Supplemental Material can be found at:

http://www.jbc.org/cgi/content/full/M708983200/DC1

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 6, pp. 3059 –3066, February 8, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Biochemical Activities of Arabidopsis RNA-dependent RNA

Polymerase 6*□ S

Received for publication, November 1, 2007, and in revised form, December 3, 2007 Published, JBC Papers in Press, December 6, 2007, DOI 10.1074/jbc.M708983200
Julien Curaba and Xuemei Chen1
From the Department of Botany and Plant Sciences, University of California, Riverside, California 92521

In Arabidopsis, genetic evidence demonstrates that RNA-de- which are then processed into siRNAs. In Arabidopsis, genetic
pendent RNA polymerase 6 (RDR6) plays a fundamental role in evidence suggests that an RNA that lacks a cap or a poly(A) tail
at least four RNA silencing pathways whose functions range is deemed “aberrant” and is preferentially recognized by the
from defense against transgenes or viruses to endogene regula- silencing machinery (8 –10). Most eukaryotic organisms also
tion in development and in stress responses. Despite its critical produce microRNAs that are similar to siRNAs in length but
role in RNA silencing, the biochemical activities of RDR6 have the biogenesis of microRNAs does not require RDRs (11).
yet to be characterized. In this study, we transiently expressed Amplification of siRNA production constitutes a major step

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Arabidopsis RDR6 in Nicotiana benthamiana and investigated that enhances the efficiency of RNA silencing and this amplifi-
the biochemical activities of immunopurified RDR6 in vitro. We cation requires RDRs (12). Secondary siRNAs produced by the
showed that RDR6 possesses terminal nucleotidyltransferase amplification process can originate from regions of the target
activity as well as primer-independent RNA polymerase activity mRNA not targeted by the primary siRNAs. The production of
on single-stranded RNAs. We found that RDR6 cannot distin- siRNAs outside of the region targeted by the initial RNA silenc-
guish RNAs with or without a cap or poly(A) tail. We also dem- ing trigger is known as transitivity (13–15). In C. elegans, the
onstrated that RDR6 has strong polymerase activity on single- amplification process generates secondary siRNAs that bear a
stranded DNA. All these activities require the conserved 5⬘ triphosphate and that are predominantly in antisense orien-
catalytic Asp867 residue. Our findings have important implica- tation to the target mRNA, suggesting that they are directly
tions on the processes involving RDR6 in vivo and provide new synthesized by RDR through a primer-independent mechanism
biochemical insights into the mechanisms of RNA silencing in (16, 17). In Arabidopsis, it was observed with a GFP transgene
Arabidopsis. containing a modified miR171 binding site that small RNA-
guided amplification can probably occur without cleavage of
the target mRNA. This led to the hypothesis that an RDR uses
RNA silencing is a universal regulatory mechanism that the small RNA as a primer to copy the target mRNA to lead to 3⬘
employs small interfering RNAs (siRNAs)2 to provide specific- to 5⬘ transitivity (18). However, the priming hypothesis cannot
ity to protein effectors that act to repress transcription or cleave explain secondary siRNAs originating from regions 3⬘ to the
an mRNA target (1). siRNAs are produced from long double- matching primary siRNAs in the target mRNA.
stranded RNAs (dsRNAs), which in some organisms are gener- In Arabidopsis, the biogenesis of several types of siRNAs
ated from single-stranded RNA (ssRNA) templates by RNA-de- requires RDRs. Transcriptional gene silencing (TGS), mediated
pendent RNA polymerases (RDRs). In Caenorhabditis elegans, by 24-nucleotide siRNAs that guide histone and DNA methyl-
fungi, and plants, genes encoding RDRs are required for RNA ation, requires RDR2 (6). PTGS initiated by a sense transgene
silencing (2– 6). Arabidopsis has six genes that encode potential (S-PTGS) or by infection with cucumber mosaic virus or cabbage
RDRs, and genetic evidence suggests that RDR1, RDR2, and leaf curl virus (CaLCuV), requires RDR6 (3, 4, 19). More
RDR6 act in different biological processes with RDR6 being the recently, two other PTGS mechanisms, also RDR6-dependent,
major RDR acting in post-transcriptional gene silencing (PTGS) were found to regulate endogene expression through two new
(3, 4, 6, 7). It is thought that RDR6 initiates RNA silencing by classes of siRNAs, the trans-acting siRNAs (ta-siRNAs) (20 –
recognizing “aberrant RNAs” and converting them to dsRNAs, 22) and the natural cis-acting siRNAs (23, 24). Whereas natural
cis-acting siRNAs are involved in stress responses, ta-siRNAs
* This work was supported by National Science Foundation Grant MCB- control developmental phase transitions such that the absence
0718029 (to X. C.). The costs of publication of this article were defrayed in of ta-siRNAs underlies the developmental defects of rdr6
part by the payment of page charges. This article must therefore be hereby mutants (25). The biogenesis of both types of siRNAs occurs via
marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
a two-step process. First, a small RNA (an microRNA or an
□S siRNA) guides the cleavage of a target mRNA. Then, the cleav-
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S6. age fragments are presumably copied into dsRNAs by RDR6
1
To whom correspondence should be addressed. Tel.: 951-827-3988; Fax:
and the dsRNAs are processed into the ta-siRNAs or the natural
951-827-4437; E-mail: [email protected].
2
The abbreviations used are: siRNA, small interfering RNA; dsRNA, double- cis-acting siRNAs.
stranded RNA; ssRNA, single-stranded RNA; RDR, RNA-dependent RNA po- Viral RDRs have been biochemically well characterized.
lymerase; CaLCuV, cabbage leaf curl virus; VIGS, virus-induced gene silenc- Many viral RDRs use a de novo mechanism of initiation in
ing; TGS, transcriptional gene silencing; PTGS, post-transcriptional gene
silencing; ta-siRNA, trans-acting siRNA; HA, hemagglutinin; MES, 4-mor- which the first phosphodiester bond is formed between two
pholineethanesulfonic acid. nucleoside triphosphates, but some use a primer-dependent

FEBRUARY 8, 2008 • VOLUME 283 • NUMBER 6 JOURNAL OF BIOLOGICAL CHEMISTRY 3059

Arabidopsis RDR6 Activities
mechanism (26 –29). A eukaryotic protein with RDR activity gel, transferred to a Zeta-Probe GT membrane (Bio-Rad), and
was first isolated through biochemical means in tomato (30 – hybridized with an end-labeled oligonucleotide probe.
32). RDR activities, however, have been detected in extracts Expression and Purification of HA-RDR6 and HA-RDR6m
from various plants such as Chinese cabbage, cauliflower, Proteins—Transient expression of HA-RdRp6 and HA-
tobacco, and wheat (7, 33–37). Except for the tomato RDR, only RdRp6m in N. benthamiana was based on the protocol estab-
QDE-1 from Neurospora crassa and Rdp-1 from Schizosaccha- lished by Popescu et al. (43). Plasmids pEG201-RDR6, pEG201-
romyces pombe are cellular RDRs that have been biochemically RDR6m, and p19 (a viral RNA silencing suppressor from the
characterized (38, 39). In the model plant Arabidopsis, genetic Tomato bushy stunt virus) were first transformed into A. tume-
evidence demonstrates the requirement for RDR6 in a number faciens GV3101. One transformed colony (verified by PCR) for
of silencing pathways (3, 4, 19, 21, 23, 25), but the RNA-depend- each plasmid was cultured at 28 °C until the A600 reached 1 to
ent RNA polymerase activity of RDR6 has been inferred based 1.5. The bacteria were spun for 15 min at 2,000 ⫻ g, resus-
on sequence homology with the tomato enzyme and has yet to pended in infiltration media (10 mM MES, 10 mM MgCl2, 200
be confirmed and characterized biochemically. ␮M acetosyringone) to a final A600 of 1, and incubated for 3 to
Here we report in vitro enzymatic activities of Arabidopsis 4 h at room temperature. Bacteria were then washed with 1
RDR6 that was overexpressed and purified from Nicotiana volume of fresh infiltration media, and mixed at a 1:1 ratio
benthamiana leaves. We show that RDR6 can indeed copy between Agrobacteria containing pEG201-RDR6 or pEG201-

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various ssRNAs into dsRNAs but, contrary to expectations, RDR6m and those containing p19. The infiltration of bacterial
the presence of a cap or poly(A) tail does not affect its activ- mixtures was performed with a 1-ml syringe on the abaxial side
ity. In addition, RDR6 possesses strong polymerase activities of leaves of 3-week-old N. benthamiana plants. After 48 h, infil-
on ssDNA templates. These findings provide biochemical trated leaves were harvested, ground in liquid nitrogen, and
insights into the molecular mechanisms of RNA silencing in stored at ⫺80 °C.
Arabidopsis. For protein extraction, 1 volume of tissue powder was mixed
with 2 volumes of protein lysis buffer (50 mM Tris-HCl, pH 7.6,
EXPERIMENTAL PROCEDURES 150 mM NaCl, 5 mM MgCl2, 0.1% (v/v) Nonidet P-40, 10% (v/v)
Plasmid Construction—The full-length cDNA of RDR6 (3.6 glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 mM dithiothre-
kb) was amplified from the ATG to the stop codon by PCR itol, 1 protease inhibitor mixture tablet/20 ml (Roche)), and
using the primers R6-1 (5⬘-ccggaattcgagaaatggggtcagagg- incubated for 20 min at 4 °C. Lysates were centrifuged one time
gaaata-3⬘) and R6-2 (5⬘-ccgcggccgcaacataaccttttagagacgtgagc- for 30 min at 1,000 ⫻ g at 4 °C, then three times for 30 min each
3⬘). The resulting amplicon was cloned into pENTR1A (Invitro- at 16,000 ⫻ g at 4 °C. 1/50 volume of anti-HA affinity matrix
gen) and the pENTR1A-RDR6 construct was verified by (Roche) was added to the supernatant and the mixture was
sequencing. The pENTR1A-RDR6 plasmid was amplified by incubated on a rotator (8 rpm) for 1 h at 4 °C. The beads were
PCR using two complementary primers R6-1m (5⬘-gacctt- then washed once with 20 volumes of protein lysis buffer, 4
gacggggccctgtactttgtg-3⬘) and R6-2m (5⬘-cacaaagtacagggc- times with 20 volumes of the washing buffer (20 mM Tris-HCl,
cccgtcaaggtc-3⬘) to substitute the putative catalytic residue Asp pH 7.6, 100 mM NaCl, 0.1 mM EDTA, 0.05% (v/v) Tween 20, 2
located at amino acid 867 by an Ala residue. The pENTR1A- mM dithiothreitol, 1 protease inhibitor tablet/20 ml) and finally
RDR6m plasmid was verified by sequencing. Both RDR6 and resuspended in 7 volumes of Storage Buffer (20 mM HEPES-
RDR6m cDNAs were introduced into the pEG201 vector (40), KOH, pH 7.6, 20 mM NaCl, 0.2 mM EDTA, 20% (v/v) glycerol, 2
which contains an HA tag for N-terminal fusion, and the read- mM dithiothreitol, 1 protease inhibitor tablet/20 ml), and stored
ing frame was verified by sequencing. in aliquots at ⫺80 °C.
Plant Growth and Transformation—Plants were grown in soil RNA Template Synthesis—Single-stranded RNA templates
in growth chambers at 22 °C under continuous illumination. Ara- were synthesized by in vitro transcription of DNA fragments
bidopsis transgenic lines were obtained by Agrobacterium-medi- obtained by PCR amplification from a luciferase/nopaline syn-
ated transformation using the floral dip procedure (41). Agrobac- thasae chimeric template (supplemental Fig. S2A). The PCR
teria containing the plasmids pEG201-RDR6 or pEG201-RDR6m primers were designed to introduce the T7 RNA polymerase
were used to transform both wild-type and rdr6 –11 (25) plants. promoter sequence in the correct orientation (supplemental
Transformants were selected on Murashige and Skoog medium Fig. S2B). PCR fragments were gel purified and then used as
with 50 ␮g/ml kanamycin. For each construct, 20 independent templates for in vitro transcription using the MEGAscript kit
transformants in both wild-type and rdr6 –11 backgrounds were (Ambion). Reactions were stopped by DNase treatment using 2
transferred into soil. T2 plants were selected again on kanamycin- units of RQ1 (Promega) for 15 min at 37 °C. The RNA products
containing medium and transferred into soil for phenotypic anal- (full-length and truncated forms) were resolved in a 6% polyac-
ysis. Young flowers were harvested from 6 and 2 individual trans- rylamide, 7 M urea gel. The bands corresponding to the full-
genic lines for 35S::HA-RDR6 and 35S::HA-RDR6m, respectively, length RNAs were excised, cut into small pieces, and incubated
quickly frozen in liquid nitrogen, and stored at ⫺80 °C for North- with 3 volumes of elution buffer (20 mM Tris-HCl, pH 7.6, 0.5 M
ern analysis. NaOAc, 10 mM EDTA, 1% (w/v) SDS) for 1 h at 37 °C. After
Small RNA Northern Blot Analysis—RNA isolation and phenol/chloroform extraction, the RNAs were precipitated and
Northern blotting were performed as described (42). Briefly, 40 resuspended in diethyl pyrocarbonate/water at a final concen-
␮g of total RNA was resolved on a 15% polyacrylamide, 7 M urea tration ranging between 0.1 and 0.5 ␮g/␮l. The purity of the

3060 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 6 • FEBRUARY 8, 2008

 Arabidopsis RDR6 Activities
RNAs was determined by analysis on a 6% polyacrylamide, 7 M
urea gel.
Capped RNAs were generated from gel-purified in vitro tran-
scripts using the ScriptCapTM m7G Capping System (Epicenter).
The reactions were stopped by phenol/chloroform extraction and
the RNAs were precipitated as previously described.
Polyadenylated RNAs (RNAs-An) were synthesized using
poly(A) polymerase (U. S. Biochemical Corp.). The reactions
were carried out with 2 ␮g of RNA, 1 ⫻ PAP (20 mM Tris-HCL
pH 7.0, 0.6 mM MnCl2, 0.02 mM EDTA, 0.2 mM dithiothreitol, 100
␮g/ml acetylated bovine serum albumin, 10% glycerol) reaction
buffer, 1 mM ATP, 600 units of PAP enzyme in a 25-␮l final volume
for 15 min at 37 °C. Reactions were stopped by incubation at 65 °C
for 10 min and were directly loaded on a 6% polyacrylamide, 7 M
urea gel and the polyadenylated RNAs were excised from the gel
and isolated.
RDR Activity Assays—RDR activity assays were performed

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according to Makeyev and Bamford (38) with some modifica-
tions. Briefly, assays were conducted in 30-␮l reaction mixtures
containing 50 mM HEPES-KOH (pH 7.6), 20 mM NH4OAc, 2%
(w/v) PEG4000, 8 mM MgCl2, 0.1 mM EDTA, 1 mM each of ATP,
CTP, and GTP, 0.1 mM UTP, and 0.8 unit/␮l of RNasin. The
final quantity of the ssRNA templates was between 5 and 15
pmol. Unless indicated otherwise, the mixture was supple-
mented with 0.15 mCi/ml of [␣-32P]UTP (⬃3000 Ci/mmol).
Reactions were initiated by adding 2 ␮l of HA-RDR6 bound
on anti-HA-agarose beads, and incubated on a rotator (8 FIGURE 1. Complementation of the rdr6 –11 mutation by the
rpm) for 2 h at room temperature (25–27 °C). Reactions were 35S::HA-RDR6 transgene. A, 23-day-old Columbia wild type (Col-0), rdr6 –11
mutant and two rdr6 –11 transgenic lines containing either 35S::HA-RDR6 or
stopped by adding 50 ␮l of buffer (20 mM Tris-HCl, pH 7.6, the 35S::HA-RDR6m transgene. rdr6 –11 mutants showed downward curling
20 mM MgCl2, 2% SDS), 90 ␮g of proteinase K (Fermentas) in of rosette leaves. This phenotype was rescued by 35S::HA-RDR6 but not
35S::HA-RDR6m. B, Northern blot analysis of two ta-siRNAs (ASRP255 and
a final volume of 100 ␮l, followed by incubation for 20 min at ASRP1511). The analysis was performed with 40 ␮g of total RNAs extracted
65 °C. The reaction mixtures were extracted with phenol- from influorescences of wild type (Col-0), rdr6 –11 mutants, and six independ-
chloroform and the RNAs were precipitated, resolved on 6% ent 35S::HA-RDR6 rdr6 –11 and two independent 35S::HA-RDR6m rdr6 –11
transgenic lines. The same membrane was used (after stripping) for the 3
polyacrylamide, 7 M urea gels, and detected by autoradiogra- hybridizations. miR173, a microRNA, served as a loading control.
phy or with a PhosphorImager.
Nuclease Treatments—For RNase I, RNase H, and RQ1 cient quantities of RDR6 from plants. To this end, full-length
(DNase) treatments, the RDR reactions were supplemented RDR6 cDNA was cloned behind the cauliflower mosaic virus
with 14 ␮l of the RNase I buffer (50 mM Tris-HCl, pH 8.0, 300 (CaMV) 35S promoter and in-frame with an N-terminal HA tag.
mM NaCl, 15 mM MgCl2), the RNase H buffer (50 mM Tris-HCl, The 35S::HA-RDR6 construct was used to generate transgenic
pH 8.0, 250 mM KCl, 10 mM dithiothreitol), or the RQ1 buffer plants in the rdr6 –11 mutant background. rdr6 –11 plants
(20 mM Tris-HCl pH 8.0, 30 mM MgSO4, 30 mM CaCl2), respec- showed downward curling of rosette leaves (Fig. 1A) (25). All 20
tively. Treatments were started by the addition of 50 units of independent 35S::HA-RDR6 rdr6 –11 transgenic lines analyzed
RNase I (New England Biolabs), 10 units of RNase H (New showed a complete restoration of the wild-type seedling phe-
England Biolabs), or 5 units of RQ1 (Promega) followed by notype (Fig. 1A), suggesting that HA-RDR6 was functional. To
incubation at 37 °C for 20 min. The reactions were stopped by further confirm this, an RNA blot analysis was performed to
proteinase K treatment as described previously. quantify the levels of two ta-siRNAs (ASRP255, ASRP1511) and
Assays for Priming Activity with an RNA Oligonucleotide—30 a microRNA, miR173, using total RNAs extracted from 6 of 20
pmol of miR173* (5⬘-rGrArUrUrCrUrCrUrGrUrGrUrA- 35S::HA-RDR6 rdr6 –11 transgenic lines (Fig. 1B). Both ta-
rArGrCrGrArArArG-3⬘) was mixed with 15 pmol of the 246R siRNAs were present in wild-type plants and were not detected
template in a 10-␮l solution containing 50 mM HEPES-KOH, in rdr6 –11 mutant plants, whereas miR173, used as a loading
pH 7.4, and 100 mM KCl. The mixture was incubated for 3 min control, was not affected by the rdr6 mutation (Fig. 1B) (21, 25).
at 95 °C and then for 10 min at 42 °C, and then used for RDR As expected, in the six 35S::HA-RDR6 rdr6 –11 transgenic lines
assay as previously described. both ta-siRNAs were restored to wild-type levels (Fig. 1B).
A previous study on two RDR6 homologs, QDE-1 (N. crassa)
RESULTS and Rdp-1 (S. pombe), showed that the conserved aspartate res-
A Conserved Catalytic Residue in Eukaryotic RDRs Is Critical idue (located at amino acid 1011 in QDE1 and amino acid 903 in
for RDR6 Function in Vivo—To study the biochemical proper- Rdp-1) is essential for RDR activity (38, 39). We introduced a
ties of Arabidopsis RDR6, we sought to immunopurify suffi- point mutation into the RDR6 cDNA to substitute the corre-

FEBRUARY 8, 2008 • VOLUME 283 • NUMBER 6 JOURNAL OF BIOLOGICAL CHEMISTRY 3061

Arabidopsis RDR6 Activities
sponding Asp (at position 867) with Ala. As expected, when the
mutant construct (35S::HA-RDR6m) was introduced into
rdr6 –11, none of 20 independent transformants showed any
rescue of the rdr6 mutant phenotype (Fig. 1A). The ASRP255
and ASRP1511 ta-siRNAs were not detected in two
35S::HA-RDR6m rdr6 –11 transgenic lines tested (Fig. 1B).
These results indicated that the Asp867 residue was essential for
RDR6 activity. The HA-RDR6m protein served as a negative
control in our biochemical analysis of RDR6 in vitro.
RDR6 Has Two Distinguishable Activities in Vitro—To inves-
tigate the biochemical properties of RDR6, we needed to immu-
nopurify relatively large amounts of RDR6 from plants.
Although the HA-RDR6 protein could be immunopurified
from 35S::HA-RDR6 rdr6 –11 plants (supplemental Fig. S1), the
amount of the protein was too little for the large numbers of
assays we wished to perform. We sought to obtain larger quan-
tities of the protein through transient expression of

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35S::HA-RDR6 in N. benthamiana leaves. We found that on
average 20-fold more HA-RDR6 was purified with anti-HA-
agarose beads when we incubated 10-fold less tissue from infil-
trated N. benthamiana leaves than from Arabidopsis transgenic
seedlings (supplemental Fig. S1). Therefore, we used immuno-
purified HA-RDR6 expressed in N. benthaniana for all our sub- FIGURE 2. RNA polymerase and nucleotidyltransferase activities of puri-
sequent biochemical assays. The purity of our RDR6 prepara- fied RDR6. A, schematic representation of ssRNAs used as templates in vari-
tion was evaluated by gel electrophoresis followed by SYPRO ous RDR assays. A dsDNA fragment (sequence in supplemental Fig. S2A) of a
luciferase/nopaline synthase chimeric gene with an engineered miR173*
RUBY staining (supplemental Fig. S1). Although the prepara- sequence was used as the template for PCR amplification of various seg-
tion had very few contaminating proteins, it was essential that ments, which in turn served as templates for in vitro transcription to generate
the ssRNA transcripts. The name of each ssRNA referred to the size (in nucle-
the RDR6m preparation was included in the activity assays as a otides) and orientation (F, forward; or R, reverse) compared with the DNA
control to ensure that the observed activity was due to RDR6 sequence. B and C, reaction products from RDR assays resolved on 6% poly-
instead of a contaminating protein. acrylamide, 7 M urea gels and visualized by phosphorimaging. B, assays were
performed with immunopurified HA-RDR6 or HA-RDR6m and different quan-
For RDR assays, we first synthesized ssRNAs of various sizes tities of the 185F RNA template (1 to 27 pmol) in the presence of all four cold
by in vitro transcription (Fig. 2A). Each ssRNA was named by its NTPs and [␣-32P]UTP. The image of the stained gel at the bottom illustrates the
size in nucleotides followed by the letter F or R to indicate its amounts of templates used. C, assays were performed with HA-RDR6, 15 pmol
of the 246R template, and [␣-32P]UTP, in the presence (⫹) or absence (⫺) of
forward or reverse orientation as compared with the DNA tem- the four cold NTPs. After the reaction, the products were treated (⫹) or not
plate (sequence in supplemental Fig. S2). The ssRNAs were (⫺) with RNase I for 20 min at 37 °C. The lower panels were images from
ethidium bromide (EtBr) staining of the PAGE gels before phosphorimaging.
then gel-purified and used as templates in RDR6 activity assays The fact that no RNAs were visible by EtBr staining in lane 9 suggested that the
in the presence of [␣-32P]UTP as well as all four cold NTPs. The amount of RDR6 products was very small.
first assays using HA-RDR6 and HA-RDR6m were performed
with an increasing quantity (from 1 to 27 pmol) of the 185F or from a 3⬘ nucleotidyltransferase activity that simply labeled
template. The reaction products, containing the radioisotope, the 3⬘ end of the RNA template, we performed an assay with 15
were resolved on denaturing polyacrylamide gels and detected pmol of the 246R template and [␣-32P]UTP with or without
by phosphorimaging (Fig. 2B). Reactions performed with HA- supplemented cold NTPs (Fig. 2C). In the absence of cold
RDR6 showed a major product, corresponding in size to the NTPs, a unique product was observed at the template size (lane
185F template, and the quantity of the product was propor- 6), which became completely degraded after RNase I treatment
tional to that of the template (Fig. 2B, lanes 1– 4). In contrast, no (lane 7). The sensitivity to RNase I treatment indicated that the
products were observed in the reaction performed with product was single stranded and likely resulted from the addi-
HA-RDR6m (Fig. 2B, lane 5). This confirmed that the products tion of one or a few nucleotides to the 3⬘ end of the template
obtained with HA-RDR6 were due to HA-RDR6 instead of a RNA. The absence of the product when the reaction was per-
contaminating protein. Therefore, RDR6 was able to use a formed with HA-RDR6m (supplemental Fig. S3) confirmed
ssRNA as a substrate to generate an RNA product of a similar that Arabidopsis RDR6 possessed a 3⬘ nucleotidyltransferase
size. To stay within the linear range, all subsequent RDR assays activity. Moreover, UTP seemed to be preferred by RDR6 over
were performed with a quantity of substrates between 5 and the other three NTPs for this activity (supplemental Fig. S3).
15 pmol. When the reaction was performed in the presence of both cold
Some viral RDRs are known to have 3⬘ nucleotidyltransferase NTPs and radioactive UTP, the products were resistant to
activity that adds one or a few nucleotides to the 3⬘ ends of the RNase I treatment, indicating that the products were in dsRNA
substrate RNA (44). To investigate whether the observed RDR6 forms (Fig. 2, lanes 8 and 9). Indeed, denaturation prior to
products resulted from a polymerase activity that led to newly RNase I treatment led to complete digestion of the RNAs (data
synthesized RNA complementary to the full-length template, not shown). These results showed that RDR6 possessed two

3062 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 6 • FEBRUARY 8, 2008

 Arabidopsis RDR6 Activities
different activities in vitro, a nucleotidyltransferase activity and
a polymerase activity, but the polymerase activity was predom-
inant in the presence of all four NTPs. The newly synthesized
RNA from the polymerase activity stayed bound to the template
RNA. The fact that no products larger than the template size
were found (Fig. 2C, lanes 8 and 9) suggested that RDR6 did not
have a back-priming activity as observed for QDE-1 (38).
Because the nucleotidyltransferase activity was undetectable in
the presence of all four NTPs, we performed all subsequent
reactions under this condition to analyze the polymerase activ-
ity of RDR6.
We tested RDR6 activity on various ssRNA templates and
found that it was able to act on RNA templates of various sizes
and sequences (supplemental Fig. S4A). We also tested the
effects of Mn2⫹ or Mg2⫹ on RDR6 activity. Maximum activity
was observed at a concentration of 8 mM for each cation (sup- FIGURE 3. RDR6 activity on RNA templates with different 5ⴕ and 3ⴕ end
structures. All enzymatic assays were performed with HA-RDR6 in the pres-
plemental Fig. S5). All subsequent reactions were performed

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ence of [␣-32P]UTP and four cold NTPs. A, assays were performed with 15
with Mg2⫹ at this concentration. pmol of four 246R templates possessing different structures at the 5⬘ ends,
RDR6 Does Not Distinguish Capped Versus Non-capped a triphosphate (246R), a hydroxyl group (OH-246R), a monophosphate
(P-246R), or a m7G cap (CAP-246R). B, assays were performed with 5 or 10
RNAs or RNAs with or without Poly(A) Tails—Molecular pmol of 206R-A40, a 206-nucleotide RNA with 40 additional adenosines at
genetic evidence suggests that RNAs lacking a cap or a poly(A) the 3⬘ end. The 246R template lacked an A tail and was used as a control.
tail are preferential targets for silencing (8 –10). This led to the
hypothesis that aberrant RNAs are preferred substrates for 395R-A40 RNAs with or without a 5⬘ cap. No difference in
RDR6. In the biogenesis of ta-siRNAs, genetic evidence sug- RDR6 activity was detected for these two templates (supple-
gests that RDR6 uses the 5⬘ or 3⬘ fragments resulting from mental Fig. S4).
microRNA-guided cleavage of an mRNA as substrates. These RDR6 Does Not Have Polymerase Activity on dsRNAs nor
RNA fragments lack either a cap or a poly(A) tail. We evaluated Does It Have Priming Activity—One potential model to explain
whether structural features at the 5⬘ and 3⬘ ends of an RNA the requirement for RDR6 in the amplification of RNA silenc-
template affect its recognition by RDR6 in vitro. ing is that RDR6 uses dsRNAs as templates to generate more
We first tested RNAs with different 5⬘ end features. In vitro dsRNAs. To test whether RDR6 was able to use a dsRNA as a
transcription products possessed a 5⬘ triphosphate. We used template, we performed an in vitro assay with two complemen-
the ScriptCapTM m7G Capping System (Epicenter) to convert tary ssRNAs, 435F and 435R (Fig. 2A), which were annealed
the 5⬘ triphosphate of the 246R RNA into a 7-methylguanosine prior to the reactions. We annealed the same amount of the
cap (CAP-246R). In parallel, the 246R RNA was also treated forward template and an increasing amount of the reverse tem-
with phosphatase to generate a 246R RNA with 5⬘ OH (OH- plate to increase the dsRNA/ssRNA ratio. In the presence of an
246R), which was further treated with T4 polynucleotide kinase equal quantity of both RNAs, when more dsRNA templates
to generate an RNA with a 5⬘ monophosphate (P-246R). The were presumably present (Fig. 4A, lane 3), the activity was
same treatments were also performed on the capping reaction greatly reduced compared with the reaction performed with
mixture followed by digestion with the 5⬘ monophosphate-spe- only the forward RNA (lane 1) or with an excess of the reverse
cific exonuclease TERMINATOR. This allowed the elimination RNA (Fig. 4A, lane 4). The small amount of products when both
of all leftover non-capped RNAs after the capping reaction templates were present at supposedly equal quantity (Fig. 4A,
(supplemental Fig. S6). RDR6 assays were performed with 246R lane 3) could be due to the presence of residual ssRNAs or due
RNAs with a 5⬘ triphosphate, 5⬘ monophosphate, 5⬘ OH, and 5⬘ to weak RDR6 activity on dsRNAs. To conclusively determine
cap. We found that these 5⬘ end modifications did not affect the activity of RDR6 on dsRNAs, we annealed equal amounts of
RDR6 activity (Fig. 3A). 435F and 435R followed by digestion with RNase I to remove
Next, we tested whether the presence of a poly(A) tail at the any residual ssRNAs. The treated dsRNAs were then used in an
3⬘ end of the template affects RDR6 activity (Fig. 3B). For this, RDR6 assay with the corresponding ssRNAs as controls. As
we compared the activity of RDR6 on a 206R ssRNA containing shown in Fig. 4B, no products were found in the reaction with
40 additional adenosines at the 3⬘ end (206R-A40) with that on dsRNAs, indicating that RDR6 was unable to use dsRNAs as
a 246R ssRNA without the 3⬘ As. No difference in RDR6 activity templates.
was seen for two different concentrations of the template RNAs Another model to explain the requirement for RDR6 in the
(Fig. 3B). We also used yeast poly(A) polymerase to add a longer amplification of RNA silencing is that RDR6 uses a primary
poly(A) tail onto the 185F RNA. RDR6 activity on this 185F-An siRNA as a primer and the target mRNA as the template to
RNA was compared with that on a similarly sized 640F RNA. synthesize dsRNAs. To investigate whether RDR6 can use a
Again, no difference in RDR6 activity was detected (supple- small RNA as a primer to copy a template RNA, we performed
mental Fig. S4B). an assay with the 246R template in the presence or absence of
To determine whether a capped and polyadenylated RNA the 21-nucleotide miR173*, which was completely complemen-
can serve as a substrate for RDR6, we performed assays on tary to a region of the template (Fig. 2A). If RDR6 were able to

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Arabidopsis RDR6 Activities

FIGURE 4. RDR6 activity on double-stranded RNAs and the absence of a

priming activity. All enzymatic assays were performed with HA-RDR6 in the
presence of [␣-32P]UTP and all four cold NTPs. A, assays were performed with FIGURE 5. DNA-dependent RNA polymerase activity of RDR6. A, all enzy-
10 pmol of the 435F template that had been hybridized with an increasing matic assays were performed with HA-RDR6 or HA-RDR6m in the presence of
quantity (0 –20 pmol) of its complementary strand, 435R. B, assays were [␣-32P]UTP and all four cold NTPs. The same PCR product was used to synthe-
formed with ssRNAs (lanes 1 and 2) and equal amounts of 435F and 435R size the ssDNA, by asymmetric PCR, and the ssRNA, by in vitro transcription. 5
annealed together and treated with RNase I to remove any residual ssRNAs pmol of a ssDNA template (d246R) and the same amount of a ssRNA template
(lane 3). A portion of the RNA templates used for the reactions were resolved (246R) were tested with HA-RDR6 (lanes 1–2) or HA-RDR6m (lane 3). B, assays

Downloaded from www.jbc.org at MEDIZINBIBLIOTHEK on December 29, 2008

on a denaturing gel and stained with ethidium bromide to demonstrate the were conducted with a ssDNA (d246R) template, HA-RDR6, and [␣-32P]UTP in
near equal amounts of templates used. C, priming activity was tested on 15 the presence (⫹) or absence (⫺) of cold NTPs. After the RDR6 reactions, the
pmol of the 246R template in the presence (⫹) or absence (⫺) of 30 pmol of reaction mixture was treated (⫹) or not (⫺) with RNase H or RQ1 (a DNase).
miR173* as a primer. The expected product synthesized from the primer (⬃80 C, assays were conducted with HA-RDR6, [␣-32P]UTP, and all four cold NTPs on
nucleotide) would migrate to the position indicated by the arrowhead. a ssDNA (d246R) and a dsDNA (ds-d246R) template. The stained gel at the
bottom illustrates near equal amounts of templates used. The larger size of
the dsDNA template as compared with the ssRNA template is due to the
presence of the T7 promoter sequence in the dsDNA.
use the small RNA as a primer, it would be predicted to generate
a product of ⬃80 nucleotides (indicated by the black arrow).
However, no product of this size was detected (Fig. 4C, lane 2). template was found (lane 1), suggesting that RDR6 had the
To exclude the possibility that the priming activity might have nucleotidyltransferase activity even on ssDNA. The fact that
been masked by the background smear (Fig. 4C, lane 2), we the product was sensitive to RQ1 but insensitive to RNase H
performed the assay with miR173* labeled at the 5⬘ end and cold confirmed that the product was ssDNA (lanes 2 and 3). When
NTPs so that only products resulting from the priming activity the reaction was performed in the presence of cold NTPs, a
would be visualized. Again, no products were found (data not product of the same size as the template was found (lane 4).
shown). RNase I treatment after the RDR6 reactions showed This product, however, was sensitive to RNase H but insensitive
that miR173* was protected from digestion, suggesting that it to RQ1 (lanes 5 and 6). This indicated that the product was
had been annealed to the template (data not shown). Therefore, newly synthesized RNA (hence insensitivity to RQ1) that stayed
the lack of priming activity was not due to lack of annealing of paired with the template DNA (hence sensitivity to RNase H).
the primer to the template. Therefore, RDR6 was able to use ssDNA as template to synthe-
RDR6 Can Use Single-stranded DNA as a Template—One of size a complementary RNA.
the first known functions of RDR6 is to mediate virus-induced It is worth noting that RDR6 was unable to incorporate
gene silencing (VIGS). However, almost all ssRNA viruses dNTPs with either DNA or RNA templates (data not shown).
tested induced VIGS in an RDR6-independent manner (3, 4), Furthermore, RDR6 was unable to use a dsDNA as template
whereas VIGS induced in response to infection by the CaLCuV, (Fig. 5C, lanes 7 and 8). Therefore, RDR6 was exclusively an
a ssDNA virus, was strongly inhibited in an rdr6 mutant (19). RNA polymerase, but it could act on either ssRNA or ssDNA
These observations led us to test whether RDR6 could also use but not dsDNA.
ssDNA as a template. We produced ssDNA using asymmetric
PCR on the dsDNA fragment that was used initially to tran- DISCUSSION
scribe the 246R template. RDR6 assay was then performed in Biochemical Properties of RDR6—Our studies demonstrate
the presence of [␣-32P]UTP and all four cold NTPs (Fig. 5A). that RDR6 indeed possesses RNA-dependent RNA polymerase
RDR6 was able to generate a product of the same size as the activity on ssRNAs. It makes a complete copy of its substrate
template (lane 1), whereas no activity was detected with RNA, and the newly synthesized RNA stays bound to the tem-
RDR6m (lane 3). Interestingly, this activity appeared at least plate RNA. Our results clearly show that RDR6 does not have
10-fold higher than the one with the same amount of a ssRNA the intrinsic capacity to distinguish capped versus non-capped
template (lanes 1 and 2), suggesting that ssDNA was a better RNAs, which was also observed with an RDR activity in wheat
template for RDR6 than ssRNA under our assay conditions. To germ extracts (37), or RNAs with or without poly(A) tails, sug-
rule out that the product was due to the 3⬘ nucleotidyl activity gesting that the recognition of aberrant RNAs involves other
that labeled the 3⬘ end of the template DNA, we performed proteins in vivo.
assays with or without cold NTPs and digested the products In the literature, conflicting observations were made regard-
with RNase H and RQ1 (a DNase) to evaluate the properties of ing the ability of RDRs to use ssDNA templates. Studies with
the products (Fig. 5B). When the reactions were performed in RDR preparations of various purities from cauliflower and
the absence of cold NTPs, a product of the same size as the tobacco showed little or no activities on dsDNA or ssDNA (34 –

3064 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 6 • FEBRUARY 8, 2008

 Arabidopsis RDR6 Activities
36). A highly pure preparation of tomato RDR, however, had However, considering that RDR6 is able to use ssDNA as tem-
similar activity on ssRNA and ssDNA templates (30). With the plates, it is tempting to suggest that instead of targeting viral
help of the catalytically inactive HA-RDR6m as a control, we RNA, the genomic ssDNA of CaLCuV is used by RDR6 to gen-
unequivocally demonstrated that Arabidopsis RDR6 acts on erate ssRNAs, which could then either base pair with the
ssDNA but not dsDNA templates in vitro. expressed viral mRNAs or be used by RDR6 as a template to
RDR6 in the Amplification of Silencing—Amplification of generate dsRNAs.
RNA silencing can occur through a number of potential mech- VIGS against cucumber mosaic virus, a double-stranded
anisms that are not mutually exclusive. The first mechanism is DNA virus, also requires RDR6 (3, 4). Because RDR6 cannot act
that an RDR copies an aberrant RNA into dsRNA and then uses on dsDNA, it is likely that a ssDNA intermediate or transcripts
the newly synthesized RNAs as substrates in a second round of from cucumber mosaic virus are targeted by RDR6.
dsRNA synthesis. We showed that the newly synthesized RNAs
stay bound to the template RNAs and that RDR6 cannot use Acknowledgments—We thank YunJu Kim for a plasmid that contains
dsRNA as substrates, which argue against this mechanism of the luciferase/NOS chimeric gene and the miR173* sequence. We are
amplification. However, in vivo the dsRNAs may be unwound grateful to Drs. Popescu and Dinesh-Kumar for suggestions on tran-
by an RNA helicase to generate ssRNAs that can serve as sub- sient expression of proteins in N. benthamiana. We thank Theresa
strates for RDR6. In fact, the production of secondary siRNAs Dinh, Vanitharani Ramachandran, Bin Yu, and Binglian Zheng for
in Arabidopsis requires SDE3, a protein with an RNA helicase comments on the manuscript.

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signature (45, 46). The second mechanism of amplification is
that a primary siRNA serves as a primer for RDR to generate
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