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R82 Current Biology Vol 12 No 3

Primer

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 . 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 ’, ‘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 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 dsRNA DICER/RISC process (Figure 2) animals are injected with dsRNA ensures that RNA species are or if they are allowed to feed on Dicer 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 . 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. protection could be targeted expect that RNA silencing will be (1999). A novel species of small 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. or chromatin. If this is the case, damaged by handling. They could Voinnet, O., Vain, P., Agnell, S., 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 There are many other applications of ectopic promoterless DNA. Cell 95, 177–187. 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, NR4 7UH, UK. systematic analysis of gene suppression of gene expression E-mail: [email protected]