RNA silencing

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					R82   Current Biology Vol 12 No 3


                                    RNA silencing
                                    David Baulcombe
                                    RNA silencing is a newly
                                    discovered mechanism of genetic
                                    regulation in eukaryotes. It is
                                    based on an immune system that
                                    protects eukaryotes against
                                    viruses and transposons and in
                                    some respects it is conceptually
                                    similar to classical humoral
                                    immunity. However RNA silencing
                                    is distinct from humoral immunity
                                    in that it only targets nucleic
                                    acids and has a specificity
                                    mechanism based on nucleic acid
                                                                           Figure 1. RNA silencing in petunia.
                                    base pairing rather than on            The petunia plant was transformed with
                                    interactions with protein              an additional copy of a gene (chalcone
                                    antibodies.                            synthase) required for pigment
                                        Five years ago the evidence for    production. The transgene activated
                                    RNA silencing was based on             RNA silencing so that expression of
                                    mysterious experimental                both the transgene and the endogenous
                                                                           gene was suppressed. The flowers
                                    observations in plants, a fungus       would normally have been purple.
                                    and in Caenorhabditis elegans.
                                    Today we know that this process
                                    has natural biological roles and we    RNA causes specific silencing of a
                                    have an outline understanding of       homologous endogenous gene.
                                    the mechanisms. In addition RNA
                                    silencing is part of powerful          Basic Mechanism of RNA Silencing
                                    enabling technologies for              The first evidence for RNA
                                    functional genomics. The story of      silencing was from plants and fungi
                                    this recent progress is revealing      carrying transgenic copies of
                                    about the way that science             endogenous genes. These plants
                                    accommodates and adapts to             and fungi were expected to
                                    unexpected results. It also            overexpress the corresponding
                                    illustrates the power of combined      protein, however the result, at least
                                    molecular, genetic and biochemical     in some of the lines, was the exact
                                    approaches to problems in biology.     opposite. For example, in plants
                                        Several terms are used to          that should have overexpressed
                                    describe RNA silencing. These          an enzyme required for floral
                                    include ‘cosuppression’, ‘RNA          pigment production, the enzyme
                                    interference’, ‘post-transcriptional   was absent and flowers were white
                                    gene silencing’, ‘sense                (Figure 1). We now know that the
                                    suppression’ and ‘quelling’. Here I    white flowers were due to an RNA
                                    use ‘RNA silencing’ generically;       silencing surveillance system that
                                    the other terms are useful in more     detects the presence of foreign
                                    specific examples of RNA               RNA in eukaryotic cells. This
                                    silencing. For example,                system recruits the foreign nucleic
                                    ‘cosuppression’ refers to the          acid as the specificity determinant
                                    specific case of RNA silencing in      of an RNA targeting mechanism.
                                    which RNAs of a transgene and          Thus, a transgene X causes
                                    homologous endogenous gene             specific degradation of X RNA and
                                    are co-ordinately suppressed;          a floral pigmentation transgene
                                    ‘quelling’ is cosuppression in         causes the degradation of the
                                    Neurospora crassa; ‘RNA                transgene and endogenous RNAs
                                    interference’ occurs when direct       encoding the floral pigmention
                                    introduction of a double stranded      enzyme. Other endogenous RNAs
                                                                                                         Magazine   R83

                                            base pairing. Thus the                 graphically by C.elegans. If these
                                            DICER/RISC process (Figure 2)          animals are injected with dsRNA
                                            ensures that RNA species are           or if they are allowed to feed on
                                            degraded if they share sequence        bacteria that produce dsRNA
                                            similarity with the dsRNA. One of      there is systemic silencing of a
                         21-25nt RNA
                                            the components of RISC is a            corresponding endogenous RNA.
                                            protein, AGO2, that shares some        Presumably the signal molecule is
                                            limited sequence similarity, in the    produced in the C. elegans cells
 Sequence specific nuclease (RISC)          PAZ domain, with DICER. It has         that receive dsRNA. This signal
                                            been suggested that interactions       then moves and causes RNA
                          Current Biology
                                            of the PAZ domains may allow           silencing in other cells that become
Figure 2. The schematic of the basic        transfer of siRNAs from RISC to        sources of secondary signal.
mechanism of RNA silencing in plants.       DICER.                                    We know that an RNA-
                                               In mammals the investigation of     dependent RNA polymerase is
are unaffected because they did             RNA silencing is complicated by        required to amplify RNA molecules
not share nucleotide sequence               the dsRNA-dependent protein            involved in RNA silencing. However,
similarity with the transgene RNA.          kinase (PKR) system in which           we do not know the nature of the
   To explain the sequence                  dsRNA is an activator of cell death.   signal although the siRNAs are a
specificity of RNA silencing it             This system, as far as we know, is     good candidate. Nor do we
seemed likely that antisense RNA            quite distinct from RNA silencing.     understand why there is the
would be involved. This antisense           However more recently it has           potential for extracellular signaling
RNA would anneal by base pairing            become apparent that RNA               and amplification of silencing. One
with the target RNA and thereby             silencing does operate in              possible explanation concerns
prevent translation or provide a            mammals. They express a homo-          antiviral defense. If silencing of
target for nuclease degradation.            logue of DICER and have an RNA         viruses occurs in the cells that are
However, in plants, the                     silencing response if dsRNA is         yet to be infected then the antiviral
transgenes with an RNA silencing            introduced in the form of siRNAs,      defense role of silencing will be
phenotype were designed to                  or into embryonic cells in which       more effective than a system that
produce sense rather than                   the PKR system is not active.          operates only after the virus has
antisense RNA. Similarly, in C.                                                    started to replicate. Other roles of
elegans, injection of sense RNA             Intercellular signaling and            systemic silencing in develop-
caused sequence-specific                    amplification of RNA silencing         mental control are also possible.
silencing of endogenous RNAs.               A remarkable feature of RNA
   These unexpected results with            silencing is its ability to act        Natural roles of RNA silencing
sense RNA were eventually                   beyond the cells in which it is           RNA silencing is more than an
explained in terms of double-               initiated. For example, in plants      artefact of transgenic plants or
stranded (ds)RNA. The sense                 carrying an expressed GFP              C. elegans injected with double-
transgenes were mosaics of                  transgene, RNA silencing can be        stranded RNA. It also has natural
multiple inserts with inverted              initiated by localized introduction    roles in antiviral defense, genome
repeats that would be transcribed           of an additional ectopic GFP           protection and developmental
into dsRNA. Similarly the sense             transgene. The RNA silencing is        regulation of gene expression. An
RNAs injected into C. elegans               initially manifested in the tissues    antiviral defense role, at least for
contained a small amount of                 containing the ectopic DNA but         RNA viruses in plants, is thought to
contaminating double-stranded               eventually becomes systemic            involve dsRNA intermediates in
(ds)RNA. Deliberate injection or            showing that a silencing signal        viral RNA replication. This dsRNA
transgenic expression of dsRNA              moves between cells and in the         is processed by DICER so that,
produces very efficient silencing.          vascular system of the plant           once the infection is established,
dsRNA is important in RNA                   (Figure 3). The production and         RISC is targeted against the viral
silencing because it is processed           movement of this signal persists       mRNAs and virus replication is
into short (21–25 nucleotide) RNAs          throughout the lifetime of the plant   slowed down. Not surprisingly
by an enzyme known as DICER.                even if the tissues containing the     plant viruses produce proteins that
DICER is a homologue of E. coli             ectopic initiator are removed.         counteract RNA silencing and this
RNAseIII with dsRNA binding and             Thus, the cells containing the         might explain why some strains are
helicase domains. The short RNAs,           ectopic DNA are not the only           highly virulent. At present it is not
known as small interfering or               source of silencing signal; as the     known whether viruses in animals
(si)RNAs, correspond to both                signal moves through the plant         or affected by RNA silencing.
sense and antisense strands of the          there must be a system that               An example of RNA silencing in
target RNA and are incorporated             amplifies the effects of the           development involves short
into a multimeric RNase complex             initiator of silencing.                temporal (st)RNAs. stRNAs were
— RNA Interference Specificity                 Animals may also have a system      discovered in C. elegans; they are
Complex- RISC — that is guided              for amplification and spread of        about 21 nucleotides long and are
to its target RNA by Watson-Crick           silencing. This is shown most          produced by DICER-mediated
R84   Current Biology Vol 12 No 3

                                             function. The principle is simple: a   by RNA silencing. Previously one
                                             fragment of a gene is introduced       of the barriers to these applications
                                             into a cell as dsRNA or as DNA         was the hit and miss nature of RNA
                                             that will give rise to dsRNA. The      silencing in transgenic plants.
                                             dsRNA activates the DICER/RISC         Normally only a few percent of
                                             process so that the properties of      lines with any one construct would
                                             the affected cell reflect a loss of    manifest the silencing phenotype.
                                             function in the corresponding          Now that we understand more
                                             gene. In order to target the           about the silencing mechanism, it
                                             genome of C. elegans a collection      is possible to produce more
                                             of E. coli strains has been            consistent silencing and,
                                             generated to produce dsRNA             conditional on public acceptance
                                             corresponding to each gene.            of transgenic plants, we can
                                             The function of each gene is           expect other products based on
Figure 3 Systemic silencing in plants.
                                             inferred from the behaviour or         this technology.
The image shows a leaf of a GFP
transgenic plant under UV light. The         properties of the C. elegans after        There may also be applications
background green fluorescence is due to      feeding on the E. coli. A parallel     of RNA silencing in transgenic
expression of the GFP transgene which is     programme is being carried out in      animals. For example, an animal
so strong that the normal red fluorescence   plants infected with viruses           could be engineered to produce a
of chlorophyll is masked. The red regions    carrying inserts corresponding to      short dsRNA corresponding to the
around the veins are due to RNA silencing
                                             each of the genes in the plant         conserved region of a virus. The
that is mediated by a signal of silencing
that has been transmitted through the        genome. The function of the gene       dsRNA would not activate the PKR
vascular system from the lower parts of      can be inferred from the               system because it was short.
the plant.                                   symptoms that develop in the           However, through RNA silencing, it
                                             infected plant.                        would condition the animal to be
processing of ds precursor RNAs.                A particularly useful property of   constitutively resistant to the virus.
stRNAs are complementary to and              RNA silencing is that it does not      There are also potential
repress the expression of longer             require complete sequence              applications in gene therapy. If
RNAs. However, unlike other                  identity in the dsRNA and the          siRNAs targeted against a gene
examples of RNA silencing, the               target RNA. There is good              required for cell proliferation could
block is at the level of translation         silencing even if there is 5% or       be introduced into cancer cells it is
rather than enhanced degradation.            10% mismatch in the initiator of       possible that the growth of a tumor
   A role for the RNA silencing in           silencing and the target RNA.          could be suppressed. Is it not
genome protection is shown by                Thus it is possible to investigate     amazing how a process that was
C. elegans mutants that are                  the function of multigene families     first described in plants only a few
defective in RNA silencing and in            in which different members have        years ago is now being considered
which there is a high frequency of           overlapping and redundant roles.       as part of anti-cancer therapy?
spontaneous mutation due to                  These families are otherwise
enhanced mobility of transposable            difficult to investigate by genetic    Additional Reading
DNA. In principle this genome                means. Over the next year or two I     Hamilton, A. J., and Baulcombe, D. C.
                                                                                         (1999). A novel species of small
protection could be targeted                 expect that RNA silencing will be
                                                                                         antisense RNA in post-transcriptional
through the DICER/RISC process               used for systematic analysis of             gene silencing. Science 286,
at mRNAs of enzymes required for             the genes in several organisms.             950–952.
DNA transposition. However, from                                                    Nishikura, K. (2001). A short primer on
work in plants we know that some             Applications of RNA Silencing               RNAi: RNA-directed RNA polymerase
                                                                                         acts as a key catalyst. Cell 107,
examples of RNA silencing lead to            One of the first commercial                 415–418.
methylation of the target DNA as             products of RNA silencing was a        Nykanen, A., Haley, B., Zanmore, P.D.
well as degradation of the target            tomato in which the target was a            (2001). ATP requirements and small
RNA. These findings indicate that            gene required for softening of              interfering RNA structure in the RNA
                                                                                         interference pathway. Cell 107,
suppression of transposable                  ripening fruit. Reduced expression          309–321.
elements in C. elegans and                   of this gene in the silenced plants    Ruvkun, G. (2001). Molecular biology -
Drosophila could be mediated by              meant that the tomatoes were firm           Glimpses of a tiny RNA world.
the effect of RNA silencing on DNA           after ripening and were not                 Science 294, 797–799.
                                                                                    Voinnet, O., Vain, P., Agnell, S.,
or chromatin. If this is the case,           damaged by handling. They could             Baulcombe, D.C. (1998). Systemic
the RNA silencing mechanism will             be left to ripen on the vine and so         spread of sequence-specific
be more complex than shown in                tasted better than conventional             transgene RNA degradation in plants
Figure 2.                                    tomatoes that are harvested green.          as initiated by localized introduction
                                                                                         of ectopic promoterless DNA. Cell 95,
                                             There are many other applications
Functional genomics and RNA                  in agriculture where crop
silencing                                    production or product quality can      The Sainsbury Laboratory, John Innes
RNA silencing is well suited to the          be enhanced by specific                Centre, Norwich NR4 7UH, UK.
systematic analysis of gene                  suppression of gene expression         E-mail: david.baulcombe@bbsrc.ac.uk