Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering

N. Baumberger and D. C. Baulcombe*

Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, United Kingdom

Contributed by D. C. Baulcombe, June 30, 2005 ARGONAUTE (AGO) RNA-binding proteins are involved in RNA at sRNA-complementary sites. The size and composition of silencing. They bind to short interfering RNAs (siRNAs) and RISC varies (8–10), but AGO proteins are a common compo- microRNAs (miRNAs) through a conserved PAZ domain, and, in nent in all systems. The crystal structure of an archaebacterial animals, they assemble into a multisubunit RNA-induced silencing AGO homologue revealed that the PIWI domain, one of the two complex (RISC). The mammalian AGO2, termed Slicer, directs signature domains of AGO proteins, adopts a fold similar to siRNA- and miRNA-mediated cleavage of a target RNA. In Arabi- RNase H (11). This finding prompted the suggestion that AGO dopsis, there are 10 members of the AGO family, and the AGO1 proteins may be the Slicer , and, consistent with this protein is potentially the Slicer component in different RNA- idea, the human AGO2 has a DDH motif that is essential for silencing pathways. Here, we show that AGO1 selectively recruits Slicer activity and is functionally equivalent to the catalytic certain classes of short silencing-related RNA. AGO1 is physically metal-coordinating triad DDE of RNase H (12, 13). Final proof associated with miRNAs, transacting siRNAs, and transgene- that hAGO2 has Slicer activity was from the finding that the derived siRNAs but excludes virus-derived siRNAs and 24-nt siRNAs bacterially expressed recombinant protein is competent for involved in chromatin silencing. We also show that AGO1 has Slicer sRNA-directed RNA cleavage (13). The PAZ domain is a activity. It mediates the in vitro cleavage of a mir165 target RNA in second signature of AGO proteins, and it has been implicated by a manner that depends on the sequence identity of amino acid structural studies as an sRNA-binding feature (14–17). residues in the PIWI domain that are predicted by homology with Many eukaryotes, with the notable exception of fission yeast, animal Slicer-competent AGO proteins to constitute the RNase have AGO multigene families whose members have specialized catalytic center. However, unlike animals, we find no evidence that biological function, as revealed by the variety of mutant phenotypes AGO1 Slicer is in a high molecular weight RNA-induced silencing (18). For example, mutants defective in RDE1, one of the 23 complex. The Slicer activity fractionates as a complex of Ϸ150 kDa Caenorhabditis elegans AGO family members, are resistant to that likely constitutes the AGO1 protein and associated RNA RNAi, but the miRNA pathway operates normally (19), whereas without any other proteins. Based on sequence similarity, we AGO-like (alg)-1 and alg-2 mutants are competent for RNAi predict that other Arabidopsis AGOs might have a similar catalytic but have heterochronic defects due to defects in a miRNA-silencing activity but recruit different subsets of siRNAs or miRNAs. pathway (20). Drosophila ago mutants also reveal diversification of siRNA and miRNA pathways: ago2 mutants are RNAi-defective posttranscriptional regulation ͉ ͉ viral RNA ͉ silencing but are able to mediate miRNA-directed RNA cleavage. The ago1 mutants, in contrast, are deficient in miRNA processing and RGONAUTE (AGO) proteins are implicated in RNA- subsequent miRNA-mediated RNA cleavage but not in RNAi (21). Asilencing processes that also involve 21- to 26-nt short RNAs The four human ARGONAUTES are equally competent for (sRNAs) (1) cleaved from double-stranded or partially double- binding siRNAs and miRNAs, but only AGO2 is able to mediate stranded (ds) RNAs by the RNase III . There are RNA cleavage of target RNAs (12, 22). Although AGO2 is essential several types of RNA-silencing mechanisms, including RNA inter- for embryo development, it is still unclear whether this requirement ference (RNAi), the micro RNA (miRNA) pathway, and RNA- is related to its unique ability to cleave mRNAs (12). directed chromatin silencing (1). RNAi is a type of RNA silencing In Arabidopsis, the AGO family comprises 10 members (18, 23) in which the Dicer substrate is fully double stranded, the sRNA of which two have been unambiguously associated with different cleavage product is short interfering RNA (siRNA), and the forms of RNA silencing. It is therefore likely that, as in animals, outcome is targeted destruction of siRNA-complementary RNAs. the functional diversification of RNA silencing is linked to the The miRNA pathway is similar except that the Dicer substrate is an variation between AGO family members. AGO1 is associated inverted repeat RNA with a partially ds structure, the sRNA is with the miRNA pathway and transgene-silencing pathway (23, referred to as a miRNA, and the target RNAs can be suppressed 24), and AGO4 with endogenous siRNAs affecting epigenetic at the translational level or degraded as in RNAi, depending on the silencing (25, 26). In addition, AGO7 and ZLL͞AGO10 have a degree of complementarity between the sRNA and its target. Plants function in the transition from juvenile to adult phases of plant possess an additional class of degradative sRNAs called transacting growth (27) and meristem maintenance (28, 29), respectively. siRNAs (ta-siRNAs) whose formation depends on the miRNA- Although a role in sRNA-mediated regulation seems likely, it is mediated cleavage of their precursor and its conversion into a not yet supported by evidence. dsRNA by RDR6 (2–4). The last pathway, RNA-directed chro- matin silencing, is similar to RNAi, but the siRNA targets are either DNA or chromatin-associated RNAs and the outcome is DNA Freely available online through the PNAS open access option. methylation or histone modification at the target . Abbreviations: sRNA, short RNA; siRNA, short interfering RNA; miRNA, microRNA; AGO, In the best understood of these RNA-silencing mechanisms, Argonaute; RNAi, RNA interference; ta-siRNA, transacting siRNA; RISC, RNA-induced si- lencing complex; CMV, Cucumber Mosaic Virus; GF-IR, GFP inverted repeat; PHV, the duplex siRNAs or miRNAs produced by Dicer are unwound PHAVOLUTA; T-DNA, portion of the Ti (tumor-inducing) plasmid that is transferred to plant in an ATP-dependent process. One strand of this RNA is then cells. preferentially assembled with an AGO protein to form an *To whom correspondence should be addressed. E-mail: david.baulcombe@sainsbury- RNA-induced silencing complex (RISC) (5–7). RISC has an laboratory.ac.uk. associated ribonuclease activity (Slicer) that cleaves its substrate © 2005 by The National Academy of Sciences of the USA

11928–11933 ͉ PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505461102 Downloaded by guest on September 29, 2021 The most studied Arabidopsis AGO protein is AGO1. It is ated on a Superose 6 column (Amersham Pharmacia). Fractions clearly implicated in miRNA silencing because strong ago1 were concentrated again and used for the Slicer assay and alleles affect miRNA accumulation and miRNAs target regula- western analysis. tion (24), as do dcl1, hen1, and hyl1 (30–33). The ago1 mutants A more detailed version of Materials and Methods is available are also impaired in spontaneous silencing of a foreign transgene as Supporting Materials and Methods, which is published as (cosupression) (23) and exhibit hypersusceptibility to Cucumber supporting information on the PNAS web site. Mosaic Virus (CMV) (34), suggesting that, in plants, miRNA- mediated silencing, transgene silencing, and virus induced si- Results and Discussion lencing share a common AGO factor. However, those observa- Epitope Tagging of Arabidopsis AGO1. If AGO1 is an RNA- tions could be explained equally well if AGO1 functions in either silencing Slicer, it would physically interact with miRNAs and the biogenesis of sRNAs or as Slicer. siRNAs and would cleave mRNA targets that are complemen- Here, we investigate the role of AGO1 in RNA silencing. We tary to these small RNAs. To test these predictions, we con- show that affinity-purified AGO1 is associated with miRNAs, structed an N-terminal FLAG-tagged version of the AGO1 endogenous ta-siRNAs, and transgene-derived siRNAs but not cDNA coupled to 1,648 bp of the AGO1 promoter (Fig. 1A). The virus-specific siRNAs or siRNAs involved in chromatin silenc- construct was transformed into an ago1 mutant (salk࿝087076, ing. We also show that, dependent on conserved amino acid named ago1-36)(Fig. 1B) in which a T-DNA [portion of the Ti residues in the PIWI domain, AGO1 mediates the in vitro (tumor-inducing) plasmid that is transferred to plant cells] cleavage of PHAVOLUTA RNA at the mir165 target site. It is insertion at the end of the PAZ domain resulted in production therefore likely that AGO1 is a Slicer that selectively recruits of a truncated protein. The ago1-36 alleles are likely null because Ϸ sRNAs. The Slicer activity fractionates in a complex of 150 the encoded protein lacked Ͼ50% of the protein sequence, kDa that likely constitutes the AGO1 protein and associated including the PIWI domain and because they show a stunted RNA without any other proteins. growth phenotype like that of strong ago1 alleles (Fig. 1C) AGO1 FLAG AGO1 Materials and Methods (39–41). The tagged transgene ( - ) fully restored a WT phenotype in ago1-36 homozygous mutants, FLAG-AGO Transgenic Arabidopsis. A FLAG-AGO1 construct was indicating that the introduced tag does not interfere with AGO1 generated by fusing the AGO1 cDNA to an N-terminal FLAG function (Fig. 1 C and D). The expression level of the transgene sequence under the regulation of the AGO1 promoter in the assessed by RT-PCR was similar to that of the endogenous binary vector pGreen0229. The construct was transformed into AGO1 gene in WT plants (Fig. 1E). heterozygous ago1-36 (Salk࿝087076) (http:͞͞signal.salk.edu) and homozygous ago1-36 transgenic seedlings were identified by AGO1 Interacts Selectively with miRNAs and siRNAs. FLAG-AGO1 AGO1 PCR. The expression levels of the gene and transgene could be immunoprecipitated from young inflorescence extracts, were assessed by RT-PCR on 2-week-old WT, ago1-36, and thereby demonstrating that the N terminus of the protein is FLAG-AGO1 seedlings. accessible under native conditions (Fig. 2A). The AGO1- associated RNA was extracted from immunoprecipitated AGO1, Immunoprecipitation and sRNA Analysis. FLAG-AGO1 immuno- and the presence of siRNAs and miRNAs was assessed by precipitation from inflorescences of transgenic FLAG-AGO1 Northern blotting. plants or WT plants was performed with ␣-FLAG M2 agarose All tested miRNA species specifically copurified with FLAG- beads (Sigma). RNA extracted either from the immunoprecipi- tate or directly from tissues as control was analyzed by Northern AGO1 and were absent from FLAG immunoprecipitates of nontransformed (WT) extracts (Fig. 2B). The associated RNAs blotting. For analysis of transgene-derived siRNAs, we used F1 plants from crosses between FLAG-AGO1 plants and two ge- include both 21-nt (miR160, 167, and 319) and 24-nt (miR163) species. The AGO1-associated miRNAs, in common with the notypes that were silencing GFP. One of these was an RNAi line ␤ in which the transgene expressed a GFP inverted repeat (GF-IR) total pool of plant miRNAs, are methylated (42). In a - (35). The second genotype (GF-amp) expressed a viral transgene elimination test, the miRNAs associated with AGO1 remained and exhibited sense RNA silencing (previously described as unmodified, consistent with the presence of an O-methyl group Ј Ј Ј GxA) (36). Virus infections were performed by rub-inoculation on the 3 terminal nucleotide either on the 2 or 3 carbon of the on 2-week-old FLAG-AGO1 plants. For the RNA methylation ribose (37), whereas a synthetic unmethylated siRNA included in test, GFP synthetic unmethylated siRNAs were added to the the samples as internal control underwent the predicted increase AGO1-associated RNA sample, and a ␤-elimination reaction in electrophoretic mobility (Fig. 2C). was performed as described in ref. 37. The ta-siRNA255 was also physically associated with AGO1 (Fig. 2D), but the 24-nt siRNAs produced by DCL3, including Slicer Assay. PHAVOLUTA (PHV) cleavage assays were per- siRNAs 02, cluster 2, and 1003, were not (Fig. 2E) (43). Thus, formed with immunoprecipitated FLAG-AGO1 and 32P-labeled AGO1 is selective for certain types of endogenous silencing- PHV or phv in vitro transcripts (mMESSAGE mMACHINE T7, related sRNAs. We could also demonstrate selectivity of AGO1 Ambion, Austin, TX). As a positive control, 20 ␮l of wheat germ with siRNAs of foreign nucleic acids. Transgene-specific extract was used. Cleavage was tested after 90 min at 25°C by siRNAs, either from an inverted repeat transgene (GF-IR)or extracting and separating the RNA on an 8 M urea͞3% poly- from a viral amplicon͞sense GFP transgene silencing system acrylamide gel. (GFP-Amp), were associated with AGO1 (Fig. 2F) but virus- specific siRNAs (CMV, Turnip Crinkle Virus, and crucifer To- PLANT BIOLOGY Nicotiana benthamiana Transient Expression. Specific mutations in bacco Mosaic Virus) were not (Fig. 2G). FLAG-AGO1 were introduced by PCR. For transient expression The AGO1 association of GF-IR siRNAs was unexpected in N. benthamiana, the constructs were transferred into the because ago1-27 plants retain the ability to carry out RNAi from pBIN61 vector (38) and Agro-infiltrated into N. benthamiana inverted repeat transgenes (44). However, the genetic test might leaves. Immunoprecipitations, Slicer assay and sRNA analysis not have been conclusive because ago1-27 is a weak allele and were as described above. might encode a protein still competent for RNAi (34, 44). Conversely, we had anticipated that viral siRNAs would be Size Exclusion Chromatography. Immunoprecipitated and eluted associated with AGO1 because Arabidopsis ago1 plants were FLAG-AGO1 were concentrated by ultrafiltration and fraction- previously reported to be hypersusceptible to CMV (34). This

Baumberger and Baulcombe PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 ͉ 11929 Downloaded by guest on September 29, 2021 mechanism by which this protein recruits sRNA. Among the different factors that might determine this specificity, we have considered sRNA size, subcellular localization, and the effects of virus-encoded suppressors of silencing. Of these factors, sRNA size can be ruled out because 21- and 24-nt sRNAs were present in both the AGO1-associated and AGO1-excluded fractions (Fig. 2). Subcellular location may be involved, but it is unlikely to be the sole determining factor in the AGO1 selection of sRNA because certain classes of both nuclear (chromatin associated siRNAs) (43, 45) and cytoplasmic sRNAs (viral siRNAs) were excluded from AGO1. A third possible factor involves the virus-encoded silencing suppressors produced in virus-infected plants. In principle, the absence of viral siRNAs in the AGO1- associated sRNA could be due to the action of these suppressors. However, this result does not seem likely, because these proteins had no effect on AGO1 association with a miRNA (miR167; Fig. 2G). An attractive alternative mechanism of selectivity involves linking the mode of sRNA biogenesis with particular AGOs. Perhaps different Dicers dock onto specific AGO proteins so that sRNAs are addressed to specialized effector complexes. Consistent with this idea, the Dicers in animal systems not only are required for the processing of sRNAs from their precursors but they also play a role in the assembly of RISC (8, 46–48). In addition, there is a direct interaction in vitro between a subregion of the PIWI domain of hAGO2 and the RNase III domain of human Dicer (49). Evidence consistent with this channeling model is from the finding that miRNAs and ta-siRNAs associ- ated with AGO1 (Fig. 2 B–D) all require DCL1 for their biogenesis (2, 4, 30) whereas the AGO1 excluded 24-nt siRNAs, and viral siRNAs are instead produced by DCL3 and possibly DCL2 (43). A prediction from this Dicer-channeling hypothesis is that the Dicer for production of transgene siRNAs would also interact Fig. 1. Generation of epitope-tagged AGO1 transgenic Arabidopsis.(A) directly with AGO1. In principle, this Dicer could be either Diagram of the FLAG-AGO1 construct. Positions of the restriction sites used for DCL1 or DCL4, whose function is unknown, that channels the cloning are given relative to the start codon. The sequence and position of transgene sRNAs into AGO1. The finding that RNAi from the FLAG epitope is indicated. Thick lines, regions encoding the PAZ and PIWI inverted repeat transgenes functions in dcl1-9 plants (50) does domains; thin broken arrow, translation start; black dot, translation stop. (B) not necessarily rule out this prediction. Functional redundancy Diagram of the AGO1 genomic locus. Gray boxes, exons; triangle, T-DNA insertion in ago1 mutant Salk࿝087076 line (ago1-36) with left border (Lb) and in the DCL family or residual function of the dcl1-9 allele could right border (Rb) orientation. Other symbols as in A.(C) FLAG-AGO1 comple- mask the role of DCL1 in production of transgene siRNAs. ments the ago1-36 phenotype. Photographs are taken 2 weeks postgermina- tion. (D) PCR genotyping of the FLAG-AGO1 line. The ago1-36 (Upper) and not FLAG-AGO1 Has Slicer Activity and Is Not in a High Molecular Weight the WT allele (Lower) is amplified from the selected FLAG-AGO1 transgenic RISC. To find out whether AGO1 is present in a RISC complex line. (E) Expression of FLAG AGO1 transcripts. ago1-36 mutants produce a or is itself Slicer, we first examined the ability of immunopurified truncated transcript comprising the sequence 5Ј (Middle) but not 3Ј (Bottom) FLAG-AGO1 to mediate in vitro cleavage of a PHV transcript, of the T-DNA insertion. Expression of a full-length AGO1 transcript is restored a target of miR165 (40, 51). The results, shown in Fig. 3, reveal in the selected FLAG-AGO1 line. Actin primers (Top) were used to confirm that PHV 5Ј and 3Ј RNA cleavage products were specifically equal loading, and reactions without reverse transcriptase were performed to exclude DNA contamination. DNA, control PCR with genomic DNA. formed in extracts from FLAG-AGO1 plants but not from control non transformed plants. No cleavage products were either observed when a G 3 A mutation was inserted into the phenotype could have been accounted for by the involvement of PHV RNA sequence at the residue complementary to positions AGO1 in virus-induced silencing. However, it is now unlikely 6ofmiR165 (phv, Fig. 3). This alteration induces a dominant that AGO1 is a major cofactor of virus-induced silencing because mutation phenotype in planta by preventing miRNA-mediated PHV PHB the viral siRNAs were not associated with AGO1 (Fig. 2G) and clearing of the and transcripts (52, 53) and abolishes cleavage in a wheat germ in vitro assay (51). we could not reproduce the hypersusceptibility phenotype even This miRNA-directed RNA cleavage could result from Slicer with plant genotype (ago1-27) and strain of CMV (I17F) used activity of either AGO1 or other associated proteins. To inves- previously (34) (data not shown). To reconcile the earlier tigate these possibilities, we modified the core PIWI domain of findings with the results presented here, we propose that there AGO1 at residues that are conserved in cleavage-competent could be subtle environmental factors affecting the experiments. AGO proteins from animals (Fig. 4A). In AGO1, the aspartate For example, there could be an environmentally influenced residue at position 760 is equivalent to the first metal coordi- miRNA that targets the mRNA of a plant-encoded suppressor nating aspartate D597 of hAGO2. Mutation of this residue to of viral defense or there could be an environmentally sensitive alanine in hAGO2 abolishes in vitro cleavage activity, and we ago1 phenotype that influences the virulence of CMV. predicted that a similar mutation in AGO1 would yield the same result if AGO1 is Slicer. The Gly-758 and His-798 are also The Molecular Basis of AGO1 Selectivity. The profile of RNA strongly conserved throughout AGOs. Gly-758 is mutated to associated with AGO1 implies that there is selectivity in the proline in the hypomorphic ago1-25 mutant allele (34) whereas

11930 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505461102 Baumberger and Baulcombe Downloaded by guest on September 29, 2021 Fig. 3. AGO1 copurifies with Slicer activity. In vitro-labeled WT PHV or mutant G 3 A phv target RNAs were incubated with immunoprecipitates from FLAG-AGO1 and WT plants or with wheat germ extracts (Wg) as positive control. The sizes of the predicted 5Ј and 3Ј PHV RNA cleavage products are indicated.

the H798P mutation in hAGO2 (12) results in loss of RNA cleavage activity. The G758S and H798P mutations were introduced into FLAG-AGO1 constructs under the cauliflower mosaic virus (CaMV)35S promoter and expressed transiently in N. benthami- ana leaves, whereas the construct carrying the D760A mutation was transformed stably in Arabidopsis under the AGO1 pro- moter. The immunoaffinity-purified FLAG-AGO1 mutant pro- teins could be detected by Western blotting, and the WT protein was correctly programmed by endogenous miR165 as it cleaved the PHV RNA in vitro into the predicted 5Ј and 3Ј fragments (Fig. 4B). The AGO1 G758S, was also able to cleave the PHV RNA target, and it is possible that the hypomorphic phenotype of the corresponding Arabidopsis mutant and its impaired cosuppres- sion ability (34) are due to altered kinetic properties of AGO1 but not to total inactivity. In contrast, D760A and H798P were totally cleavage-deficient (Fig. 4 B and C) despite their ability to recruit miRNAs. We interpret the effect of changes at conserved catalytic site residues as a strong indication that AGO1 is Slicer. Slicer in animal cells is part of a high molecular weight RISC that includes accessory proteins including TSN nuclease, Gemin, Fragile X syndrome-associated protein, and other proteins (8– 10, 54–57). However, if such a large complex exists in plants, the accessory proteins are not required for Slicer activity because size exclusion chromatography revealed that the FLAG-AGO1 and the associated Slicer activity from Arabidopsis inflorescences eluted together close to the 158-kDa molecular mass standard (Fig. 5). The predicted molecular mass of the tagged version of AGO1 is 116 kDa, the associated siRNA would be Ϸ7 kDa and, given the imprecision of size determination by gel filtration, it

recruited by AGO1. (E) Endogenous chromatin siRNAs are not recruited by AGO1. In B, D, and E, the IP samples were derived from 7-fold more tissue than directly extracted RNA. (F) Transgene-derived siRNAs are associated with Fig. 2. AGO1 recruitment of small RNAs. (A) Immunoprecipitation of FLAG- AGO1. FLAG-AGO1 plants were crossed with plants coexpressing the GFP AGO1. FLAG-AGO1 was immunoprecipitated from crude inflorescence extract together with a GF inverted repeat silencer construct (GF-IR line) or with plants as described in Materials and Methods. Input, crude extract before immuno- coexpressing the GFP together with a Potato Virus X-GFP silencer construct precipitation; unbound, supernatant after immunoprecipitation; IP, immuno- (GFP-Amp). Blots show GFP-derived siRNAs (Top)ormiR167 (Middle) from PLANT BIOLOGY precipitate. AGO1(ϩ), FLAG-AGO1 transgenic extracts; AGO(-), WT extracts. parent and F1 plants. The IP samples were derived from 20-fold more tissue (B) miRNAs are recruited by AGO1. sRNA blots were hybridized with DNA than directly extracted RNA. (Bottom) A Western blot of FLAG-AGO1. (G) Viral oligonucleotide probes complementary to the indicated miRNAs. RNAs were siRNAs are not associated with AGO1. FLAG-AGO1 or WT plants were infected either extracted directly from plant material (RNAtot) or from ␣-FLAG immu- with Cucumber Mosaic Virus strain I17F (CMV), Turnip Crinkle Virus (TCV), and noprecipitate (␣FLAG IP). (C) AGO1-recruited miRNAs are methylated at the 3Ј Cruciferae Tobacco Mosaic Virus (CrTMV). siRNAs (Upper) were detected by terminus. A synthetic unmethylated GFP 21-nt RNA oligomer mixed with the hybridization with an in vitro transcribed sense probe corresponding to the immunoprecipitated RNAs (Upper) displays an increased electrophoretic mo- coat protein sequence of the virus (CMV and TCV) or the full-length cDNA bility after a ␤-elimination reaction (␤-El ϩ) whereas miR167 (Lower) does not, (TMV). miR167 (Lower) was detected with a complementary DNA oligonucle- indicating that its 3Ј end is protected by methylation. Both GFP and miR167 otide probe on the same blot after stripping. The IP samples were derived from hybridization were done on the same blot after stripping. (D) ta-siRNAs are 10-fold more tissue than directly extracted RNA.

Baumberger and Baulcombe PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 ͉ 11931 Downloaded by guest on September 29, 2021 Fig. 4. Mutations of conserved residues of the AGO1 PIWI domain affect Slicer activity. (A) Alignment of the catalytic center of the Arabidopsis and human ARGONAUTE PIWI domains. The positions of the Mg2ϩ coordinating residues in the DDH catalytic triad are indicated above the alignment (arrows), as well as the mutations introduced in FLAG-AGO1 (*). (B and C) Slicer activity of mutant AGO1 proteins. FLAG-AGO1, FLAG-AGO1G758S, and FLAG-AGO1H798P were expressed transiently in N. benthamiana by Agrobacterium infiltration, and Slicer activity of the immunoprecipitated proteins was assayed in vitro (B top). FLAG-AGO1D760A could not be assayed because it was unstable in the transient assay, but it did accumulate and its PHV Slicer activity could be assayed in extracts of transgenic Arabidopsis (C Top). Recruitment of miR165 was verified by Northern blotting of sRNAs extracted from immunoprecipitates (Bottom). The level of immunopurified FLAG-AGO1 proteins was tested by Western blotting (Middle).

seems that, as with hAGO2 (13, 58), the minimal Arabidopsis much less stable in vitro than the equivalent complexes in RISC contains little more than AGO1 and an associated sRNA. animals. Alternatively, the high molecular weight complex might These findings do not rule out that a high molecular weight have a structure that impairs accessibility of the epitope tag on complex, for example including Dicer, HYL1, and other pro- the N terminus of AGO1 and prevents the purification of the teins, such as TSN nuclease, is formed during the assembly of a native complex. However, only a minor fraction of the total miRNA͞siRNA-programmed AGO1 Slicer. Such a complex AGO1 pool would be present in a high molecular weight complex could be difficult to detect because it exists only transiently or is because most AGO1 can be immunoprecipitated from a crude extract (Fig. 2A).

Selective sRNA Recruitment and Slicer Activity in Other Arabidopsis AGO Proteins? All of the 10 Arabidopsis AGO proteins have PAZ domains and so are potentially able to recruit siRNAs and miRNAs. All 10 also resemble the RNA cleavage-active hAGO2 in that they have conserved aspartates as two of the putative metal- coordinating residues in the catalytic site (Fig. 4A) (11, 12). However, AGO2 and -3 differ from the other AGOs in that they have an additional aspartate as the third coordinating residues in place of the conserved histidine. This third carboxylic acid residue is not likely to impair Slicer activity because two other related , RNaseH1 and Tn5 integrase, can use aspartate efficiently Fig. 5. FLAG-AGO1 Slicer is present in low molecular weight complexes. in that position (13, 59, 60). Most of the AGO proteins, also like ␣ FLAG-AGO1 was immunopurified and eluted from -FLAG M2 agarose beads hAGO2, have a conserved histidine at the equivalent of AGO1 by competition with 3XFLAG peptides. The concentrated eluate was fraction- ated on a Superpose 6 column, and the fractions were tested for cleavage of position 798, and it is likely that they selectively recruit siRNAs or PHV target RNA (Upper) and for presence of AGO1 by Western blotting a subset of miRNAs and use them as guides in RNA cleavage (Lower). The elution profile of the molecular weight markers is indicated. Vo, reactions. The exceptions are AGO4, -6, -8, and -9. AGO6 and void volume; In, input; Wg, wheat germ. AGO9 have a proline residue aligned with AGO1 position 798 and,

11932 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505461102 Baumberger and Baulcombe Downloaded by guest on September 29, 2021 based on the in vitro phenotype of the H798P mutant, it is probable We thank the Salk Institute and the Nottingham Arabidopsis Stock that they are not active Slicer proteins. Perhaps these more variant Centre for providing the Salk࿝087076 line and Attila Molnar and AGO proteins recruit siRNAs but regulate their targets by way of Alan Herr for critical reading of the manuscript and fruitful discus- translation interference or DNA methylation. For example, AGO4, sions. Herve´ Vaucheret (Institut National de la Recherche which has a serine residue at position 798, has been implicated in Agronomique, Versaille, France) is thanked for ago1-27 mutant plants and CMV I17N. The use of imported strains of virus was under license chromatin modification and DNA methylation and so may be (DEFRA 161A͞4391 (01͞03). This work was partly supported by a directly targeted to DNA (25, 26). It will be interesting to find out European Molecular Biology Organization fellowship and a Swiss to what extent selective recruitment of siRNA and miRNA plays a National Science Foundation fellowship (to N.B.). We are grateful to role in the functional diversification of AGO protein function and the Gatsby Charitable Foundation for supporting the Sainsbury of RNA silencing pathways. Laboratory.

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