKLFKWKLVGRPDLQZDV¿UVWUHFRJQL]HG3LZL$UJRQDXWHDQG=ZLOOH D
helicase domain and a dsRNA-binding domain. It binds to the ends of dsRNA substrates
and introduces a staggered double-stranded break further along the dsRNA.14 The catalytic
DFWLYLW\ LV YHU\ FKDUDFWHULVWLF DQG DOZD\V OHDYHV D ƍK\GUR[\O JURXS D ƍSKRVSKDWH
JURXSDQGDWZREDVHRYHUKDQJDWWKHƍHQG7KHOHQJWKRIWKHVPDOO51$JHQHUDWHG
can vary, but usually is between 20 and 25 bases. Within one organism, different Dicer
microRNA BIOGENESIS AND FUNCTION: AN OVERVIEW 3

Figure 1. Schematic comparison between RNAi and miRNA mechanisms. For a more detailed scheme
of miRNA action see Figure 2. “RdRP activity” refers to RNA-dependent RNA polymerase activity
that in plants and yeast can turn ssRNA into dsRNA that is subsequently cleaved by Dicer. Likely, this
is a major source for the dsRNA trigger in PTGS. The scissors indicate passenger strand and target
cleavage. The dashed lines crossing from RNAi to miRNA and vice versa indicate that the separation
between these pathways is not absolute: side effects from siRNAs in RNAi experiments can be triggered
through miRNA like activities and miRNAs are capable of inducing target cleavage if presented with a
properly matching target RNA. The type of silencing induced is also strongly dependent on the sub-type
of Argonaute protein involved.

JHQHVFDQEHSUHVHQWHDFKHQFRGLQJDSURWHLQJHQHUDWLQJUDWKHUVSHFL¿FVXEVHWVRIVPDOO
RNA products.15 Mammals, however, only have one Dicer gene.
4 REGULATION OF microRNAs

ARGONAUTE

*HQHWLFH[SHULPHQWVLGHQWL¿HGDQ$UJRQDXWHSURWHLQDVDPDMRUSOD\HULQ51$LLQ
C. elegans soon after the discovery of RNAi itself.16%LRFKHPLFDOGDWDVXSSRUWHGWKLV¿QGLQJ
by identifying an Argonaute protein as an essential component of RISC.17 Argonaute
proteins had no known biochemical activities at that time. The only thing that was clear
from Argonaute protein sequences is that they contained two characteristic domains:
a PAZ domain (also found in Dicer) and a Piwi domain (named after the Drosophila Piwi
SURWHLQLQZKLFKLWZDV¿UVWUHFRJQL]HG ,WWRRNDQXPEHURI\HDUVEHIRUHLWEHFDPHFOHDU
that Argonaute proteins actually form the catalytic center of RISC. The PAZ domain was
VKRZQWRELQGWRWKHƍHQGRIWKHVPDOO51$18,19 while the Mid domain, in between the
3$=DQG3LZLGRPDLQVDSSHDUHGWRLQWHUDFWZLWKWKHƍHQGRIWKHVPDOO51$20 Finally,
the structure of the Piwi domain revealed an RNaseH-like structure,21 consistent with the
ELRFKHPLFDOFKDUDFWHULVWLFVRI5,6&WKDWKDGE\WKHQEHHQZHOOGH¿QHGHQGRQXFOHRO\WLF
K\GURO\VLVOHDYLQJDƍSKRVSKDWHDQGDƍK\GUR[\OJURXS22,23
7KLV51$FOHDYDJHDFWLYLW\LV¿UVWXVHGIRU5,6&DFWLYDWLRQ,QLWLDOO\$UJRQDXWH
proteins are loaded with double-stranded siRNAs and in order to become active, one of
the strands has to be removed. This can be done through endonucleolytic cleavage.24-26
The discarded strand is referred to as the passenger strand, while the strand remaining
bound to the Argonaute is known as the guide strand. It is this strand that guides the
Argonaute protein to a target. At the target the very same catalytic activity used for RISC
activation now can induce target RNA destabilization. However, this scenario is most
OLNHO\DQRYHUVLPSOL¿FDWLRQDVWKHUHDUHLQGLFDWLRQVWKDWWKHFDWDO\WLFDFWLYLW\RI$UJRQDXWH
proteins on small RNA duplexes differs from that on target RNA.27,28
It should also be noted that many Argonaute proteins contain a Piwi domain that is
not compatible with nucleolytic activity. This has implications for both the mechanism of
RISC activation as well as for the mechanism through which the targeted RNA is silenced.
Passenger strand displacement in these Argonautes depends on weakened basepairing
interactions within the small RNA duplex,29 while target RNA silencing depends on
additional cofactors recruited by the Argonaute (see below).

THE FIRST microRNAs AND LINKS TO RNAi

Already in 1993, years before the discovery of RNAi and siRNAs, two papers were
published by the Ambros and Ruvkun labs describing a small RNA molecule in C. elegans
WKDWWXUQHGRXWWREHWKH¿UVWPLFUR51$HYHUWREHGHVFULEHGlin-4.30,31 lin-4 was named
as such because mutants display lineage defects during development. The molecular basis
of the lineage defects was the capability of lin-4 to repress the activity of another gene,
named lin-14WKURXJKLPSHUIHFWEDVHSDLULQJLQWHUDFWLRQVZLWKWKHƍ875RIWKHlin-14
mRNA. It was also clear that lin-4 came in two forms: a small 22 nucleotide version
and a longer 61 nucleotide version that could fold into an imperfect hairpin structure.
The small form of lin-4 contained all the bases required for the basepairing interaction
with lin-14 and hence was likely the active, or mature form. In a follow-up study it was
proposed that lin-4 represses lin-14 at the translational level.32 The broader relevance of
WKHVH¿QGLQJVUHPDLQHGXQFOHDUXQWLOZKHQDQRWKHUVPDOO51$JHQHZDVFORQHG
let-7.33 This second small RNA had many features in common with lin-4 but, in contrast to
lin-4, turned out to be extremely well conserved across bilaterian animals.34 This sparked