RNA interference and double-stranded-RNA- activated pathways C.A. Sledz and B.R.G. Williams

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RNA interference and double-stranded-RNAactivated pathways
C.A. Sledz and B.R.G. Williams1
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, U.S.A.
Abstract
RNAi (RNA interference) has become a powerful tool to determine gene function. Different methods of
expressing the short ds (double-stranded) RNA intermediates required for interference in mammalian
systems have been developed, including the introduction of si (short interfering) RNAs by direct transfection
or driven from transfected plasmids or lentiviral vectors encoding sh (short hairpin) RNAs. Although RNAi
relies upon a high degree of specificity, recent findings suggest that off-target non-specific effects can
be encountered. We found that transfection of siRNAs can results in an interferon-mediated activation
of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway and global upregulation of interferon-stimulated genes. This effect is mediated in part by the dsRNA-dependent protein
kinase PKR, as this kinase is activated by the 21 bp siRNA, and is required in response to the siRNAs.
However, the transcription factor IRF3 (interferon-regulatory factor 3) is also activated by siRNA as a
primary response, resulting in the stimulation of genes independent of an interferon response. In cells
deficient in IRF3, this response is blunted, but can be restored by re-introduction of IRF3. Thus siRNAs induce
complex signalling responses in target cells, leading to effects beyond the selective silencing of specific
genes.
Intense interest in the field of RNAi (RNA interference) has
facilitated its rapid movement from a biological phenomenon
to a valuable research tool used to silence target gene expression. This application is now widely used to determine
gene function and has potential as a therapeutic agent. Although the mechanism has only recently been uncovered, the
concept of expression interference has been a recurring
theme in biology for over 20 years. Schmidt and colleagues
identified a non-toxigenic strain of Aspergillus flavus and
found ds (double-stranded) RNA to be the cause of this attenuation (described in [1]). More than a decade ago, a posttranscriptional silencing event in petunias that was attributed
to dsRNA was reported [2]. In yet another experimental
system, Romano and Maciano [3] described a similar silencing
mechanism that they termed quelling, in the fungus Neurospora crassa. These studies hinted at the existence of a
well-conserved mechanism of dsRNA-mediated specific gene
silencing. However, it was the elucidation of the mechanism
responsible for this response that led to the realization of its
potential as a powerful research tool for manipulating gene
expression.
Drawing upon these previous studies, Fire and Mello
determined that dsRNA molecules, like those produced
during viral replication, induce a potent and specific geneKey words: double-stranded RNA (dsRNA), interferon, RNA interference (RNAi), RNA
polymerase, short interfering RNA (siRNA), transcription.
Abbreviations used: ds, double-stranded; ISG, interferon-stimulated gene; miRNA, microRNA; PKR, dsRNA-dependent protein kinase; RNAi, RNA interference; shRNA, short-hairpin RNA;
siRNA, short interfering RNA; UTR, untranslated region.
1
To whom correspondence should be addressed (email williab@ccf.org).
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silencing response in the nematode, Caenorhabditis elegans
[4]. Subsequent work by multiple groups showed that the
potentially harmful viral dsRNA is recognized and cleaved
into 21–23 nucleotide siRNAs (short interfering RNAs) [5].
The siRNA is then incorporated into the RISC (RNA-induced silencing complex), which targets the mRNA of an
homologous sequence for degradation much more efficiently
than antisense-mediated silencing (reviewed in [6]). Since
organisms such as C. elegans and Drosophila use the specific
RNAi pathway to respond to dsRNA-containing challenges,
it was relatively easy to apply this concept to artificial assays
set up to determine gene function.
It was initially thought that this process would not work
in mammalian systems due to the presence of an innate
immune defence mechanism directed against dsRNA-containing challenges, such as virus infection. Introduction of
dsRNA molecules into most mammalian cells causes global,
non-specific suppression of gene expression.
Although Toll-like receptor 3 has been identified as
a dsRNA-response protein [7], the two best characterized
dsRNA pathways found in most cell types signal through
the dsRNA recognition proteins PKR (dsRNA-dependent
protein kinase) and 2 ,5 -oligoadenylate synthetase and have
been recognized for over 20 years. Activation of both of
these pathways by dsRNA results in the general inhibition
of protein synthesis. PKR autophosphorylates in response
to dsRNA and subsequently phosphorylates its substrates,
one of which is eIF2 (eukaryotic initiation factor 2), leading to translation inhibition [8]. The activation of 2 ,5 oligoadenylate synthetase results in the formation of
Genes: Regulation, Processing and Interference
2 ,5 -oligoadenylates that bind to and activate RNaseL. This
enzyme then non-specifically cleaves both cellular and viral
RNAs, also causing translation inhibition [9].
In addition to its role as a translational inhibitor, PKR
also acts as a signalling transducer. Following activation,
PKR initiates a signalling cascade that results in the production of interferons. Interferon, in turn, activates cellular
signalling pathways that culminate in the nucleus with the
up-regulation of interferon-stimulated genes, mediators of
antiviral and antiproliferative and pro-apoptotic activity [10].
Cells exposed to interferon are sensitive to very low levels of
dsRNA, eventually leading to cell death. These properties
initially precluded the use of RNAi based on long (>100 bp)
dsRNA targets of Dicer as an effective research tool in these
systems.
In 2001, Tuschl and colleagues found that they could initiate RNAi in mammalian systems by the intracellular introduction of artificially synthesized mimics of Dicer products
of 21–23 bp [11]. By excluding long dsRNAs from the
process, researchers hoped to also eliminate non-specific side
effects, resulting in activation of the interferon system.
The enthusiasm that greeted this successful application in
the mammalian system led to its widespread application
and an underlying assumption of specificity. However, there
was an accompanying failure to take into account the multiple
roles that short dsRNAs can have in regulating signal transduction and gene expression. In particular, while a large
number of dsRNA-binding proteins had been identified and
the structural requirements for activation of enzymes such
as PKR were known, these were overlooked [12,13]. The
alternative pathways turn out to be a source of non-specific
off-target effects. Some of these are not apparent unless
global gene expression studies are performed that look at the
effects of siRNAs beyond the expected suppression of target gene expression. A siRNA-specific, as opposed to a targetspecific, trend emerged from these studies and up-regulation
of interferon-stimulated genes, as well as non-specific effects
on non-targeted genes are frequently observed.
Studies into a distinct type of short dsRNAs, miRNAs
(micro-RNAs), have added to our understanding of how
cells deal with short dsRNA molecules. miRNAs make up a
group of small non-translated RNAs that regulate the timing
of developmental events in an organism. Similar to siRNAs,
miRNAs are 21–25 nucleotides long, mediate the downregulation of target genes, and are produced as a result of
Dicer activity. Despite these similarities, miRNAs downregulate target gene expression after translation initiation by
binding to a region of partial complementarity in the 3 UTR
(untranslated region) of the target mRNAs and appear to
be the primary products of Dicer action during unstressed
conditions [14].
Since such similar formation pathways exist for the two
types of short RNAs, it is feasible that a subset of siRNAs
will have miRNA activity. Gene expression studies aimed
at exploring this possibility have found that this is the case.
A subset of siRNAs have been shown to induce post-translational suppression of target gene expression, as seen with
miRNA activity. In addition, the non-specific inhibitory
effects are seen at low siRNA concentrations and only partial
complementarity to the suppressed gene is required. While a
single mismatch between an siRNA and its target will reduce
specific silencing efficiency, that same siRNA may still be able
to down-regulate the expression of non-targeted genes that
contain regions of partial complementarity [13,14].
When computational analyses were performed on the
siRNAs, multiple potential 3 UTR targets were identified
that contained partial sequence similarity to each strand of
a given siRNA [14]. Even a relatively stringent algorithm,
allowing for target matches with a minimum of eight complementary nucleotides at either end of the siRNA and G:U
wobble base pairing between the siRNA and the 3 UTR
target, produced multiple hits in nine out of 26 siRNA strands
tested [14]. If siRNAs can indeed act as miRNAs and control
the expression of multiple genes, global expression profiles
must be analysed to ensure siRNA specificity, especially when
these studies are used to determine gene function.
Although siRNAs were initially thought to be too short to
induce a dsRNA-initiated response initiated by PKR or 2 ,5 oligoadenylate synthetase, early assays (using what would
now be considered non-optimized siRNA conditions)
resulted in up-regulation of certain classic ISGs (interferonstimulated genes), including GBP1, CCL2, FGF2, CXCL11
and ISG20. The observed up-regulation was not seen in
the control or mock-transfected samples, proving that the
effect was due to the intracellular presence of the ds siRNA.
These non-specific effects were concentration-dependent and
appeared to be consistent for all chemically synthesized
siRNAs tested. Microarray experiments revealed that chemically synthesized siRNAs trigger the activation of signalling components that overlap, but are not identical with,
those regulated by interferon [15]. Similarly, we reported
up-regulation of the ISG Stat 1, in addition to activation of
the dsRNA-response protein PKR, in response to multiple
chemically synthesized siRNAs [16]. While recent optimization studies of siRNAs through efficacy-determining
algorithms (as discussed later in this review) can alleviate
these effects, significant ISG up-regulation by siRNAs has
been noted. However, not all chemically synthesized siRNAs
cause ISG up-regulation, although in most cases this has been
determined by measuring the level of only OAS2, a highly
inducible ISG [14,17]. There are a number of possibilities
to explain this result, including siRNA sequence specificity
and/or cell-type specificity.
In addition, it was determined that not all siRNAs targeting
a particular gene are effective to the same degree at silencing its expression. Because no obvious trend could be determined that ruled an siRNA’s level of efficiency, such as
secondary structure at the target sequence site or location
of the target region within the gene, a systematic analysis was
needed to define specific determinants of siRNA efficiency.
Researchers from different reagent supplier companies,
including Dharmacon and Ambion, have expended considerable effort determining the siRNA characteristics that
increase functionality while minimizing the non-specific
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effects. These two ideals may not be mutually exclusive; a
highly efficient siRNA can be used at low concentrations,
reducing the potential for siRNA concentration-dependent,
non-specific side effects. Dharmacon’s study in particular
identified eight characteristics associated with siRNA
functionality and the application of an algorithm that incorporates these criteria to siRNA design greatly improves
the success rate of siRNA selection [18]. Applying these
constraints to siRNA design not only increases the silencing
efficiency of the target, but also decreases the number of test
siRNAs that must be designed before finding one of desired
efficiency.
Under these optimized conditions, RNAi assays using
chemically synthesized siRNAs have been optimized and
non-specific side effects are much reduced. However, there
are drawbacks. Assays using this method are obviously transient in nature, and the suppressed phenotype is lost within
approx. 1 week. While this approach is reliable for shortterm studies of gene expression, it cannot substitute for the
utility of knockout mouse models. This is not a concern in
organisms such as C. elegans, because they express an RNAdependent RNA polymerase that facilitates the propagation
of siRNA expression. In these systems, the suppressed
phenotype is not only maintained, but is also passed on to
future generations [19]. Another consideration is that siRNA
synthesis is very costly, and this limits the number and scale
of experiments that can benefit from this technique.
To address these issues, a system for the stable expression
of siRNAs has been developed. Mammalian expression vectors were designed to direct the synthesis of siRNAs
from integrated vectors [20,21]. In most cases, the targetspecific insert is made up of a 19-nucleotide sequence complementary to the target, followed by a short spacer and
the reverse complement of the same target sequence. Once
transcribed, a 19 bp stem-loop structure, termed shRNA
(short-hairpin RNA), forms that is able to induce the downregulation of target gene expression via elements of RNAi
machinery. A polymerase III promoter was initially used in
these constructs, as they produced RNAs that mimic the
requirements for an efficient siRNA. These requirements
include, but are not limited to, the absence of a polyadenylate
tail and a termination signal that yields transcripts with a 3
overhang.
This idea has been applied to kits that are commercially
available which enzymically synthesize large amounts of
siRNAs in vitro using a T7 RNA polymerase-based system.
Clearly, the siRNAs produced by this method will also
produce a transient phenotype, but it allows for the rapid,
and relatively inexpensive, production of large amounts
of siRNAs. While very effective at silencing target gene expression, the siRNAs synthesized from T7 RNA polymerases
have moved farther away from the natural biological process
of RNAi and non-specific side effects have emerged.
It was initially noted that siRNAs produced from both
the expression of the endogenous vectors [17] and from the
transfection of in vitro transcribed siRNAs [16] induce a
robust interferon response. Microarray studies illustrate the
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Figure 1 Up-regulation of ISGs in response to GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) siRNA transfection
(A) Section of ISG array [16] showing specific down-regulation (green
spot colour) of GAPDH and up-regulation (red spot colour) of three
unique ISGs in response to 50 nM siRNA transfection. (B) Gene tree representing ISGs induced over a factor of 2 in at least one of the two
siRNA-transfected samples, stable expression of housekeeping genes,
and the expression of the targeted GAPDH. All expression levels are
relative to an untreated, control sample. M, mock transfection; 50 and
100 refer to siRNA concentration (in nM) relative to the final transfection
volume; HSKP, housekeeping genes.
concentration-dependent, global up-regulation of ISGs in
response to T7-synthesized siRNAs (Figures 1 and 2).
These non-specific effects represent a robust interferon
response, unlike that seen in earlier studies using chemically
synthesized siRNAs, where only a subset of ISGs were
up-regulated. While the direct cause of these side effects
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Figure 2 Primary and secondary signalling by siRNA
In addition to interaction with RISC (RNA-induced silencing complex) to mediate RNA silencing, siRNA can activate TLR3 (Tolllike receptor 3) and/or PKR to induce both primary (as exemplified by interferon β and p56) and secondary (protein-synthesisdependent ISGs) transcriptional events. The transcription factor IRF3 (interferon-regulatory factor-3) is a key mediator for
both TLR3-dependent and -independent signalling by siRNAs. IFN, interferon.
is still being explored, evidence suggests that the pol III
promoters, which produce siRNAs that effectively mimic
natural siRNAs, may be the source of the problem. In a recent
paper, Rossi and colleagues presented evidence showing that
the 5 -triphosphate, characteristic of all RNAs transcribed
from pol III promoters, is required for interferon induction
[22]. Removing the 5 -triphosphate from the siRNAs greatly
attenuates the interferon response. The molecular basis for
this effect remains to be explored. However, another report
suggested an alternative mechanism as the source of interferon
induction: Iggo and colleagues determined that interferon induction in response to RNAi vectors could be traced
to the presence of an AA dinucleotide near the transcription
start site [23]. Again, although much is known about
the signalling mechanisms triggering interferon production,
exactly how this transcription start site feature would interact
with components involved in interferon induction is not
clear. Regardless of the mechanism, it does appear that some
component near the 5 end of the pol III-driven transcript
contributes to interferon induction by shRNA-producing
vectors.
These observations beg the question of whether shRNA
vectors driven by a pol II promoter, such as those currently
being developed, will have the same adverse, non-specific
effects. While pol III is responsible for transcribing many
RNA genes (such as tRNA, 5 S rRNA, and U6 snRNA
genes), pol II is generally responsible for transcribing proteinencoding genes. RNAs produced by pol II activity are posttranscriptionally processed into a product that does not
contain a 5 -triphosphate. This difference in the putative
interferon-stimulating region of siRNAs may help to
attenuate, if not eliminate, ISG activation.
The results reviewed here clearly emphasize the need for
stringent controls used in siRNA experiments. Measuring
only the mRNA levels of the target compared with a nontargeted control gene such as GAPDH (glyceraldehyde-3phosphate dehydrogenase) is not sufficient. In certain cases,
non-specific effects that include both the up-regulation and
suppression of non-targeted genes are observed. The problem
lies in the fact that these ‘certain cases’ can not yet be
clearly defined before experiments using these techniques are
performed. Newly developed algorithms help in this process,
but our current understanding of the role that short, noncoding RNAs play in the regulation of gene expression is far
from complete. While these algorithms take into account the
currently accepted dogma of RNAi, there is still a lot to learn
about the non-specific effects of siRNAs. Because the benefit
of RNAi applications to both basic and medical research is
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substantial, the focus is not to discredit siRNA experiments,
but to develop strategies that maintain a high level of siRNA
efficiency while minimizing the non-specific off-target effects
that have been associated with siRNA expression.
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Received 30 July 2004