Gene Silencing by RNA

1 Interference and the Role

of Small Interfering RNAs
Natasha J. Caplen

CONTENTS

1.1 The Identification of Post-Transcriptional Gene-Silencing Mechanisms

1.2 RNA Silencing, siRNAs, and RISC
1.3 Species-Specific Aspects of RNA-Triggered Gene Silencing
1.4 RNAi and Epigenetic Endogenous RNA Silencing
1.5 Applications of RNAi-Based Technologies
References

The completion or near completion of several whole genome sequence projects has
highlighted two important issues: (1) a far higher percentage of transcription results in
the generation of non-coding RNAS than was originally predicted, and (2) there is
now a need to rapidly translate genomic information into functional data. The recent
identification of RNA interference (RNAi) an endogenous post-transcriptional gene-
silencing mechanism that requires formation of a non-coding RNA containing protein
complex, and the rapid development of technologies that exploit this mechanism to
determine gene function illustrates the need to characterize basic cellular processes so
that these can be exploited as scientific tools. This chapter summarizes RNA-mediated
epigenetic gene-silencing mechanisms, the identification and role of key players in
RNAi and how this work has impacted on both our understanding of the role of non-
coding RNAs in regulating endogenous gene expression and on the development of a
whole new research field utilizing RNAi-generated knockdowns, even up to a genome-
wide scale to study gene function.

1.1 THE IDENTIFICATION OF POST-TRANSCRIPTIONAL

GENE SILENCING MECHANISMS

Post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) has been

observed in a wide range of organisms including plants, with Nicotina and Arabidopsis
the species most widely studied; fungi, in particular Neurospora crassa; several

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 2 Gene Silencing by RNA Interference

Metazoan and Protozoan species, for example, Planarian, Hydra, and Trypanosoma;
and more complex organisms, particularly Caenorhabditis elegans, Drosophila, and
most recently mammalian cells both human and mouse.1–17 The term PTGS has been
used mostly to indicate epigenetic post-transcriptional gene-silencing in plants and
fungi (also known as co-suppression and quelling, respectively), whereas RNAi has
been mostly applied to a similar epigenetic gene-silencing effect seen in invertebrates
and mammals, though it has been suggested that the collective term of RNA silencing
is more appropriate for all of these processes.18 Central to PTGS and RNAi in all
species studied to date is the role of small RNA molecules (20 to 25 nts in length) and
the formation of a ribonucleoprotein complex called the RNA-induced silencing
complex or RISC.19–22 In plants, RNA silencing can be observed following the intro-
duction of multiple copies of a transgene and in cells infected with cytoplasmically
replicating RNA viruses.23 Plants defective for PTGS, show increased susceptibility
to infection and viruses produce proteins that block PTGS, leading to the hypothesis
that PTGS acts as an antiviral response in plants.24,25 The term RNAi was first used to
describe the inhibition of gene expression seen in C. elegans following the direct
injection into embryos of double-stranded RNA (dsRNA) of a sequence cognate to
the gene that was silenced.6 As in plants and fungi the silencing induced by dsRNA in
C. elegans is highly potent, suggesting that an intracellular process both amplifies and
spreads this effect.6,7,26 Double-stranded RNA was quickly shown to mediate a similar
inhibition of gene expression in other invertebrate species, including Drosophila.8,9
Though dsRNA generated through aberrant transcription from multiple transgenes
(particularly those arranged as an inverted repeat), or from a viral intermediate, or
introduced exogenously appeared to be the triggers of both PTGS and RNAi, it was
unclear for some time how degradation of the target RNA was mediated. The first
evidence suggesting a role for small RNA molecules in PTGS and RNAi emerged
from a plant study that showed that in cells where PTGS had been triggered (by a
transgene or virus) there was an accumulation of a small species of RNA estimated to
be approximately 25 nucleotides (nts) in size.19 Subsequent in vitro analysis of RNAi
in Drosophila cell extracts showed that a small species of RNA, now termed small or
short interfering RNAs (siRNAs), are enzymatically derived from the input double-
stranded RNA.20,22,27–29 Cleavage of the target mRNA was observed at a site that corre-
sponded to approximately the middle of the siRNA, suggesting that the siRNA acts as
guide for an enzyme-containing complex that leads to degradation of the target
mRNA.22,30 Since the identification of siRNAs, their central role in PTGS and RNAi
has been more fully defined and their relationship to endogenous small non-coding
RNAs has begun to be elucidated.

1.2 RNA SILENCING, SIRNAS, AND RISC

siRNAs processed from dsRNA within a cell are typically of 20 to 25 nts in length,
though most have been sized as 21 to 23 nt.20,22 The enzyme responsible for the pro-
cessing of siRNAs from dsRNA is an RNase III-like endonuclease termed Dicer.29 The
siRNAs are duplex RNA molecules with 2 or 3 nt 3' overhangs of ssRNA and carry a

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 Gene Silencing by RNA Interference and the Role of Small Interfering RNAs 3

5' phosphate group.20,22,28 It is unclear at exactly what point an individual siRNA is

unwound, a process that is ATP dependent, but stable RISC complexes appear to con-
tain just one strand of the siRNA; ssRNA can enter the RNAi pathway but do so fair
less efficiently.20,31–33 siRNAs can be generated from all parts of the input dsRNA but
there may be an accumulation of those sequences that most effectively induce silenc-
ing.34,35 This selection may relate to certain features of the sequence of siRNAs that
influence the effectiveness of a particular siRNA. An important feature may be the
internal energy of the siRNA molecule, particularly the internal stability of the 5' end
of the antisense strand compared to the target sequences, which in most effective siRNAs
seems to be lower than other portions of the molecule; it is assumed that this allows
the siRNA to be efficiently unwound.34 The internal free energy around the predicted
cleavage site also appears to need to be relatively low in comparison with adjacent
regions of the molecule.34 There is evidence from studies of RNAi in Drosophila cells
that at least one protein, called R2D2, is involved in the transfer of siRNAs as they are
generated by Dicer onto proteins that from part of the RISC complex.36 R2D2 may
influence the polarity (sense/antisense orientation) with which the siRNA interacts
with components of RISC.
Several protein components of RISC have been isolated. The first to be identified,
Argonaute 2 (Ago2) appears to form the core of RSIC.37 Ago2 and Dicer are both
members of the PAZ (PIWI, Argonaute, and Zwille/Pinhead) domain-containing family
of proteins.38,39 Recent structural analysis of the Drosophila Ago2 protein has shown
that the PAZ domain can form a nucleic acid-binding fold.40,41 The affinity of the inter-
action of this domain and RNA is relatively weak, but the presence of a 3' single-
stranded RNA overhang appears to be critical to the interactions.40,41 RNA, single- and
double-stranded, can also bind, though in the case of duplex DNA the binding is inde-
pendent of a 3' overhang.41 Other proteins associated with RISC include in Droso-
phila, the Vasa intronic gene product (a p68 RNA helicase homolog) and the fragile X
mental retardation protein, and in human cells the homolog of Ago2–E2FC and Gemin3
and Gemin4.31,42–46 The antisense single-stranded siRNA component of RISC leads to
localization of the RISC complex through perfect sequence alignment of the two RNA
molecules. Cleavage of the target occurs at a position ~10 nts from the first nucleotide
that represented the first based pair from the 5' end of the original siRNA.28 The
ribonuclease(s) responsible for this cleavage is not defined but an enzyme with ho-
mology to Tudor staphylococcal nuclease is associated with RISC and effective RNAi.47

1.3 SPECIES-SPECIFIC ASPECTS OF RNA-TRIGGERED

GENE SILENCING

The identification of siRNAs as intermediate molecules in the RNAi pathway was

essential to establishing the presence of RNAi in mammalian cells. Many viruses pro-
duce dsRNA intermediates during their life cycle, and thus as part of an antiviral response
mammalian cells have evolved potent responses to dsRNA that result in a nonspecific
down-regulation in gene expression. The best-characterized responses involve the
triggering RNase L-dependent pathways through activation of 2'5'-oligoadenylate

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 4 Gene Silencing by RNA Interference

synthetase and the interferon-associated dsRNA-dependent kinase (PKR) but there is

also evidence of additional dsRNA-sensitive pathways. Though only limited data exist
maximal activation of these pathways may be dependent on the size and concentration
of the triggering dsRNA molecule. To test if RNA molecules modeled on the structure
of siRNAs could mediate sequence-specific cleavage in mammalian cells, two groups
transfected siRNA molecules made from chemically synthesized oligoribonucleotides
into a variety of different cell lines.16,22 Initially, using a plasmid/siRNA co-transfection
model system of RNAi sequence-specific inhibition was detected at both RNA and
protein level.16,22 Endogenous gene expression could also be downregulated using
siRNAs against Lamin A/C.22
In the last two years these findings have been utilized and extended by many
groups using cell lines derived from a very broad range of cells and against transcripts
involved in a broad range of cellular processes. Double-stranded RNAs of over 30 nts
can be used in cells that have an attenuated response to larger dsRNAs such as those
derived from embryonic cells, but in the vast majority of studies molecules are used
based on a siRNA structure.48,49 In most cases the expression of gene expression has
been determined to be specific; however, non-specific effects have been reported that
may reflect some low level triggering of non-RNAi dsRNA dependent pathways,
concentration-dependent saturation of RISC which may effect its cellular role, and
off-target effects due to imperfect sequence matches that can trigger alternative RNA
silencing mechanisms.50–56 Mammalian cells show no amplification or spread of the
inhibition of gene expression mediated by synthetic siRNAs.30,31,54,57–59 To overcome
this limitation when applying RNAi as a functional genomics tool in mammalian cells,
many groups have developed siRNAs that are expressed as a single transcript that
forms a stem–loop structure where the stem corresponds to the siRNA.60–64 A key
advantage of these short hairpin RNAs (shRNAs) is that they can be expressed from
plasmids, usually from RNA polymerase III promoters, enabling the generation of
cell lines expressing a shRNA against a particular transcript.61,63,64
An early and important observation unique to gene silencing in plants, Neuro-
spora and C. elegans is the amplification and spread of the gene-silencing effect. The
local (cell-to-cell plasmodesmata-based movement) and distant (phloem-based trans-
port) spread of RNA silencing seen in plants can be observed following the use of an
exogenous nucleic acid trigger and transgenic plants that induce RNA silencing in
grafts.65–67 The molecule(s) that mediate this spread have not been formally identified,
but candidates include siRNAs, larger dsRNAs, and an RNA/protein complex.68 Re-
cent data suggests that plants generate two different species of siRNA, one of ~21 to
22 nts in length and another group of ~24 to 26 nts that are probably generated by
different Dicer enzymes.69,70 The 21 to 22 nt siRNAs appear to be critical for mediat-
ing localized RNA silencing, with one model suggesting that short siRNAs can move
directly to ~10 to 15 cells but for more extensive spread synthesis of additional siRNAs
is required.71 The intracellular generation of secondary siRNAs through the activity of
an RNA-dependent RNA polymerase (RdRp) has been identified in plants (Arabidopsis,
SGS2/SDE1), N. crassa (QDE-1) and C. elegans (Ego-1, RRF-1).72–76 It is presumed
that transitive RNA silencing or transitive RNAi, as this amplification process is termed,
uses the homologous ssRNA transcript as a template to produce additional dsRNA

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 Gene Silencing by RNA Interference and the Role of Small Interfering RNAs 5

that is then subjected to Dicer to generate further siRNA. In plants, the nature of the
siRNA generated following amplification is unclear. One study suggests that only
siRNAs of 21 nts are produced, whereas another study observed the generation of only
the longer species of siRNA.70,71 This difference may reflect species or model system-
specific variations. The role of the larger siRNAs is unclear but in one study these
have been linked to expression from endogenous retrotransposons that resulted in sys-
temic and stable silencing and it has been suggested that it is this species that could
act as a mobile silencing signal through the vascular (phloem) system of plants that
could then trigger the intracellular production of small siRNAs at a distant site.69,71
Additional evidence that the systemic spread of RNA silencing in plants could be
mediated in different ways depending on the nature of the initiating molecule comes
from data using grafting models that showed a difference in the induction of distant
RNA-silencing effects depending if the trigger was transgene or amplicon derived.77
In Neurospora the QDE-1 RdRp catalyzes at least two activities that could relate
to the mediation of RNA silencing; the generation of full length RNA duplexes from
ssRNA templates (with no requirement for a primer) that could then act as substrate
for Dicer, and shorter < 21 nt ssRNA molecules corresponding to different parts of the
template that may interact directly with RISC.78 In C. elegans not only is there evidence
for the presence of an RdRp-dependent amplification that generates secondary siRNAs,
there is also evidence for the active transport of dsRNA through the transmembrane
protein SID-1 which was identified by analysis of C. elegans mutants that were sensitive
to local RNAi but that were defective for systemic RNAi.75,76,79–81 Expression of SID-1
as a transgene in Drosophila cells can induce these cells to take up dsRNA from medium
that can then mediate RNAi.81 Drosophila cells naturally show no cell-to-cell movement
of RNA silencing and most studies suggest that there is no RdRP amplification of
RNAi in Drosophila, though one study has reported synthesis of dsRNA in Drosophila
cell extracts.30,82,83

1.4 RNAI AND EPIGENETIC ENDOGENOUS RNA SILENCING

A highly coordinated control of gene expression is essential for normal development

and differentiation and for the maintenance of appropriate cellular function. In addition,
there is a need for the cell to protect itself from the potential effect of aberrant expres-
sion from the large amount of selfish genetic material that is present within the ge-
nome. Until recently most research on the control of gene expression has focused on
gene regulation at a genomic, transcriptional or translational level; however, with the
identification of RNA silencing the role of RNA both as a mediator and as a target for
controlling expression has become increasingly apparent. The first evidence for a direct
link between RNAi and the control of endogenous gene expression came with the
identification of a role for Dicer in the processing what are now known to be an impor-
tant group of endogenous non-coding RNAs termed micro RNAS (miRNAs).84–90 Micro
RNAs are 21–23 nt ssRNA molecules that are processed by Dicer from large stem-
loop single-stranded RNAs termed precursor miRNAs in an asymmetric manner.91–94
Precursor miRNAs are themselves processed from the initially expressed transcript

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 6 Gene Silencing by RNA Interference

by another PAZ domain containing RNase III nuclease called Drosha.95,96 Micro RNAs
also interact with many, if not all, of the same components of the RISC complex that
siRNAs interact with.31,42–46 However, miRNA-RISC complexes appear to mediate
silencing by a somewhat different manner in that the interaction between miRNAs and
the putative target transcripts seems to require only minimal homology between the
sequences of the two RNA species that leads to a structural perturbation of the mRNA
and/or a locking of the RISC complex at the site of binding so that protein translation
is blocked.97–101 The action of miRNAs may be different in plants in that the miRNAs
in plants have exact sequence matches with their putative target mRNAs. This sug-
gests that here either an siRNA-like cleavage or an miRNA-like protein translational
blockade could occur.70,86,102–105 Very few target transcripts that miRNAs regulate are
known, but of those that have been characterized many are associated with develop-
mentally regulated genes which may explain, at least in part, the embryonic lethal
phenotype seen in Dicer transgenic knockout mice.105–108
A second role for RNAi in regulating endogenous expression links it to modifi-
cations of genomic DNA that may induce repression of transcription from a variety of
sources including coding sequences, heterochromatin regions and other repetitive se-
quences including transposons. In plants methylation of genomic DNA is often seen
within the transcribed region that encodes the transcript that is the subject of RNA
silencing.109,110 This has been best studied when an RNA virus carrying a sequence
homologous to the endogenous transcript triggers RNA silencing; however, it is un-
clear how the RNA–DNA interaction triggers methylation or which methylase is in-
volved.111–113 A link between heterochromatin formation and RNAi has currently only
been studied in Schizosaccharomyces pombe where Dcr (Dicer), Ago2, and RdRp knock-
out mutant yeast, show transcription from previously silent regions as a result of the
loss of histone H3 lysine methylation, which allows for Swi6 localization and thus the
spreading of heterochromatin formation.114,115 Overlapping non-coding RNAs expressed
from both strands of the repeat sequences within heterochromatin regions have been
purified and siRNAs corresponding to these repeat regions have been identified.114,116
Loss of heterochromatin RNAi in S. pombe leads to disruption in centromere and
telomere function, leading to disruption of both mitosis and meiosis.114,117 Another set
of repeat sequences that may be silenced endogenously by RNAi is selfish genetic
material such as integrated retroviral and retrotransposons. In C. elegans some RNAi
mutants show enhanced activation of transposition and in S. pombe transcribed long
terminal repeats can induce RNAi-dependent chromatin silencing that can extend to
closely neighboring transcribed sequence.118–120

1.5 APPLICATIONS OF RNAI BASED TECHNOLOGIES

Sequencing and gene expression studies on a whole genome scale have generated
enormous amounts of data that imply a function for a particular gene, but a direct
functional analysis on a similar scale has not been possible. Determining the function
of a gene through knockout transgenics has been of enormous value but this has not
been applicable on a high throughput basis and is often restricted by aspects of the

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 Gene Silencing by RNA Interference and the Role of Small Interfering RNAs 7

biology of the organism. In contrast technologies based on knockdowns generated on

PTGS and RNAi have been rapidly developed on a scale from single gene to whole
genome, with application both at cell culture and whole organism levels using tran-
sient and stable induction of an RNAi phenotype. Subsequent chapters in this book
will describe in detail the different technologies that have been developed that exploit
the RNAi pathway. Some of these technologies are species specific, whereas the use of
others will be dependent on the biological question that is being asked. Key applica-
tions for RNAi-based technologies include direct gene analysis on a gene-by-gene
basis, but this has also been extended to examine whole pathways and to examine
broad RNAi phenotypes on a high throughput basis in whole organisms.121–127 One
advantage of RNAi-based knockdowns is that multiple gene knockdowns can be si-
multaneously or consecutively analyzed and, particularly in mammalian cells because
different siRNA/shRNAs sequences can produce different levels of knockdown, the
effect of modulating expression by inducing different degrees of inhibition can be
studied.128 In addition, RNAi has the potential to generate new model systems both
cell line- and whole organism-based.129–131 From a clinical perspective, an important
application of RNAi knockdowns in mammalian cells is for drug target validation and for
determining the specificity with which a particular drug interacts with a particular target
molecule. In addition, as result of RNAi functional studies many new drug targets will
be identified for which small molecule drugs can be developed, or it may be possible
to develop the RNAi molecule itself as an inhibitor, leading to the concept of RNAi as
a therapeutic approach.132–134

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