RNA Interference and Gene Silencing
History and Overview
May 20, 2002
Post-transcriptional gene silencing (PTGS), which was
initially considered a bizarre phenomenon limited to petunias and a few
other plant species, is now one of the hottest topics in molecular biology
(1). In the last few years, it has become clear that
PTGS occurs in both plants and animals and has roles in viral defense
and transposon silencing mechanisms. Perhaps most exciting, however, is
the emerging use of PTGS and, in particular, RNA interference (RNAi)
PTGS initiated by the introduction of double-stranded RNA (dsRNA)
as a tool to knock out expression of specific genes in a variety of organisms
(reviewed in 1-3).
How was RNAi discovered? How does it work? Perhaps more
importantly, how can it be harnessed for functional genomics experiments?
This article will briefly answer these questions and provide you with
resources to find in depth information on PTGS and RNAi research.
A Bizarre Phenomenon is Discovered:
Cosuppression and PTGS in Plants
More than a decade ago, a surprising observation
was made in petunias. While trying to deepen the purple color of these flowers,
Rich Jorgensen and colleagues introduced a pigment-producing gene under
the control of a powerful promoter. Instead of the expected deep purple
color, many of the flowers appeared variegated or even white. Jorgensen
named the observed phenomenon "cosuppression", since the expression of both
the introduced gene and the homologous endogenous gene was suppressed (1-5).
First thought to be a quirk of petunias, cosuppression
has since been found to occur in many species of plants. It has also been
observed in fungi, and has been particularly well characterized in Neurospora
crassa, where it is known as "quelling" (1-3).
But what causes this gene silencing effect? Although
transgene-induced silencing in some plants appears to involve gene-specific
methylation (transcriptional gene silencing, or TGS), in others silencing
occurs at the post-transcriptional level (post-transcriptional gene silencing,
or PTGS). Nuclear run-on experiments in the latter case show that the
homologous transcript is made, but that it is rapidly degraded in the
cytoplasm and does not accumulate (1, 3,
Introduction of transgenes can trigger PTGS, however
silencing can also be induced by the introduction of certain viruses (2,
3). Once triggered, PTGS is mediated by a diffusible,
trans-acting molecule. This was first demonstrated in Neurospora,
when Cogoni and colleagues showed that gene silencing could be transferred
between nuclei in heterokaryotic strains (1, 7).
It was later confirmed in plants when Palauqui and colleagues induced
PTGS in a host plant by grafting a silenced, transgene-containing source
plant to an unsilenced host (8). From work done in nematodes
and flies, we now know that the trans-acting factor responsible for PTGS
in plants is dsRNA (1-3).
Gene Silencing by dsRNA:
- Silencing of an endogenous gene caused by the
introduction of a transgene or infection by a virus.
This term, which can refer to silencing at the post-transcriptional
(PTGS) or transcriptional (TGS) level, has been primarily
adopted by researchers working with plants.
Gene Silencing (PTGS) - Silencing of an endogenous
gene caused by the introduction of a homologous dsRNA,
transgene or virus. In PTGS, the transcript of the silenced
gene is synthesized but does not accumulate because
it is rapidly degraded. This is a more general term
than RNAi, since it can be triggered by several different
- PTGS in Neurospora crassa induced by
the introduction of a transgene.
- RNA-induced silencing complex. A nuclease complex,
composed of proteins and siRNA (see below), that targets
and destroys endogenous mRNAs complementary to the siRNA
within the complex.
interference (RNAi) - Post-transcriptional gene
silencing (PTGS) induced by the direct introduction
of dsRNA. The term "RNA interference" was first used
by researchers studying C. elegans.
- Small interfering RNAs. Current models of PTGS
indicate that these 21-23 nucleotide dsRNAs mediate
PTGS. Introduction of siRNAs can induce PTGS in mammalian
cells. siRNAs are apparently produced in vivo by cleavage
of dsRNA introduced directly or via a transgene or virus.
Amplification by an RNA-dependent RNA polymerase (RdRP)
may occur in some organisms. siRNAs are incorporated
into the RNA-induced silencing complex (RISC), guiding
the complex to the homologous endogenous mRNA where
the complex cleaves the transcript.
RNAi Is Discovered in Nematodes
The first evidence that dsRNA could lead to gene
silencing came from work in the nematode Caenorhabditis elegans.
Seven years ago, researchers Guo and Kemphues were attempting to use antisense
RNA to shut down expression of the par-1 gene in order to assess
its function. As expected, injection of the antisense RNA disrupted expression
of par-1, but quizzically, injection of the sense-strand control
did too (9).
This result was a puzzle until three years later. It
was then that Fire and Mello first injected dsRNA a mixture of
both sense and antisense strands into C. elegans (10).
This injection resulted in much more efficient silencing than injection
of either the sense or the antisense strands alone. Indeed, injection
of just a few molecules of dsRNA per cell was sufficient to completely
silence the homologous gene's expression. Furthermore, injection of dsRNA
into the gut of the worm caused gene silencing not only throughout the
worm, but also in its first generation offspring (10).
The potency of RNAi inspired Fire and Timmons to try
feeding nematodes bacteria that had been engineered to express dsRNA homologous
to the C. elegans unc-22 gene. Surprisingly, these worms
developed an unc-22 null-like phenotype (11-13).
Further work showed that soaking worms in dsRNA was also able to induce
silencing (14). These strategies, whereby large numbers
of nematodes are exposed to dsRNA, have enabled large-scale screens to
select for RNAi-defective C. elegans mutants and have led to large
numbers of gene knockout studies within this organism (15-18).
RNAi in Drosophila
RNAi has also been observed in Drosophila. Although a strategy
in which yeast were engineered to produce dsRNA and then fed to fruit
flies failed to work, microinjecting Drosophila embryos with dsRNA
does effect silencing (2). Silencing can also be induced
by "shooting" dsRNA into Drosophila embryos with a "gene gun" or
by engineering flies to carry DNA containing an inverted repeat of the
gene to be silenced. Over the last few years, these RNAi strategies have
been used as reverse genetics tools in Drosophila organisms, embryo
lysates, and cells to characterize various loss-of-function phenotypes
The Biochemical Mechanism of RNAi
So how does injection of dsRNA lead to gene silencing?
Many research groups have diligently worked over the last few years to
answer this important question. A key finding by Baulcombe and Hamilton
provided the first clue. They identified RNAs of ~25 nucleotides in plants
undergoing cosuppression that were absent in non-silenced plants. These
RNAs were complementary to both the sense and antisense strands of the
gene being silenced (24).
Further work in Drosophila using embryo
lysates and an in vitro system derived from S2 cells shed more
light on the subject (3, 25, 26).
In one notable series of experiments, Zamore and colleagues found that
dsRNA added to Drosophila embryo lysates was processed to 21-23
nucleotide species. They also found that the homologous endogenous mRNA
was cleaved only in the region corresponding to the introduced dsRNA and
that cleavage occurred at 21-23 nucleotide intervals (26).
Rapidly, the mechanism of RNAi was becoming clear.
Current Models of the RNAi Mechanism
Both biochemical and genetic approaches (see "The Genes and Enzymes
Involved in PTGS and RNAi" below for a discussion of genetic approaches
used to undersand RNAi) have led to the current models of the RNAi mechanism.
In these models, RNAi includes both initiation and effector steps (27,
see also a Flash animation of "How
Does RNAi Work?", from reference 3).
In the initiation step, input dsRNA is digested into
21-23 nucleotide small interfering RNAs (siRNAs), which have also been
called "guide RNAs" (reviewed in 3, 18,
27). Evidence indicates that siRNAs are produced when
the enzyme Dicer, a member of the RNase III family of dsRNA-specific ribonucleases,
processively cleaves dsRNA (introduced directly or via a transgene or
virus) in an ATP-dependent, processive manner. Successive cleavage events
degrade the RNA to 19-21 bp duplexes (siRNAs), each with 2-nucleotide
3' overhangs (27, 28).
In the effector step, the siRNA duplexes bind to a nuclease
complex to form what is known as the RNA-induced silencing complex, or
RISC. An ATP-depending unwinding of the siRNA duplex is required for activation
of the RISC. The active RISC then targets the homologous transcript by
base pairing interactions and cleaves the mRNA ~12 nucleotides from the
3' terminus of the siRNA (3, 18, 27,
29). Although the mechanism of cleavage is at this date
unclear, research indicates that each RISC contains a single siRNA and
an RNase that appears to be distinct from Dicer (27).
Because of the remarkable potency of RNAi in some organisms,
an amplification step within the RNAi pathway has also been proposed.
Amplification could occur by copying of the input dsRNAs, which would
generate more siRNAs, or by replication of the siRNAs themselves (see
"Possible Role for RNA-dependent RNA Polymerase" below). Alternatively
or in addition, amplification could be effected by multiple turnover events
of the RISC (3, 18, 27).
The Genes and Enzymes Involved in PTGS and RNAi
Possible Role for RNA-dependent
Genetic screens in Neurospora, C. elegans, and Arabidopsis
have identified several genes that appear to be crucial for PTGS and RNAi.
Several of these, including Neurospora qde-1, Arabidopsis SDE-1/SGS-2
and C. elegans ego-1, appear to encode RNA-dependent RNA polymerases
(RdRPs). At first glance, it might be assumed that this is proof that
an RdRP activity is required for RNAi. Certainly the existence of an RdRP
might explain the remarkable efficiency of dsRNA-induced silencing if
it amplifed either the dsRNA prior to cleavage or the siRNAs directly.
But mutants of these genes have varying phenotypes, which makes the role
of RdRP in RNAi difficult to discern (1, 3,
In C. elegans ego-1 mutants ("ego" stands for
"enhancer of glp-1"), RNAi functions normally in somatic cells,
but is defective in germline cells where ego-1 is primarily expressed.
In Arabidopsis SDE-1/SGS-2 mutants ("SGS" stands for suppressor
of gene silencing), siRNAs are produced when dsRNA is introduced via an
endogenously replicating RNA virus, but not when introduced by a transgene.
It has been proposed that perhaps the viral RdRP is substituting for the
Arabidopsis enzyme in these mutants (1, 3,
17, 18). Although no homolog of an
RdRP has been found in flies or humans, an RdRP activity has recently
been reported in Drosophila embryo lysates (30).
One model of amplification, termed the "random degradative PCR"
model, suggests that an RdRP uses the guide strand of an siRNA as a primer
for the target mRNA, generating a dsRNA substrate for Dicer and thus more
siRNAs (27, 30). Evidence supporting
this model has been found in worms, whereas experimental results refuting
the model have been obtained from Drosophila embryo lysates (26,
Two C. elegans genes, rde-1 and rde-4 ("rde" stands
for "RNAi deficient"), are believed to be involved in the initiation step
of RNAi. Mutants of these genes produce animals that are resistant to
silencing by injection of dsRNA, but silencing can be effected in these
animals by the transmission of siRNA from heterozygous parents that are
not silencing deficient. The C. elegans rde-1 gene is a member
of a large family of genes and is homologous to the Neurospora qde-2
("qde" stands for "quelling deficient") and the Arabidopsis
AGO1 genes ("AGO" stands for "argonaute"; AGO1 was previously
identified to be involved in Arabidopsis development). Although
the function of these genes in PTGS is unclear, a mammalian member of
the RDE-1 family has been identified as a translation initiation factor.
Interestingly, Arabidopsis mutants of AGO1, which are defective
for cosuppression, also exhibit defects in leaf development. Thus some
processes or enzymes involved in PTGS may also be involved in development
(1, 3, 17, 18).
Important genes for the effector step of PTGS include the C. elegans
rde-2 and mut-7 genes. These genes were initially identified
from heterozygous mutant worms that were unable to transmit RNAi to their
homozygous offspring (16). Worms with mutated rde-2
or mut-7 genes exhibit defective RNAi, but interestingly, they
also demonstrate increased levels of transposon activity. Thus, silencing
of transposons appears to occur by a mechanism related to RNAi and PTGS.
Although the rde-2 gene product has not yet been identified, the
mut-7 gene encodes a protein with homology to the nuclease domains
of RNase D and a protein implicated in Werner syndrome (a rapid aging
disease) in humans (1, 3, 17,
18, 31). Perhaps this protein is a
candidate for the nuclease activity required for target RNA degradation.
PTGS Has Ancient Roots
Discoveries from both genetic and biochemical approaches point to the
fact that PTGS has deep evolutionary roots. Proposals have been put forth
that PTGS evolved as a defense mechanism against transposons or RNA viruses,
perhaps before plants and animals diverged (1, 3,
Interestingly, it was noted by many researchers that
disruption of genes required for RNAi often causes severe developmental
defects. This observation suggested a link between RNAi and at least one
A group of small RNA molecules, known as small temporal
RNAs (stRNAs), regulates C. elegans developmental timing through
translational repression of target transcripts. Research indicates that
the C. elegans lin-4 and let-7 stRNAs are generated
from 70-nt transcripts following the folding of these longer transcripts
into a stem-loop structure. The folded RNA molecules are cleaved to produce
22-nt stRNAs by the enzyme Dicer (called DCR-1 in C. elegans).
Thus Dicer generates both siRNAs and stRNAs, and represents an intersection
point for the RNAi and stRNA pathways (32-34).
Recently, nearly 100 additional ~22 nt RNA molecules,
termed microRNAs (miRNAs), were identified in Drosophila, C.
elegans, and HeLa cells (35-38). Much like lin-4
and let-7, these miRNAs are formed from precursor RNA molecules
that fold into a stem-loop secondary structure. The newly discovered ~22
nt miRNAs are believed to play a role in regulation of gene expression,
and at least two of them are known to require Dicer for their production
(37). It appears that the use of small RNAs for both
gene regulation and RNAi is a common theme throughout evolution.
Inducing RNAi in Mammalian Cells From Mechanism
Non-specific Gene Silencing by Long
While the natural presence of RNAi had been observed in a variety of organisms
(plants, protozoa, insects, and nematodes), evidence for the existence
of RNAi in mammalian cells took longer to establish. Transfection of long
dsRNA molecules (>30 nt) into most mammalian cells causes nonspecific
suppression of gene expression, as opposed to the gene-specific suppression
seen in other organisms. This suppression has been attributed to an antiviral
response, which takes place through one of two pathways.
In one pathway, long dsRNAs activate a protein kinase,
PKR. Activated PKR, in turn phoshorylates and inactivates the translation
initiation factor, eIF2a, leading to repression of translation. (39)
In the other pathway, long dsRNAs activate RNase L, which leads to nonspecific
RNA degradation (40).
A number of groups have shown that the dsRNA-induced
antiviral response is absent from mouse embryonic stem (ES) cells and
at least one cell line of embryonic origin. (41, 42)
It is therefore possible to use long dsRNAs to silence specific genes
in these specific mammalian cells. However, the antiviral response precludes
the use of long dsRNAs to induce RNAi in most other mammalian cell types.
siRNAs Bypass the Antiviral Response
Interestingly, dsRNAs less than 30 nt in length do not activate the PKR
kinase pathway. This observation, as well as knowledge that long dsRNAs
are cleaved to form siRNAs in worms and flies and that siRNAs can induce
RNAi in Drosophila embryo lysates, prompted researchers to test
whether introduction of siRNAs could induce gene-specific silencing in
mammalian cells (43). Indeed, siRNAs introduced by transient
transfection were found to effectively induce RNAi in mammalian cultured
cells in a sequence-specific manner. The effectiveness of siRNAs varies
the most potent siRNAs result in >90% reduction in target RNA
and protein levels (44-46). The most effective siRNAs
turn out to be 21 nt dsRNAs with 2 nt 3' overhangs. Sequence specificity
of siRNA is very stringent, as single base pair mismatches between the
siRNA and its target mRNA dramatically reduce silencing (44,
47). Unfortunately, not all siRNAs with these characteristics
are effective. The reasons for this are unclear but may be a result of
positional effects (46, 48, 49).
For current recommendations on designing siRNAs, see "siRNA
RNAi as a Tool for Functional Genomics
Although the history and mechanism of RNAi and PTGS
are fascinating, many researchers are most excited about RNAi's potential
use as a functional genomics tool. Already RNAi has been used to ascertain
the function of many genes in Drosophila, C. elegans, and
several species of plants. With the knowledge that RNAi can be induced
in mammalian cells by the transfection of siRNAs, many more researchers
are beginning to use RNAi as a tool in human, mouse and other mammalian
cell culture systems.
In early experiments with mammalian cells, the siRNAs
were synthesized chemically (Ambion is one of several companies that offer
custom siRNA synthesis).
Recently, Ambion introduced a kit (the Silencer
siRNA Construction Kit) to produce siRNAs by in vitro transcription,
which is a less expensive alternative to chemical synthesis, particularly
when multiple different siRNAs need to be synthesized. Once made, the
siRNAs are introduced into cells via transient transfection. Due to differences
in efficacy, most researchers will synthesize 34 siRNAs to a target
gene and perform pilot experiments to determine the most effective one.
Transient silencing of more than 90% has been observed with this type
of approach (44-46, 48, 49).
So far, injection and transfection of dsRNA into cells
and organisms have been the main method of delivery of siRNA. And while
the silencing effect lasts for several days and does appear to be transferred
to daughter cells, it does eventually diminish. Recently, however, a number
of groups have developed expression vectors to continually express siRNAs
in transiently and stably transfected mammalian cells (50-56).
Some of these vectors have been engineered to express small hairpin RNAs
(shRNAs), which get processed in vivo into siRNAs-like molecules capable
of carrying out gene-specific silencing (50, 53,
54, 56). The vectors contain the shRNA
sequence between a polymerase III (pol III) promoter and a 4-5 thymidine
transcription termination site. The transcript is terminated at position
2 of the termination site (pol III transcripts naturally lack poly(A)
tails) and then folds into a stem-loop structure with 3' UU-overhangs.
The ends of the shRNAs are processed in vivo, converting the shRNAs into
~21 nt siRNA-like molecules, which in turn initiate RNAi (50).
This latter finding correlates with recent experiments in C. elegans,
Drosophila, plants and Trypanosomes, where RNAi has been induced
by an RNA molecule that folds into a stem-loop structure (reviewed in
Another siRNA expression vector developed by a different
research group encodes the sense and antisense siRNA strands under control
of separate pol III promoters (52). The siRNA strands
from this vector, like the shRNAs of the other vectors, have 5 thymidine
termination signals. Silencing efficacy by both types of expression vectors
was comparable to that induced by transiently transfecting siRNA.
The recent studies on RNAi have taken the research world
by storm. The ability to quickly and easily create loss-of-function phenotypes
has researchers rushing to learn as much as they can about RNAi and the
characteristics of effective siRNAs. In the future, RNAi may even hold
promise for development of gene-specific therapeutics. Much has been learned
about this powerful technique, but additional information becomes available
on an almost daily basis (see The RNA
Interference Resource to learn about the very latest RNAi research
and tools). It is not an understatement to say that the field of functional
genomics is being revolutionized by RNAi.
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Flash Animation: How Does RNAi Work?
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