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Crystal structure of p19 – a universal suppressor of RNA silencing
Update
TRENDS in Biochemical Sciences
Vol.29 No.6 June 2004
| Research Focus
Crystal structure of p19 – a universal suppressor of RNA silencing David C. Baulcombe and Attila Molna´r The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK
RNA silencing in plants has an antiviral role and, consequently, plant viruses encode counter-defensive suppressor proteins that block this process. The recently reported crystal structure of two Tombusvirus suppressor proteins reveals a novel RNA-binding structure and illustrates precisely how the silencing mechanism is blocked. These suppressor protein structures, combined with molecular analyses of their effects in animal and plant cells, are informative about RNA silencing mechanisms. They also suggest various ways that Tombusvirus suppressors can be used to investigate RNA silencing in plants and animals. Eukaryotic cells can silence gene expression at the transcriptional or post-transcriptional level through processes that involve double-stranded (ds) and short [21 –25 nucleotide (nt)] RNAs. An RNase III-like enzyme DICER (DCR) [1] releases the short RNAs from longer dsRNAs of viruses, transposons, inverted-repeat DNA or from complementary pairs of sense and antisense RNAs. The short RNA is then incorporated into effector complexes of post-transcriptional or transcriptional silencing known as RNA interference specificity complex (RISC) [1] and RNA-induced initiation of transcriptional gene silencing (RITS), respectively [2]. RISC contains a ribonuclease that is guided by base pairing of the short single-stranded (ss) RNAs to a target mRNA. RITS contains short silencing RNAs that are likely to base pair with DNA or with chromatin-associated RNA and that directs chromatin modifications including histone methylation [2]. Short silencing RNAs – small interfering RNAs (siRNAs) – are derived from viral, transgene or transposon sequences and might exist in both single- and double-stranded forms. The most abundant forms of siRNAs are double stranded with a two-nucleotide overhang at each 30 end [3,4]. Depending on the nature of the dsRNA precursor, the siRNAs act to target either RISC or RITS at nucleic acids of viruses, transgenes or transposons. Micro RNAs (miRNAs) are a special case of short silencing RNA in which the precursor is an inverted-repeat RNA with partially doublestranded regions and its target is a ssRNA transcribed from a separate part of the genome. Like siRNA, the miRNA is incorporated into RISC although, in some instances, the silencing is due to interference with translation rather than RNA cleavage. The miRNAs are Corresponding author: David C. Baulcombe ([email protected]). Available online 30 April 2004 www.sciencedirect.com
unlike siRNAs in that they are predominantly single stranded and are often conserved in distantly related organisms [5,6]. p19 – a viral suppressor of silencing Viral suppressor proteins that counteract RNA silencing were first identified in plant cells [7– 9]. This, together with the discovery of siRNAs in virus-infected plant cells [10], led to the notion that RNA silencing is an antiviral defence system of plants. However, it is now known that viruses of insect and mammalian cells, including influenza virus [11,12], also encode silencing suppressor proteins. It seems that RNA silencing could have been an antiviral defence in primitive eukaryotes and that its function has been conserved during the evolution of plants and animals. One of the plant virus suppressors of silencing is a 19-kDa protein (p19) from the Tomato bushy stunt (Tombus) group of positive-strand RNA viruses. Previously, p19 was identified as a Tombusviral symptom determinant and a suppressor of silencing that binds siRNAs in vitro [13]. Now, from the crystal structures of Carnation Italian ringspot and Tomato bushy stunt virus p19 proteins bound to an siRNA duplex [14,15], we can explain precisely how it blocks silencing. The p19 structures have four [15] or five [14] a-helical regions and four b-strand regions. A tail-to-tail homodimer (Figure 1) is formed with hydrogen bonds between the fourth b strands and with hydrophobic and salt-bridge interactions between a helices at the C termini. The homodimer forms a concave b sheet that makes contact with phosphates and sugar 20 hydroxyls of the siRNA. Because these contacts are with the sugar phosphate backbone rather than the bases, the binding of p19 is not affected by the nucleotide sequence of the siRNA [14,15]. The involvement of the 20 hydroxyls also correlates with the ability of p19 to bind RNA rather than DNA duplexes [14,15]. The p19 dimers also have a pair of N-terminal a-helical structures that account for binding of p19 to 20 –22nt duplex siRNAs in preference to those that are longer or shorter [14,15]. These helices each have a pair of tryptophan residues that form a bracket around the ends of the siRNA base-paired region. Mutation analysis has confirmed the functional importance of these tryptophan residues for suppressor activity [14]. The p19 homodimer easily accommodates the 21-nt siRNA between these brackets. However, because the a helix is anchored through sidechain interactions with the b-sheet core, the siRNA duplexes bind less efficiently if they are . 22 nt or
Update
280
TRENDS in Biochemical Sciences
Vol.29 No.6 June 2004
α-helical bracket (dimer subunit 1)
α-helical bracket (dimer subunit 1)
Dimer subunit 1
siRNA
C terminus (dimer subunit 1)
siRNA
Dimer subunit 2
C terminus (dimer subunit 2)
Dimer subunit 2 α-helical bracket (dimer subunit 2) Ti BS
Figure 1. Crystal structure of Carnation Italian ringspot virus (CIRV) p19 in complex with 21-nucleotide small interfering RNA (siRNA). The two diagrams show different views of the p19 dimer with N-terminal (blue) and C-terminal (green) sub-domains and the siRNA rendered as ball-and-stick. Tryptophan residues involved in bracketing of the siRNA are added to the ribbon diagram and shown more clearly in the view on the right hand side. The disordered regions between the sub-domains are shown as a dotted line.
, 20 nt in length. The affinity of p19 for 19-nt and 25-nt siRNAs is 320- and 37-fold lower than for the 21-nt species, respectively [14]. This preference for 20 – 22-nt siRNA duplexes might be significant because plant cells contain different size classes of siRNA. There are the 21 – 22-nt siRNAs, as in animals, and a longer 24 – 25-nt class that has been implicated in RNA-directed epigenetic changes to the chromatin [16,17]. These 24 – 25-nt siRNAs are less affected by p19 than the 21-nt siRNAs [18], presumably because their duplexed form does not fit efficiently between the two a-helical brackets of the p19 dimer. The structures of p19 are unusual amongst dsRNAbinding proteins – but they are not unique. A C-terminal sub-domain of p19 is topologically similar to domain I of the 50S ribosomal protein L1 that binds, as a monomer, to a short double-stranded stem – loop in rRNA [14]. Strikingly, the RNA-binding domain in both proteins is a b sheet and it seems likely that it represents a general dsRNAbinding domain. Perhaps the structure of p19 is the signature of an ancient evolutionary event in which a virus acquired a host gene. It will be interesting, in due course, to find out whether other siRNA-binding proteins have a related structure. How does p19 binding to a siRNA duplex result in suppression of silencing? One scenario is that it enhances the activity of host-encoded suppressors of silencing that have now been identified in both plants and animals [19,20]. However, a simpler and perhaps more plausible explanation does not require additional host proteins. It www.sciencedirect.com
requires only that there is direct binding of p19 to the siRNA duplex. The presence of p19 would interfere with assembly of RISC because it would prevent an RNA helicase from gaining access to the unpaired ends of a siRNA duplex (Figure 2). In the absence of p19 this helicase selects one strand of the siRNA duplex to be the guide RNA in the mature RISC [21]. There are three lines of evidence to support this model of p19 action. First, there is the recent finding that p19 suppresses silencing in both animal and plant cells [12,13,22,23]. If other host proteins were involved it might be expected that p19 would be effective only in plant hosts. Second, it has been reported that p19 has no effect on the activity of DCR or on pre-formed RISC in a Drosophila cellfree extract, but that it does suppress recruitment of exogenously added siRNA into an active RISC [23]. Third, transgenic Arabidopsis expressing p19 [22] accumulate both miRNA and its complement (miRNA*), whereas in the control plants – without p19 – the miRNA* is undetectable [22]. Presumably, the miRNA – miRNA* duplex, being a product of DCR, has 30 overhangs like those in the duplex siRNA. It seems that this miRNA –miRNA* duplex is normally a short-lived precursor of miRNA RISC [21] but that it is stabilized in the presence of p19 because it cannot be unwound by an RNA helicase. p19 as a tool for the analysis of silencing An implication of this model (Figure 2) is that p19 can be used as a tool for the investigation of duplex siRNA and miRNA precursors in almost any cell type. It should be
Update
TRENDS in Biochemical Sciences
Vol.29 No.6 June 2004
281
References miRNA precursor
dsRNA
DICER Duplex miRNA or siRNA RNA helicase
p19
No silencing RISC
RNA silencing Ti BS
Figure 2. A model for RNA interference specificity complex (RISC) assembly in the presence or absence of p19. Dicing of double-stranded RNA (dsRNA) or of a micro RNA (miRNA) precursor is proposed to generate duplex small interfering (siRNA) or miRNA intermediates. The duplex RNA is a substrate for an RNA helicase that selects one of the two strands for incorporation into RISC; the other strand is degraded. In the presence of p19 the helicase is unable to access the duplex RNA and it is proposed that strand separation and RISC assembly does not occur.
possible, for example, to use p19 as a probe for affinity purification of siRNA or siRNA nucleoprotein. Complexes containing p19 could be purified by immunoprecipitation so that the siRNAs and the associated proteins could be characterized in detail. It might also be possible to modify p19, using information from the crystal structure, so that it preferentially recognizes the 25-nt siRNA and can be used to interfere with RITS rather than RISC. A further potential application involves targeted expression of p19 to tease out the natural role of RNA silencing. One example of how this can be achieved involves targeting p19 to the nucleus and cytoplasm of transgenic Arabidopsis [18]. In future, this approach could be refined by more precise targeting of p19 to different subcellular complexes or by regulated expression at particular times in development. The use of p19 in targeted suppression of silencing could have advantages over conventional genetic approaches because generalized loss of silencing, as implied by the phenotype of DCR mutants in plants and animals [24 – 26], is either lethal or results in severe growth defects.
1 Hannon, G.J. (2002) RNA interference. Nature 418, 244– 251 2 Verdel, A. et al. (2004) RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672 – 676 3 Elbashir, S.M. et al. (2001) RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev. 15, 188– 200 4 Nykanen, A. et al. (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309– 321 5 Ambros, V. et al. (2003) A uniform system for microRNA annotation. RNA 9, 277 – 279 6 Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281– 297 7 Anandalakshmi, R. et al. (1998) A viral suppressor of gene silencing in plants. Proc. Natl. Acad. Sci. U. S. A. 95, 13079 – 13084 8 Brigneti, G. et al. (1998) Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17, 6739 – 6746 9 Kasschau, K.D. and Carrington, J.C. (1998) A counterdefensive strategy of plant viruses: suppression of post-transcriptional gene silencing. Cell 95, 461 – 470 10 Hamilton, A.J. and Baulcombe, D.C. (1999) A species of small antisense RNA in post-transcriptional gene silencing in plants. Science 286, 950 – 952 11 Li, H. et al. (2002) Induction and suppression of RNA silencing by an animal virus. Science 296, 1319 – 1321 12 Li, W-X. et al. (2004) Interferon antagonist proteins of influenza and vaccinia virsus are suppressors of RNA silencing. Proc. Natl. Acad. Sci. U. S. A. 101, 1350 – 1355 13 Silhavy, D. et al. (2002) A viral protein suppresses RNA silencing and binds silencing-generated, 21-to 25-nucleotide double-stranded RNAs. EMBO J. 21, 3070 – 3080 14 Vargason, J.M. et al. (2003) Crystal structure of CIRV p19 bound to siRNA. Cell 115, 799– 811 15 Ye, K. et al. (2003) Recognition of small interfering RNA by a viral suppressor of RNA Silencing. Nature 426, 874– 878 16 Hamilton, A.J. et al. (2002) Two classes of short interfering RNA in RNA silencing. EMBO J. 21, 4671– 4679 17 Zilberman, D. et al. (2002) ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299, 716– 719 18 Papp, I. et al. (2003) Evidence for nuclear processing of plant micro RNA and short interfering RNA precursors. Plant Physiol. 132, 1382– 1390 19 Anandalakshmi, R. et al. (2000) A calmodulin-related protein that suppresses posttranscriptional gene silencing in plants. Science 290, 142– 144 20 Kennedy, S. et al. (2004) A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature 427, 645– 649 21 Schwarz, D.S. et al. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199 – 208 22 Dunoyer, P. et al. Probing the miRNA and siRNA pathways with virusencoded suppressor of RNA silencing. Plant Cell (in press) 23 Lakatos, L. et al. (2004) Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombsviruses. EMBO J. 23, 876– 884 24 Schauer, S.E. et al. (2002) DICER-LIKE1: blind men and elephants in Arabidopsis development. Trends Plant Sci. 7, 487– 491 25 Bernstein, E. et al. (2003) Dicer is essential for mouse development. Nat. Genet. 35, 215 – 218 26 Wienholds, E. et al. (2003) The microRNA-producing enzyme Dicer1 is essential for zebrafish development. Nat. Genet. 35, 217 – 218
Acknowledgements We are grateful to Traci M. Tanaka Hall (National Institute of Environmental Health Sciences) for preparation of Figure 1.
www.sciencedirect.com
0968-0004/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2004.04.007
TRENDS in Biochemical Sciences
Vol.29 No.6 June 2004
| Research Focus
Crystal structure of p19 – a universal suppressor of RNA silencing David C. Baulcombe and Attila Molna´r The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK
RNA silencing in plants has an antiviral role and, consequently, plant viruses encode counter-defensive suppressor proteins that block this process. The recently reported crystal structure of two Tombusvirus suppressor proteins reveals a novel RNA-binding structure and illustrates precisely how the silencing mechanism is blocked. These suppressor protein structures, combined with molecular analyses of their effects in animal and plant cells, are informative about RNA silencing mechanisms. They also suggest various ways that Tombusvirus suppressors can be used to investigate RNA silencing in plants and animals. Eukaryotic cells can silence gene expression at the transcriptional or post-transcriptional level through processes that involve double-stranded (ds) and short [21 –25 nucleotide (nt)] RNAs. An RNase III-like enzyme DICER (DCR) [1] releases the short RNAs from longer dsRNAs of viruses, transposons, inverted-repeat DNA or from complementary pairs of sense and antisense RNAs. The short RNA is then incorporated into effector complexes of post-transcriptional or transcriptional silencing known as RNA interference specificity complex (RISC) [1] and RNA-induced initiation of transcriptional gene silencing (RITS), respectively [2]. RISC contains a ribonuclease that is guided by base pairing of the short single-stranded (ss) RNAs to a target mRNA. RITS contains short silencing RNAs that are likely to base pair with DNA or with chromatin-associated RNA and that directs chromatin modifications including histone methylation [2]. Short silencing RNAs – small interfering RNAs (siRNAs) – are derived from viral, transgene or transposon sequences and might exist in both single- and double-stranded forms. The most abundant forms of siRNAs are double stranded with a two-nucleotide overhang at each 30 end [3,4]. Depending on the nature of the dsRNA precursor, the siRNAs act to target either RISC or RITS at nucleic acids of viruses, transgenes or transposons. Micro RNAs (miRNAs) are a special case of short silencing RNA in which the precursor is an inverted-repeat RNA with partially doublestranded regions and its target is a ssRNA transcribed from a separate part of the genome. Like siRNA, the miRNA is incorporated into RISC although, in some instances, the silencing is due to interference with translation rather than RNA cleavage. The miRNAs are Corresponding author: David C. Baulcombe ([email protected]). Available online 30 April 2004 www.sciencedirect.com
unlike siRNAs in that they are predominantly single stranded and are often conserved in distantly related organisms [5,6]. p19 – a viral suppressor of silencing Viral suppressor proteins that counteract RNA silencing were first identified in plant cells [7– 9]. This, together with the discovery of siRNAs in virus-infected plant cells [10], led to the notion that RNA silencing is an antiviral defence system of plants. However, it is now known that viruses of insect and mammalian cells, including influenza virus [11,12], also encode silencing suppressor proteins. It seems that RNA silencing could have been an antiviral defence in primitive eukaryotes and that its function has been conserved during the evolution of plants and animals. One of the plant virus suppressors of silencing is a 19-kDa protein (p19) from the Tomato bushy stunt (Tombus) group of positive-strand RNA viruses. Previously, p19 was identified as a Tombusviral symptom determinant and a suppressor of silencing that binds siRNAs in vitro [13]. Now, from the crystal structures of Carnation Italian ringspot and Tomato bushy stunt virus p19 proteins bound to an siRNA duplex [14,15], we can explain precisely how it blocks silencing. The p19 structures have four [15] or five [14] a-helical regions and four b-strand regions. A tail-to-tail homodimer (Figure 1) is formed with hydrogen bonds between the fourth b strands and with hydrophobic and salt-bridge interactions between a helices at the C termini. The homodimer forms a concave b sheet that makes contact with phosphates and sugar 20 hydroxyls of the siRNA. Because these contacts are with the sugar phosphate backbone rather than the bases, the binding of p19 is not affected by the nucleotide sequence of the siRNA [14,15]. The involvement of the 20 hydroxyls also correlates with the ability of p19 to bind RNA rather than DNA duplexes [14,15]. The p19 dimers also have a pair of N-terminal a-helical structures that account for binding of p19 to 20 –22nt duplex siRNAs in preference to those that are longer or shorter [14,15]. These helices each have a pair of tryptophan residues that form a bracket around the ends of the siRNA base-paired region. Mutation analysis has confirmed the functional importance of these tryptophan residues for suppressor activity [14]. The p19 homodimer easily accommodates the 21-nt siRNA between these brackets. However, because the a helix is anchored through sidechain interactions with the b-sheet core, the siRNA duplexes bind less efficiently if they are . 22 nt or
Update
280
TRENDS in Biochemical Sciences
Vol.29 No.6 June 2004
α-helical bracket (dimer subunit 1)
α-helical bracket (dimer subunit 1)
Dimer subunit 1
siRNA
C terminus (dimer subunit 1)
siRNA
Dimer subunit 2
C terminus (dimer subunit 2)
Dimer subunit 2 α-helical bracket (dimer subunit 2) Ti BS
Figure 1. Crystal structure of Carnation Italian ringspot virus (CIRV) p19 in complex with 21-nucleotide small interfering RNA (siRNA). The two diagrams show different views of the p19 dimer with N-terminal (blue) and C-terminal (green) sub-domains and the siRNA rendered as ball-and-stick. Tryptophan residues involved in bracketing of the siRNA are added to the ribbon diagram and shown more clearly in the view on the right hand side. The disordered regions between the sub-domains are shown as a dotted line.
, 20 nt in length. The affinity of p19 for 19-nt and 25-nt siRNAs is 320- and 37-fold lower than for the 21-nt species, respectively [14]. This preference for 20 – 22-nt siRNA duplexes might be significant because plant cells contain different size classes of siRNA. There are the 21 – 22-nt siRNAs, as in animals, and a longer 24 – 25-nt class that has been implicated in RNA-directed epigenetic changes to the chromatin [16,17]. These 24 – 25-nt siRNAs are less affected by p19 than the 21-nt siRNAs [18], presumably because their duplexed form does not fit efficiently between the two a-helical brackets of the p19 dimer. The structures of p19 are unusual amongst dsRNAbinding proteins – but they are not unique. A C-terminal sub-domain of p19 is topologically similar to domain I of the 50S ribosomal protein L1 that binds, as a monomer, to a short double-stranded stem – loop in rRNA [14]. Strikingly, the RNA-binding domain in both proteins is a b sheet and it seems likely that it represents a general dsRNAbinding domain. Perhaps the structure of p19 is the signature of an ancient evolutionary event in which a virus acquired a host gene. It will be interesting, in due course, to find out whether other siRNA-binding proteins have a related structure. How does p19 binding to a siRNA duplex result in suppression of silencing? One scenario is that it enhances the activity of host-encoded suppressors of silencing that have now been identified in both plants and animals [19,20]. However, a simpler and perhaps more plausible explanation does not require additional host proteins. It www.sciencedirect.com
requires only that there is direct binding of p19 to the siRNA duplex. The presence of p19 would interfere with assembly of RISC because it would prevent an RNA helicase from gaining access to the unpaired ends of a siRNA duplex (Figure 2). In the absence of p19 this helicase selects one strand of the siRNA duplex to be the guide RNA in the mature RISC [21]. There are three lines of evidence to support this model of p19 action. First, there is the recent finding that p19 suppresses silencing in both animal and plant cells [12,13,22,23]. If other host proteins were involved it might be expected that p19 would be effective only in plant hosts. Second, it has been reported that p19 has no effect on the activity of DCR or on pre-formed RISC in a Drosophila cellfree extract, but that it does suppress recruitment of exogenously added siRNA into an active RISC [23]. Third, transgenic Arabidopsis expressing p19 [22] accumulate both miRNA and its complement (miRNA*), whereas in the control plants – without p19 – the miRNA* is undetectable [22]. Presumably, the miRNA – miRNA* duplex, being a product of DCR, has 30 overhangs like those in the duplex siRNA. It seems that this miRNA –miRNA* duplex is normally a short-lived precursor of miRNA RISC [21] but that it is stabilized in the presence of p19 because it cannot be unwound by an RNA helicase. p19 as a tool for the analysis of silencing An implication of this model (Figure 2) is that p19 can be used as a tool for the investigation of duplex siRNA and miRNA precursors in almost any cell type. It should be
Update
TRENDS in Biochemical Sciences
Vol.29 No.6 June 2004
281
References miRNA precursor
dsRNA
DICER Duplex miRNA or siRNA RNA helicase
p19
No silencing RISC
RNA silencing Ti BS
Figure 2. A model for RNA interference specificity complex (RISC) assembly in the presence or absence of p19. Dicing of double-stranded RNA (dsRNA) or of a micro RNA (miRNA) precursor is proposed to generate duplex small interfering (siRNA) or miRNA intermediates. The duplex RNA is a substrate for an RNA helicase that selects one of the two strands for incorporation into RISC; the other strand is degraded. In the presence of p19 the helicase is unable to access the duplex RNA and it is proposed that strand separation and RISC assembly does not occur.
possible, for example, to use p19 as a probe for affinity purification of siRNA or siRNA nucleoprotein. Complexes containing p19 could be purified by immunoprecipitation so that the siRNAs and the associated proteins could be characterized in detail. It might also be possible to modify p19, using information from the crystal structure, so that it preferentially recognizes the 25-nt siRNA and can be used to interfere with RITS rather than RISC. A further potential application involves targeted expression of p19 to tease out the natural role of RNA silencing. One example of how this can be achieved involves targeting p19 to the nucleus and cytoplasm of transgenic Arabidopsis [18]. In future, this approach could be refined by more precise targeting of p19 to different subcellular complexes or by regulated expression at particular times in development. The use of p19 in targeted suppression of silencing could have advantages over conventional genetic approaches because generalized loss of silencing, as implied by the phenotype of DCR mutants in plants and animals [24 – 26], is either lethal or results in severe growth defects.
1 Hannon, G.J. (2002) RNA interference. Nature 418, 244– 251 2 Verdel, A. et al. (2004) RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672 – 676 3 Elbashir, S.M. et al. (2001) RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev. 15, 188– 200 4 Nykanen, A. et al. (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309– 321 5 Ambros, V. et al. (2003) A uniform system for microRNA annotation. RNA 9, 277 – 279 6 Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281– 297 7 Anandalakshmi, R. et al. (1998) A viral suppressor of gene silencing in plants. Proc. Natl. Acad. Sci. U. S. A. 95, 13079 – 13084 8 Brigneti, G. et al. (1998) Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17, 6739 – 6746 9 Kasschau, K.D. and Carrington, J.C. (1998) A counterdefensive strategy of plant viruses: suppression of post-transcriptional gene silencing. Cell 95, 461 – 470 10 Hamilton, A.J. and Baulcombe, D.C. (1999) A species of small antisense RNA in post-transcriptional gene silencing in plants. Science 286, 950 – 952 11 Li, H. et al. (2002) Induction and suppression of RNA silencing by an animal virus. Science 296, 1319 – 1321 12 Li, W-X. et al. (2004) Interferon antagonist proteins of influenza and vaccinia virsus are suppressors of RNA silencing. Proc. Natl. Acad. Sci. U. S. A. 101, 1350 – 1355 13 Silhavy, D. et al. (2002) A viral protein suppresses RNA silencing and binds silencing-generated, 21-to 25-nucleotide double-stranded RNAs. EMBO J. 21, 3070 – 3080 14 Vargason, J.M. et al. (2003) Crystal structure of CIRV p19 bound to siRNA. Cell 115, 799– 811 15 Ye, K. et al. (2003) Recognition of small interfering RNA by a viral suppressor of RNA Silencing. Nature 426, 874– 878 16 Hamilton, A.J. et al. (2002) Two classes of short interfering RNA in RNA silencing. EMBO J. 21, 4671– 4679 17 Zilberman, D. et al. (2002) ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299, 716– 719 18 Papp, I. et al. (2003) Evidence for nuclear processing of plant micro RNA and short interfering RNA precursors. Plant Physiol. 132, 1382– 1390 19 Anandalakshmi, R. et al. (2000) A calmodulin-related protein that suppresses posttranscriptional gene silencing in plants. Science 290, 142– 144 20 Kennedy, S. et al. (2004) A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature 427, 645– 649 21 Schwarz, D.S. et al. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199 – 208 22 Dunoyer, P. et al. Probing the miRNA and siRNA pathways with virusencoded suppressor of RNA silencing. Plant Cell (in press) 23 Lakatos, L. et al. (2004) Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombsviruses. EMBO J. 23, 876– 884 24 Schauer, S.E. et al. (2002) DICER-LIKE1: blind men and elephants in Arabidopsis development. Trends Plant Sci. 7, 487– 491 25 Bernstein, E. et al. (2003) Dicer is essential for mouse development. Nat. Genet. 35, 215 – 218 26 Wienholds, E. et al. (2003) The microRNA-producing enzyme Dicer1 is essential for zebrafish development. Nat. Genet. 35, 217 – 218
Acknowledgements We are grateful to Traci M. Tanaka Hall (National Institute of Environmental Health Sciences) for preparation of Figure 1.
www.sciencedirect.com
0968-0004/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2004.04.007