Advanced Information on The Nobel Prize in Physiology or Medicine 2006

RNA INTERFERENCE
This year’s Nobel Prize in Physiology or Medicine is shared by Professor Andrew Z. Fire at Stanford
University, California, USA, and Professor Craig C. Mello at the University of Massachusetts Medical
School in Worcester, USA. They receive the prize for their discovery that double-stranded RNA trig-
gers suppression of gene activity in a homology-dependent manner, a process named RNA interference
(RNAi). Their discovery revealed a new mechanism for gene regulation, and the biochemical machinery
involved plays a key role in many essential cellular processes. Double-stranded RNA synthesized within
the cell can reduce or abolish gene activity by RNAi-like mechanisms. This control system for gene
expression has proven to be important for both the development of an organism and the physiological
functions of cells and tissues. Furthermore, RNAi protects against RNA virus infections, especially in
plants and invertebrate animals, and secures genome stability by keeping mobile elements silent. Today,
double-stranded RNA is used as a powerful tool to experimentally elucidate the function of essentially
any gene in a cell. The discovery of RNAi has already had an immense impact on biomedical research
and will most likely lead to novel medical applications in the future.

Introduction
The gene expression process is of fundamental importance for all living organisms. Most genes reside in the
chromosomes located in the cell nucleus and express themselves via proteins synthesised in the cytoplasm.
The genetic material was identified as deoxyribonucleic acid (DNA) in 1944 (ref. 1) and the double-helical
nature of DNA was revealed in 1953 (by Francis Crick, James Watson and Maurice Wilkins; Nobel Prize in
Physiology or Medicine in 1962). At that time, the main problem outstanding was how DNA in the cell
nucleus could govern protein synthesis in the cytoplasm. It was proposed that another nucleic acid, single-
stranded ribonucleic acid (RNA), acts as an intermediary in the process, and the so-called Central Dogma
was formulated, i.e. the idea that the genetic information is transcribed from DNA to RNA and then trans-
lated from RNA into protein. The RNA carrying the genetic information was first believed to be the RNA
in ribosomes; for several years the hypothesis was formulated as “one gene-one ribosome-one protein”. In
1961, Francois Jacob and Jacques Monod presented a visionary gene control model, for which they received
the Nobel Prize in Physiology or Medicine in 1965 together with André Lwoff. In their model, they proposed
that the gene is transcribed into a specific RNA species, messenger RNA (mRNA). Soon afterwards it was
demonstrated that a short-lived, non-ribosomal RNA directs the synthesis of proteins. Subsequently, Marshall
Nirenberg and Gobind Khorana broke the genetic code and could assign code words (codons; triplets of nucleo-
tides) to the twenty amino acids (they received the Nobel Prize in Physiology or Medicine in 1968 together with
Robert Holley). Francis Crick predicted that an RNA molecule could act as an adaptor between mRNA and the
amino acid, and a short, stable RNA, transfer RNA (tRNA) was soon identified as the predicted adaptor.

For many years, messenger RNA was believed to correspond to an uninterrupted nucleotide sequence in the
DNA. It therefore came as a complete surprise when Phillip Sharp and Richard Roberts showed in 1977 that
the mRNA sequence could be distributed discontinuously in the genome (the split gene concept; Nobel Prize in
1993). It was known that long RNA molecules (pre-mRNA, heterogeneous nuclear RNA) are trimmed to much
shorter mature mRNAs, and Sharp and Roberts therefore suggested that the mRNA sequences, the exons, are
likely to be cut out from the primary transcript and spliced, while the intervening sequences, the introns, are
degraded. It was immediately realised that the discontinuous arrangement of the mRNA sequences in DNA had
important evolutionary implications. In addition, the RNA splicing process could generate different mRNAs,
and more than one protein can emanate from the primary transcript (alternative splicing).

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The discovery that RNA can act as a catalyst gave a radically new perspective on the roles of RNA
(Nobel Prize in Chemistry to Sidney Altman and Thomas Cech in 1989). It was soon revealed that
RNA is able to catalyse its own replication and the synthesis of other RNA molecules (the ribozyme
concept), which led to the idea that RNA was the first genetic material on earth. An “RNA world”
is believed to have existed before DNA took over the role of being the key genetic material, and
RNA was relegated to the role of messenger between DNA and protein. Not only did the discovery
of catalytic RNA have evolutionary implications, but it also suggested that RNA could play a more
active role in gene expression than earlier realised. It is now well established that ribosomal RNA
catalyses peptide bond formation during translation.

A large number of small RNA molecules work in conjunction with proteins in ribonucleoprotein
(RNP) complexes. There are non-coding RNA molecules that affect transcription (e.g. human
7SK snRNA bound to elongation factors), translation (e.g. SRP RNA in the signal recognition
particle), replication (e.g. telomerase RNA) and chromosome structure (e.g. XIST RNA causing X
chromosome inactivation). Others regulate RNA processing (e.g. M1 RNA in RNase P, snRNAs and
snoRNAs) and RNA editing (guide RNAs). These various RNP particles are now being extensively
investigated to understand their specific roles in the cell.

In the early 1980s it was revealed in Escherichia coli that small RNA molecules (about 100 nucleo-
tides in length) can bind to a complementary sequence in mRNA and inhibit translation2,3. Today,
about 25 cases of regulatory trans-acting antisense RNAs are known in E. coli4. Regulation of trans-
lation by antisense RNA also occurs in eukaryotes as was first demonstrated in 1993 when genes
governing the development of the worm Caenorhabditis elegans were studied5,6. For many years,
this thoroughly documented case of posttranscriptional regulation was regarded as an oddity. The
mechanism received more attention when a second example of a small regulatory RNA was found
in C. elegans7, because in this case similar sequences were also present in other species. However,
the situation changed drastically when a largenumber of small RNA molecules, called microRNAs
(miRNAs), were revealed in 2001 (reff. 8-10).

Prior to the discovery of RNA interference, a phenomenon called gene (or RNA) silencing was de-
scribed in plants. It was noted in experiments around 1990 that a cloned gene incorporated into the
genome (a transgene) could not only induce or stimulate gene activity but could also inhibit the ex-
pression of homologous sequences, a phenomenon called homology-dependent gene silencing. The
inhibition of gene activity could take place at the transcriptional level (transcriptional gene silenc-
ing, TGS)11-13, or at the posttranscriptional level (posttranscriptional gene silencing, PTGS)14-18. A
PTGS-like process called quelling was also established in the fungus Neurospora crassa19. Analyses
of viral infection in plants gave further insight into the mechanism of PTGS20-21. However, although
it was evident that RNA played a key role in gene silencing, the phenomenon remained enigmatic
until the discovery of RNA interference provided a most unexpected explanation with many pro-
found consequences.

The discovery of RNA interference

Andrew Fire and Craig Mello published their break-through study on the mechanism of RNA inter-
ference in Nature in 1998 (ref. 22). It was earlier known that antisense RNA23, but remarkably also
sense RNA24 could silence genes, but the results were inconsistent and the effects usually modest.
However, due to the fact that both sense and antisense RNA could cause silencing Mello argued that
the mechanism could not just be a pairing of antisense RNA to mRNA, and he coined the term RNA
interference for the unknown mechanism25.

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In their Nature paper, Fire and Mello tested the phenotypic effect of RNA injected into the worm
C. elegans. They established that annealed sense/antisense RNA, but neither antisense nor sense RNA
alone, caused the predicted phenotype (Fig. 1). Furthermore, only injection of double-stranded RNA
(dsRNA) led to an efficient loss of the target mRNA (Fig. 2). Fire and Mello could present a series of
straightforward conclusions in their study. The main results can be summed up as follows: First, silenc-
ing was triggered efficiently by injected dsRNA, but weakly or not at all by sense or antisense single-
stranded RNAs. Second, silencing was specific for an mRNA homologous to the dsRNA; other mRNAs
were unaffected. Third, the dsRNA had to correspond to the mature mRNA sequence; neither intron nor
promoter sequences triggered a response. This indicated a posttranscriptional, presumably cytoplasmic
mechanism. Fourth, the targeted mRNA disappeared suggesting that it was degraded. Fifth, only a few
dsRNA molecules per cell were sufficient to accomplish full silencing. This indicated that the dsRNA
was amplified and/or acted catalytically rather than stoichiometrically. Sixth, the dsRNA effect could
spread between tissues and even to the progeny, suggesting a transmission of the effect between cells.
Furthermore, Fire and Mello made the remark that RNAi could provide an explanation for a phenomenon
studied in plants for several years: posttranscriptional gene silencing (PTGS). Finally, they ended their
paper by speculating about the possibility that “dsRNA could be used by the organism for physiological
gene silencing”.
Sense RNA Antisense RNA Double-stranded RNA

Wild type Wild type Twitcher

C. elegans

Figure 1. Phenotypic effect after injection of single-stranded or double-stranded unc-22 RNA

into the gonad of C. elegans. The unc-22 gene encodes a myofilament protein. Decrease in
unc-22 activity is known to produce severe twitching movements. Injected double-stranded
RNA, but not single-stranded RNA, induced the twitching phenotype in the progeny.

Antisense RNA Double-stranded RNA

Uninjected

C. elegans embryos

Figure 2. The effect on mex-3 mRNA content in embryos after injection of single-stranded or
double-stranded mex-3 RNA into the gonad of C. elegans. mex-3 mRNA is abundant in the
gonads and early embryos. The mRNA was lost after injection of double-stranded RNA, while
injection of antisense RNA only reduced the content of mRNA to some extent. The extent of
brown colour reflects the amount of mRNA present.

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In their Nature paper, Fire and Mello did not take a firm stand on the issue of whether dsRNA acts
via a transcriptional or posttranscriptional mechanism. However, in a follow-up study published in
PNAS the same year, Fire provided good evidence for the view that mRNA is the target for dsRNA
(recognition via complementary strands), and that the targeted mRNA is degraded prior to transla-
tion, i.e. dsRNA exerts its effect at the posttranscriptional level26. He also presented a specific model
showing how double-stranded RNA could function in a catalytic manner to target homologous
mRNAs for degradation. This model differed strikingly from the simple antisense model of that
time, which only predicted interaction between an interfering single-stranded RNA molecule and
mRNA. It can be added that in this PNAS paper, Fire also indicated the possibility that the RNAi
mechanism could be a specific “tactical” approach to viral defence in lower organisms (to be com-
pared with the global interferon response in mammals).

Within a year, the presence of RNAi had been documented in many other organisms, including fruit
flies, trypanosomes, plants, planaria, hydra and zebrafish27. In initial experiments with mammalian
cultured cells, it was not possible to elicit a potent and specific RNAi response because of a predomi-
nant nonspecific physiological reaction of these cells to long dsRNA. However, when the cells were
exposed to a short, 21 nucleotide long, dsRNA, an efficient targeted silencing was also obtained in
these cells28. Thus, the generality of the RNAi phenomenon among eukaryotes was proven very
rapidly; a remarkable exception is the budding yeast, Saccharomyces cerevisiae.

The revelation of the RNAi mechanism

Shortly after the discovery of RNAi, it was shown that PTGS in plants is correlated to the presence
of a population of small RNAs (about 25 nucleotides long), and that this RNA contains both sense
and antisense RNA sequences29. It was proposed that this RNA is the determinant of PTGS. Earlier,
but subsequent to the discovery of RNAi in animal cells, it had been shown that RNA in double-
stranded form also initiates PTGS in plants30.

The biochemistry of RNAi was further elucidated in an in vitro system based on Drosophila embryo
extracts27. It could be shown that dsRNA is processed to 21-23 nucleotide long dsRNA fragments31,
which was in good agreement with the data for PTGS in plants29. It was proposed that this short
dsRNA, siRNA (small interfering RNA), guided the cleavage of mRNA. Subsequently, Fire and
Mello were able to follow the process in vivo32. They established that long dsRNA is cleaved to
small RNA (about 25 nucleotides long), and that antisense RNA triggers degradation of mRNA via
base-pairing to mRNA. Thus, the trigger, dsRNA, had then been connected to the low molecular
weight effector.

The molecular machinery involved in RNAi was subsequently revealed (Fig. 3). In an in vitro
system, built on Drosophila cultured cells, it was demonstrated that a large complex, called RISC
(RNA-induced silencing complex) is targeted to the mRNA via a short antisense RNA, and that
mRNA is cleaved and subsequently degraded33. Subsequently, it has been shown that RISC contains
at least one member of the argonaute protein family, which is likely to act as an endonuclease and cut
the mRNA (nowadays often referred to as the Slicer function). It was also demonstrated that a ribo-
nuclease III-like nuclease, called Dicer, is responsible for the processing of dsRNA to short RNA34.
In certain systems, in particular plants, worms and fungi, an RNA dependent RNA polymerase
(RdRP) plays an important role in generating and/or amplifying siRNA35.

Thus, in just a few years a vast amount of information accumulated on the specific proteins and
protein complexes involved in RNAi and on the molecular details of the particular steps in the
process36-39.

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 Figure 3. The RNA interference process and the bio-
dsRNA
chemical machinery involved. Double-stranded RNA
is cut into short pieces (siRNA) by the endonuclease
Dicer. The antisense strand is loaded into the RISC
Dicer
complex and links the complex to the mRNA strand by
siRNA base-pairing. The RISC complex cuts the mRNA strand,
and the mRNA is subsequently degraded.

RISC

RISC
RISC

mRNA

Significance of the discovery of RNAi

It was evident from the very beginning that the significance of the discovery of RNAi would be
exceptional. The far-reaching consequences of the discovery can be summed up as follows (Fig. 4):

1. RNAi protects against viral infections: The finding of Fire and Mello that cells can process injected
dsRNA and eliminate homologous single-stranded RNA suggested that RNAi could constitute a
defence mechanism against viral attacks. It had earlier been shown that plant cells have an efficient
defence against viruses based on the PTGS phenomenon40,41. When it became apparent that PTGS
is the plant equivalent to RNAi, this early work in plants supported the proposition that RNAi is
involved in protecting cells from viral attacks. Today, we know that this anti-viral mechanism is at
work in plants, worms and flies, whereas it is still unclear how relevant it is for vertebrates, includ-
ing man.

2. RNAi secures genome stability by keeping mobile elements silent: It was proposed early on that
RNAi/PTGS in C. elegans and plants could block the action of transposons (mobile elements in
the genome). Subsequently, it could be shown that when components of the RNAi machinery are
mutated in C. elegans, transposons are activated and the mobile elements cause disturbances in
the function of the genome42,43. It has been proposed that in transposon-containing regions of the
genome both DNA strands are transcribed, dsRNA is formed, and the RNAi process eliminates
these undesirable products. As short dsRNAs can also operate directly on chromatin and suppress
transcription (see below), this would be another mode to keep transposons inactive (see below under 4).
Even if the mechanisms are not yet fully revealed, it is clear that if the RNAi machinery is not
efficient, the transposons are not kept under control and can start to jump and cause deleterious
effects in the genome.

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It has been argued that RNA silencing could represent an “immune defence” of the genome44. Close
to 50% of our genome consists of viral and transposon elements that have invaded the genome in the
course of evolution. The RNAi machinery can recognize invading double-stranded viral RNA (or
the double-stranded replicative form of the viral RNA) and suppress the infection by degradation of
the RNA. The RNAi system thus shares important features with the vertebrate immune system: it
recognizes the invading parasite (dsRNA), raises an initial response and subsequently amplifies the
response to eliminate the foreign element.

Viral RNA m
plas
Cyto

le us
Nuc

Virus dsRNA

dsRNA
Exogenous RNA dsRNA

Dicer
pre-
miRNA 2 RNA
polymerase
5 siRNA
Dicer
Transposon

siRNA Dicer

pri-miRNA
miRNA
siRNA 3 RNA
polymerase DNA
RISC

Micro RNA gene

RISC
mRNA
4
RISC
mRNA
RNA
polymerase
ILLUSTRATION: ANNIKA RÖHL

mRNA cleavage mRNA

and degradation DNA

Suppression of Inhibition of RNA synthesis

protein synthesis Chromatin condensation

Figure 4. Cellular processes dependent on the RNAi machinery. The Dicer and RISC complexes
play a central role in the destruction of invading viral RNA (1), the elimination of transcripts from
mobile elements (transposons) and repetitive DNA (2), the block of protein synthesis brought about
by small RNAs generated within the cell (3), and the RNAi-mediated suppression of transcription
(4). The machinery is also utilized when siRNA is introduced into the cell experimentally to inhibit
the activity of specific genes (5). The figure is schematic, and the Dicer and RISC complexes can
vary dependent on cellular process.

3. RNAi-like mechanisms repress protein synthesis and regulate the development of organisms:
Soon after the discovery that short RNA is the effector of RNAi, it was shown that there is a class
of endogenous RNA molecules of the same size in worms, flies, mice and humans; this small RNA
was called microRNA (miRNA) 8-10. Plants also contain this class of endogenous RNA45. The revela-
tion of miRNA led to intense research on the nature of these RNA molecules. The C. elegans lin-4
(ref. 5) and let-7 (ref. 7) RNAs were regarded as prototypes, and examples of similar cases were

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soon revealed in several organisms46. The small miRNAs are processed from larger hairpin-like
precursors by an RNAi-like machinery47,48 (Fig. 4). The miRNAs can regulate gene expression by
base-pairing to mRNA, which results in either degradation of the mRNA or suppression of trans-
lation. Today, it is estimated that there are about 500 miRNAs in mammalian cells, and that
about 30% of all genes are regulated by miRNAs. It is known that miRNAs play an important role
during development in plants, C. elegans and mammals. Thus, the miRNA-dependent control of
gene expression represents a new major principle of gene regulation. However, the full significance
of small regulatory RNAs is probably still not apparent.

4. RNAi-like mechanisms keep chromatin condensed and suppress transcription: It was known from
work in plants that gene silencing could take place at the transcriptional level (TGS). After the dis-
covery of RNAi, it was soon shown that TGS in plants operates via RNAi-like mechanisms49,50. In
the fission yeast Schizosaccharomyces pombe51,52, and later on in Drosophila and vertebrates, it was
found that similar processes keep heterochromatic regions condensed and transcriptionally suppres-
sed. In addition, the RNAi-like machinery regulates the activity of genes in the immediate vicinity
of the condensed blocks of chromatin. The phenomenon is still not understood at the molecular level
although histone modifications, binding of specific chromatin condensing proteins (HP1), and DNA
methylation all play important roles46. It is, however, evident that this action on chromatin is most
important for proper functioning of the genome and for maintenance of genome integrity.

5. RNAi offers a new experimental tool to repress genes specifically. The targeted action of RNAi
immediately suggested that this phenomenon could be utilized as a general method to suppress spe-
cific genes and look for the resulting phenotypic effect. It was also soon evident that this could be
accomplished in such an efficient manner that essentially any gene in an organism could be studied
functionally. After the initial work in C. elegans, this technical approach could be applied to cells
from almost all organisms, including mammalian cells. This targeted gene silencing by RNAi has
already had a tremendous impact in studies of the function of individual genes. It is now exploited
not only in cultured cells but also in transgenic organisms. DNA constructs are introduced into
the organisms under appropriate promoter control, and dsRNA hairpin structures are produced and
further processed to achieve specific effects on gene regulation.

6. RNAi might be a useful approach in future gene therapy. The possibility to achieve RNAi-
governed gene regulation in transgenic organisms has stimulated many explorations of whether this
would be a useful option for medical therapy53,54. Promising results have been reported in several
animal models55-58 and even in recent clinical trials, but it is too early to predict the outcome of these
challenging efforts.

Conclusions

The discovery that cells have a special mechanism for suppressing the expression of homologous
genes by recognizing and processing double-stranded RNA was totally unexpected and has drama-
tically expanded our knowledge of gene control. Remarkably, the RNAi machinery can handle dou-
ble-stranded RNA entering the cell as well as double-stranded RNA generated within the cell. The
development of an organism and proper function of its cells and tissues are dependent on an intact
RNAi machinery. Infection by RNA viruses can be blocked by RNAi, especially in plants and lower
animals, and foreign elements in the genome (viruses and transposons) can be kept silent. Finally,
the discovery of RNAi has not only provided us with a powerful new experimental tool to study the
function of genes but also raises expectations about future applications of RNAi in medicine.

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Acknowledgements. I am grateful to Annika Röhl for designing the figures

and to Adam Smith for helpful comments on the text.

Bertil Daneholt
Professor of Molecular Genetics, Karolinska Institutet, Stockholm
Chairman of the Nobel Assembly

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