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`REVIEW
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`RNA interference—2001
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`Phillip A. Sharp1
`Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology,
`Cambridge, Massachusetts 02139-4307, USA
`
`In the few years since the discovery of RNA interference
`(RNAi; Fire et al. 1998), it has become clear that this
`process is ancient. RNAi, the oldest and most ubiquitous
`antiviral system, appeared before the divergence of
`plants and animals. Because aspects of RNAi, known as
`cosuppression, also control the expression of transpos-
`able elements and repetitive sequences (Ketting et al.
`1999; Tabara et al. 1999), the interplay of RNAi and
`transposon activities have almost certainly shaped the
`structure of the genome of most organisms. Surprisingly,
`we are only now beginning to explore the molecular pro-
`cesses responsible for RNAi and to appreciate the
`breadth of its function in biology. Practical applications
`of this knowledge have allowed rapid surveys of gene
`functions (see Fraser et al. 2000 and Gönczey et al. 2000
`for RNAi analysis of genes on chromosome I and III of
`Caenorhabditis elegans) and will possibly result in new
`therapeutic interventions.
`Genetic studies have expanded the biology of RNAi to
`cosuppression, transposon silencing, and the first hints
`of relationships to regulation of translation and develop-
`ment. The possible roles of RNA-dependent RNA poly-
`merase (RdRp) in RNAi have been expanded. Many ex-
`periments indicate that dsRNA directs gene-specific
`methylation of DNA and, thus, regulation at the stage of
`transcription in plants. Cosuppression may involve regu-
`lation by polycomb complexes at the level of transcrip-
`tion in C. elegans and Drosophila. This article will re-
`view these topics and primarily summarize advances in
`the study of RNAi over the past year.
`
`Sequence and strand specificity of RNAi
`
`Restriction of virus growth in plants is mediated by post-
`transcriptional gene silencing (PTGS), which can be ini-
`tiated by production of dsRNA replicative intermediates.
`This silencing of expression is gene specific, and Hamil-
`ton and Baulcombe (1999) discovered that tissue mani-
`festing PTGS contained small RNAs (25 nt) complemen-
`tary to both strands of the gene. Using extracts of Dro-
`sophila embryos that had been shown previously to be
`active for RNAi (Kennerdell and Carthew 1998), Tuschl
`
`1Corresponding author.
`E-MAIL sharppa@mit.edu; FAX (617) 253-3867.
`Article and publication are at www.genesdev.org/cgi/doi/10.1101/
`gad.880001.
`
`et al. (1999) were able to reproduce RNAi in a soluble
`reaction. dsRNA added to this reaction is cleaved into
`21–23-nt RNAs, which leads to cleavage of the target
`mRNA at 21–23-nt intervals (Zamore et al. 2000). Ham-
`mond et al. (2000) also concluded that small RNAs di-
`rected cleavage of mRNAs in Drosophila extracts pre-
`pared from Schneider cells. These experiments are best
`explained by a model for RNAi where dsRNA is pro-
`cessed to 21–23-nt RNAs that direct the cleavage of
`mRNA through sequence complementarity. These
`RNAs are referred to as siRNAs, or short interfering
`RNAs (see below).
`Fire and Mello have continued their collaboration
`studying the functional anatomy of dsRNA for induction
`of RNAi (Parrish et al. 2000). They first concluded, using
`short RNAs synthesized chemically and assayed by in-
`jection into C. elegans, that any dsRNA segment greater
`than ∼26 bp can generate RNAi. Thus, the process for
`generation of siRNAs is probably sequence nonspecific.
`This was confirmed by the observation that individual
`short dsRNA formed from sequences that did not con-
`tain adenosine, uridine, or cytidine were active for
`RNAi. Long dsRNAs were more active than short
`dsRNAs; a 250-fold higher concentration of 26-bp
`dsRNA generated the equivalent gene silencing activity
`as an 81-bp dsRNA. dsRNA from a related but not iden-
`tical gene can be used to target a gene for silencing if the
`two share segments of identical and uninterrupted se-
`quences of significant length, probably >30–35 nt in
`length. Silencing was inefficient when the largest unin-
`terrupted segments were 14 and 23 nt in length but effi-
`cient when 41 nt of such sequences of identity were
`shared. These results suggest that silencing will probably
`occur if long dsRNAs are used and the two related genes
`are >90% homologous. Assuming that dsRNA is pro-
`cessed to 21–23-nt segments, these results indicate that
`single basepair mismatches between the siRNA and tar-
`get RNA dramatically reduce gene targeting and silenc-
`ing.
`In the C. elegans assay used by Mello and Fire, it is
`likely that the injected dsRNA is directly processed to
`the targeting siRNAs and that these are not replicated by
`an endogenous RNA-dependent RNA polymerase. This
`conclusion rests on the effects of asymmetric modifica-
`tions of the input dsRNA. Substitution of either 2⬘-
`amino uracil for uracil or 2⬘-amino cytidine for cytidine
`in the sense strand of the dsRNA had little effect on the
`
`GENES & DEVELOPMENT 15:485–490 © 2001 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/01 $5.00; www.genesdev.org
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`Sharp
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`RNAi activity, while the same substitutions in the an-
`tisense strand rendered the RNA inactive. If the input
`dsRNA were replicated before targeting, it would be ex-
`pected to lose this asymmetry. As the above assays were
`done in somatic tissue of C. elegans, it is possible that
`the long-term RNAi observed through multiple genera-
`tions (Grishok et al. 2000) could involve replication in
`the germ-line tissues. Mutations in a C. elegans gene
`with sequence relationship to RdRp, EGO-1, have been
`reported to affect some aspects of RNAi (Smardon et al.
`2000).
`
`Genesis of RNAi
`
`The structure of siRNAs is probably the same in all or-
`ganisms, as the 21–23-nt length of siRNAs seems to be
`universal. Furthermore, siRNAs might be the best can-
`didates for use in targeted gene silencing because their
`structure would match the biochemical components of
`the RNAi system. The complex generating the siRNAs
`from short dsRNAs primarily recognizes the 3⬘ termini
`of the duplex (Elbashir et al. 2001). Internal cleavage of
`the dsRNA occurs at a distance of ∼22 nt, and a complex
`of siRNA and proteins targets cleavage of the comple-
`mentary target RNA at a position ∼10–12 nt from the
`terminus of the original dsRNA (see top panel of Fig. 1).
`The siRNA duplex probably remains associated with the
`initial complex because it asymmetrically targets a
`strand for cleavage and not its partner (the sense strand
`in the example illustrated in Fig. 1). This asymmetry was
`not observed when symmetric siRNAs with 2-nt tails on
`both strands were added to the reaction. Both strands of
`the target RNAs were cleaved within the region covered
`by the siRNA duplex, indicating that the siRNA duplex
`can bind to the complex responsible for cleavage in ei-
`ther orientation (see bottom panel of Fig. 1). In general, a
`
`siRNA duplex with 2-nt 3⬘ tails is thought to be the
`primary intermediate of RNAi. In fact, addition of RNAs
`with this structure to reactions in vitro can silence trans-
`lation of a target mRNA with a similar efficiency (within
`10-fold) on a molar basis to dsRNAs of >50 bp. Addition
`of either one of the two single strands constituting a
`siRNA duplex generates no activity.
`Tuschl’s lab developed methods for cloning of siRNAs
`using T4 RNA ligase to add linker segments to their 5⬘
`and 3⬘ termini (Elbashir et al. 2001). The predominant
`structure is a 19–20-bp duplex RNA with both termini
`possessing 2-nt 3⬘ single-strand segments, and the total
`length of each strand is predominantly 21–22 nt. RNase
`III–type endonucleases cleave dsRNA releasing RNA
`with 2-nt 3⬘ tails, indicating that this type of activity is
`probably involved in generating siRNAs (a possibility
`first suggested by Bass [2000]). Although the results were
`not described in the paper, Elbashir et al. (2001) reported
`the cloning of siRNAs that were endogenous to the Dro-
`sophila extract. This foretells future studies where
`analysis of the sequence of siRNAs in cells will indicate
`which genes are naturally silenced by RNAi.
`How are the siRNAs related to the site of cleavage on
`the target mRNA? As shown in Figure 1, the siRNAs
`direct cleavage of the target RNA in the middle of the
`paired segments, ∼12 bp from the 3⬘ terminus of the
`siRNA. This positions the site of cleavage of the target
`RNA about one turn of an A-type duplex helix from the
`cleavages that generated the siRNAs. This could indicate
`a rearrangement of the RNase III-type domains contact-
`ing the siRNA duplex before the second cleavage.
`
`Genetic analysis of RNAi
`
`Several groups are actively pursuing the identification
`and characterization of enzymes implicated in RNAi and
`
`Figure 1. Comparison of the cleavage
`patterns on sense and antisense target
`strands when either a short double strand
`RNA (top) or a siRNA (bottom) are added
`to a reaction in vitro. The dsRNA gener-
`ates a siRNA complex which only cleaves
`the sense strand. Processing of siRNAs
`from the opposite end of the dsRNA would
`only cleave the antisense strand (not
`shown). Addition of a siRNA with the
`same structure as that processed from the
`dsRNA generates cleavage of both the
`sense and antisense strands, suggesting
`that the siRNA can bind the complex in
`either orientation.
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`cosuppression. In C. elegans, initial mutant screens have
`generated ∼80 candidates, of which five have been spe-
`cifically identified: RDE-1, RDE-2, RDE-3, RDE-4, and
`Mut-7 (Ketting et al. 1999; Tabara et al. 1999; Ketting
`and Plasterk 2000; Grishok et al. 2000). Selection of mu-
`tations in cosuppression in Arabidopsis have identified
`homologs of the same genes (Dalmay et al. 2000; Fagard
`et al. 2000; Mourrain et al. 2000). Testing of previously
`identified mutations for defects in RNAi in C. elegans
`and other organisms has expanded this list.
`
`Enzymes of RNAi
`
`RNase III proteins and RNAi
`
`What type of RNase III–like activity might be active in
`RNAi? Bacterial RNase III and its homologs in Saccha-
`romyces cerevisiae and Schizosaccharomyces pombe
`function in processing of rRNA and other structural
`RNAs (Chanfreau et al. 2000). There are two general
`families of RNase III homologs in plants and animals.
`One family is represented by the drosha Drosophila
`gene, which is composed of two RNase III domains and
`one dsRNA binding domain (Filippov et al. 2000). Anti-
`sense experiments suggest that a ubiquitously expressed
`human family member closely related to drosha is im-
`portant for rRNA processing (Wu et al. 2000). The second
`family of RNase III proteins contains an N-terminal
`ATP-dependent helicase-type domain as well as two
`RNase III–type domains and a dsRNA motif (Filippov et
`al. 2000). Perhaps these represent the best candidates for
`the RNase III activity in RNAi (Elbashir et al. 2001).
`Recent results from Bernstein et al. (2001) describe the
`cleavage of dsRNA into 22-nt segments by a Drosophila
`protein of the RNase III type. Furthermore, RNA inter-
`ference was used to indicate that this protein is impor-
`tant for RNAi activity. Mutations in an Arabidopsis
`gene in this family result in unregulated cell division in
`floral meristems (Jacobsen et al. 1999). This would be
`consistent with a relationship between RNAi and devel-
`opment. Interestingly, the presence of two RNase III do-
`mains in this family of proteins suggests that it might
`cleave dsRNA as a monomer. The dsRNA-binding do-
`main could position the enzyme on the substrate, and
`the two catalytic domains could hydrolyze bonds in both
`strands.
`
`RNA-dependent RNA polymerase
`
`Mutations in genes encoding a protein related to RNA-
`dependent RNA polymerase (RdRp) affect RNAi-type
`processes in Neurospora (QDE-1), C. elegans (EGO-1),
`and plants (SGS2, Mourrain et al. 2000; and SDE-1, Dal-
`may et al. 2000). It has been generally assumed that this
`type of polymerase would replicate siRNAs as epigenetic
`agents permitting their spread throughout plants and be-
`tween generations in C. elegans. This may still be the
`case; however, results from Arabidopsis indicate that
`SDE-1 is important for gene silencing mediated by the
`presence of transgenes but not for posttranscriptional
`gene silencing (PTGS), induced by a replicating RNA vi-
`
`Perspective
`
`rus (Dalmay et al. 2000). The efficient generation of
`siRNAs from transgenes was dependent upon SDE-1,
`whereas siRNAs were generated in SDE-1 mutant plants
`by viral replication, which generates dsRNA. The au-
`thors conjecture that aberrant RNAs from the transgenes
`are recognized by the RNA-dependent RNA polymerase,
`SDE-1, generating dsRNA that is processed to siRNAs.
`
`RNA-dependent RNA helicase
`
`Another type of RNA helicase of the DEAH-box helicase
`super family has also recently been shown to be impor-
`tant for RNAi or PTGS in Chlamydomonas reinhardtii
`(Wu-Scharf et al. 2000). Mutations in this gene, Mut-6,
`relieve silencing by a transgene and also activate trans-
`posons. Helicases of the same family are important for
`RNA splicing in yeast; however, Mut-6 is not thought to
`be involved in RNA splicing. A closely related yeast gene
`that is involved in RNA splicing, PRP16, has been shown
`to have ATP-dependent RNA helicase activity (Wang et
`al. 1998). Perhaps Mut-6 unwinds duplex RNA in some
`step of RNAi.
`
`Processes related to RNAi
`
`Nonsense-mediated decay of mRNA
`
`A link between RNAi and nonsense-mediated decay was
`revealed by screening of mutants in the latter process
`(Domeier et al. 2000). mRNAs containing nonsense mu-
`tations upstream of an intron are rapidly degraded in
`organisms as diverse as worms and vertebrates. Seven
`genes, SMG 1–7, are important for this process in C.
`elegans (Page et al. 1999). Surprisingly, mutants of C.
`elegans with lesions in either smg-2, smg-5, or smg-6
`failed to efficiently maintain RNAi over the course of 4
`d following injection of dsRNA. Both mutant and wild-
`type animals showed equivalent levels of RNAi on the
`first day, and this level was essentially unchanged in the
`wild-type animals over the same 4-d interval. Smg-1, and
`probably smg-3 and smg-4, are not important for main-
`tenance of RNAi over the 4-d interval. Smg-2, based on
`homology, is thought to encode an ATPase with RNA
`binding and helicase activity (Page et al. 1999). Its spe-
`cific role in nonsense-mediated decay of mRNA is un-
`known.
`
`Regulation of translation during development
`
`RDE-1, which is important for RNAi in C. elegans , is a
`member of a family of 23 related genes in this organism
`(Tabara et al. 1999). There are four family members in
`Drosophila and several in humans. In Drosophila, two of
`the most closely related genes have unknown functions,
`whereas the other two, piwi and aubergine (aub) func-
`tion in oogenesis (Wilson et al. 1996; Cox et al. 1998).
`Specifically, aub is required for translation of two
`mRNAs, oskar and gurken. Arabidopsis encodes eight
`genes related to RDE-1. Mutations in two of these genes,
`Argonaute 1 (AGO1) and ZWILLE/PINHEAD (ZLL/
`PNH), result in defects in development. Mutations in the
`two genes have distinct phenotypes although they are ex-
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`pressed in many of the same tissues. A relationship be-
`tween RNAi and development is suggested by the obser-
`vation that mutants of AGO1 are also defective for
`cosuppression (Fagard et al. 2000). These results strongly
`suggest that multiple RDE-1 family members are likely
`to be involved in RNAi, perhaps in different tissues and
`in a redundant fashion. They also suggest that RNAi will
`share some processes in common with regulation of de-
`velopment.
`Interestingly, the C. elegans small RNAs lin-4 and let-
`7, which are 22 and 21 nt long, respectively, are known
`to regulate translation during development in C. elegans.
`These RNAs are possibly processed from dsRNA regions
`of a precursor RNA and are thought to pair with the 3⬘
`UTR of their targets in regulation of translation. The
`let-7 RNA is conserved between C. elegans, Drosophila,
`and humans (Pasquinelli et al. 2000). The similarity in
`lengths of siRNAs and lin-4 and let-7 suggests that these
`systems might share components.
`
`Regulation of transcription
`
`Three gene-silencing phenomena, cosuppression, trans-
`poson silencing, and DNA methylation, are related to
`RNAi by dependence on a common set of genes. For
`example, in C. elegans, both transposon silencing and
`cosuppression depend on RDE-2, RDE-3, and Mut-7,
`which are critical for RNAi (Ketting et al. 1999; Tabara
`et al. 1999; and Ketting and Plasterk 2000). Cosuppres-
`sion is generally defined as suppression of an endogenous
`locus following introduction of homologous transgenes.
`This trans-suppression requires transcription of the
`transgenes but is independent of the specific-promoter
`sequence used to direct transcription (Dernburg et al.
`2000). Loss of a transgene array from the germ line of C.
`elegans by deletion results in reactivation of the endog-
`enous locus after a few generations. Thus, the endog-
`enous locus is not mutated during silencing by cosup-
`pression as it is during a related phenomenon, called
`
`quelling, in Neurospora. There is no evidence for pairing
`of the transgenic array and the endogenous locus during
`cosuppression in C. elegans (Dernburg et al. 2000). Thus,
`the silencing of the endogenous locus is probably medi-
`ated by a trans-acting factor that is sequence specific and
`dependent on transcription. This, and its dependence
`upon the RNAi related genes RDE-2, RDE-3, and Mut-7,
`strongly indicates that cosuppression is mediated by
`trans-acting RNA, probably siRNAs (see Fig. 2).
`Cosuppression and the polycomb complex The silenc-
`ing of tandem arrays in C. elegans is dependent on the
`set of mes genes (maternal-effect sterile; Holdeman et al.
`1998; Kelly and Fire 1998; Korf et al. 1998). Two of these
`genes are homologs of enhancer of zeste and extra sex-
`combs in Drosophila and are in the polycomb group of
`genes. In Drosophila, endogenous loci silenced by cosup-
`pression are bound by a polycomb complex (Pal-Bhadra
`et al. 1997, 1999), indicating that this process directs the
`gene-specific binding of this epigenetic regulatory ma-
`chine. Polycomb complexes are thought to silence genes
`at the stage of transcription by forming inactive chroma-
`tin. Once associated with a gene, the polycomb complex
`and the transcriptionally suppressed state are stable
`through DNA replication and cell division. This suggests
`a model where siRNAs target specific genomic DNA se-
`quences, probably by base pairing, thus directing the
`binding of the polycomb complex to adjacent sites, re-
`sulting in silencing of the locus. This attractive but
`speculative model awaits direct evidence that dsRNA or
`siRNAs can silence endogenous genes at the stage of
`transcription with concomitant association of polycomb
`complexes.
`Double-strand RNA-directed methylation of DNA
`Double-strand RNA-initiated gene-specific methylation
`of endogenous loci is a well-established phenomenon in
`plants. An early observation of the specific methylation
`of chromosomal DNA dependent on RNA replication in
`plants was described in Wessenegger et al. (1994). This
`work has been extended to demonstrate that genomic
`
`Figure 2. Proposal that siRNAs might be
`a regulatory intermediate in mRNA cleav-
`age, mRNA translation, DNA methyl-
`ation, and suppression of transcription by
`the polycomb group. See text for discus-
`sion of evidence for these potential rela-
`tionships.
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`sequences as short as 30 bp can be specifically methyl-
`ated when present in cells with replicating viral RNA
`containing homologous sequences (Pélissier and Wes-
`senegger 2000). Replicating recombinant viral RNA vec-
`tors containing different segments of an expressed gene
`have been used to demonstrate homology-based RNA-
`directed methylation (Jones et al. 1999; Merrett et al.
`2000). Methylation was directed to different portions of
`either the body of the gene or to the promoter when the
`corresponding segment was part of the replicating RNA.
`This would be consistent with conversion of the dsRNA
`of the replicating intermediate into siRNAs and target-
`ing of methylation by these short RNAs (Merrett et al.
`2000). Interestingly, a viral protein (Hc-Pro) that sup-
`presses PTGS (RNAi) when introduced into cells inhib-
`ited the maintenance of siRNAs, and a concomitant de-
`crease in methylation of the corresponding specific ge-
`nome sequence was observed (Llave et al. 2000).
`DNA methylation results in suppression of transcrip-
`tion probably by recruitment of histone deacetylases.
`The modified and silenced state is epigenetically trans-
`mitted, reducing expression of the gene in daughter cells.
`This is strikingly similar to the conjectured role of poly-
`comb proteins in cosuppression in C. elegans and Dro-
`sophila. At present, there is no known relationship be-
`tween polycomb suppression of gene expression and sub-
`sequent DNA methylation, but the possibility does not
`seem unreasonable. The analysis to date of cosuppres-
`sion, RNAi, and PTGS strongly indicates that RNAs can
`specify regulation of transcription of genomic sequences.
`These processes probably account for suppression of ex-
`pression of repetitive sequences in genomes, such as
`transposons and retroelements. RNAi/cosuppression has
`been demonstrated to be active in germ-line tissue and
`should be considered a ubiquitous process shaping the
`sequence content and structure of the genome of eukary-
`otic organisms.
`
`Acknowledgments
`
`I thank Tom Tuschl for the preprint; Michael McManus, Carl
`Novina, Tom Tuschl, Hristo Houbaviy, and Chris Burge for
`comments; and Helen Cargill for illustrations.
`
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