molecular cell short article - pikweb.sitehost.iu.edu
TRANSCRIPT
Molecular Cell
Short Article
In Vitro Transcription Activities of Pol IV, Pol V,and RDR2 Reveal Coupling of Pol IV and RDR2for dsRNA Synthesis in Plant RNA SilencingJeremy R. Haag,1,2 Thomas S. Ream,3,5 Michelle Marasco,1 Carrie D. Nicora,4 Angela D. Norbeck,4 Ljiljana Pasa-Tolic,4
and Craig S. Pikaard1,2,*1Department of Biology and Department of Molecular and Cellular Biochemistry2Howard Hughes Medical InstituteIndiana University, 915 E. Third Street, Bloomington, IN 47405, USA3Department of Biology, Washington University, One Brookings Drive, St. Louis, MO 63130, USA4Pacific Northwest National Laboratory, Richland, WA 99352, USA5Present address: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.molcel.2012.09.027
SUMMARY
In Arabidopsis, RNA-dependent DNA methylationand transcriptional silencing involves three nuclearRNA polymerases that are biochemically undefined:the presumptive DNA-dependent RNA polymerasesPol IV and Pol V and the putative RNA-dependentRNA polymerase RDR2. Here we demonstrate theirRNA polymerase activities in vitro. Unlike Pol II,Pols IV and V require an RNA primer, are insensitiveto a-amanitin, and differ in their ability to displacethe nontemplate DNA strand during transcription.Biogenesis of 24 nt small interfering RNAs (siRNAs),which guide cytosine methylation to correspondingsequences, requires both Pol IV and RDR2, whichphysically associate in vivo. Whereas Pol IV doesnot require RDR2 for activity, RDR2 is nonfunctionalin the absence of associated Pol IV. These resultssuggest that the physical and mechanistic couplingof Pol IV and RDR2 results in the channeled synthe-sis of double-stranded precursors for 24 nt siRNAbiogenesis.
INTRODUCTION
Eukaryotes have three essential multisubunit DNA-dependent
RNA polymerases, abbreviated as Pol I, Pol II, and Pol III (Cramer
et al., 2008; Werner and Grohmann, 2011). In plants, two addi-
tional multisubunit RNA polymerases, Pol IV and Pol V, evolved
as specialized forms of Pol II (Huang et al., 2009; Ream et al.,
2009) and play important roles in development, transposon
taming, transgene silencing, and pathogen defense (Haag and
Pikaard, 2011).
Pols IV and V are best understood with respect to their roles
in RNA-directed DNA methylation (RdDM) in Arabidopsis thali-
ana (Herr et al., 2005; Kanno et al., 2005; Onodera et al., 2005;
Molec
Pontes et al., 2006; Pontier et al., 2005) (Figure 1A). Genetic
and cytological evidence suggests that Pol IV acts early in the
pathway, upstream of RNA-DEPENDENT RNA POLYMERASE
2 (RDR2) (Pontes et al., 2006). RDR2 is thought to transcribe
Pol IV transcripts to generate double-stranded RNAs (dsRNAs)
that are then cleaved into 24 nt siRNAs by DICER-LIKE 3
(DCL3) (Xie et al., 2004), 30 end methylated by HUA-ENHANCER
1 (HEN1) (Li et al., 2005), and loaded into ARGONAUTE 4 (AGO4),
or a related Argonaute protein (Havecker et al., 2010; Qi et al.,
2006). Independent of 24 nt siRNA biogenesis, Pol V generates
RNA transcripts to which AGO4-siRNA complexes bind (Wierz-
bicki et al., 2009), facilitating recruitment of the de novo DNA
methyltransferase, DRM2, and other chromatin-modifying activ-
ities that repress Pol I, Pol II, or Pol III transcription (Haag and Pi-
kaard, 2011; Law and Jacobsen, 2010; Zhang and Zhu, 2011).
Detection of Pol IV or Pol V polymerase activities has proven
elusive using standard promoter-independent transcription
assays or nuclear run-on assays (Erhard et al., 2009; Huang
et al., 2009; Onodera et al., 2005). These negative results have
suggested that Pols IV and V might require unconventional
templates, or possibly lack RNA polymerase activity, consistent
with the divergence, or absence, in Pols IV and V, of amino acids
that are invariant in Pols I, II, or III (Haag et al., 2009; Herr, 2005;
Landick, 2009). However, Pols IV and V retain key amino acids of
the magnesium-binding Metal A and Metal B sites that are
invariant at the active sites of all multisubunit RNA polymerases
(Haag et al., 2009; Herr, 2005; Landick, 2009). Mutagenesis of
these sites abolishes Pol IV or Pol V functions in vivo, including
siRNA biogenesis, de novo cytosine methylation, and trans-
poson silencing (Haag et al., 2009; Lahmy et al., 2009). More-
over, Pol V transcripts detectable in vivo are lost upon mutation
of Pol V’s Metal A site (Wierzbicki et al., 2008).
Here we demonstrate RNA-primed transcription of DNA tem-
plates by Pols IV and V and differences in Pol IV, Pol V, and Pol
II with respect to their sensitivities to the fungal toxin a-amanitin
and their abilities to transcribe RNA-RNA templates or displace
nontemplateDNAduring transcription.Wefind thatRDR2activity
is Pol IV dependent, suggesting that RNAs are channeled from
Pol IV to RDR2 to generate dsRNAs for subsequent dicing.
ular Cell 48, 811–818, December 14, 2012 ª2012 Elsevier Inc. 811
Pol IV Pol V
RDR2
DCL3
CLSY1
HEN1AGO4
AGO4DRM2
M
M
MM
M
M
M
M
RDR2-generateddsRNA product siRNA
duplexmethylated
siRNA duplexAGO-RISC
Pol IV RNA transcript
Pol V RNA transcript
M M M
5'
5'
5'
5'
3'
3'
5'5'3'
3'
DNA
A Model for the RNA-directed DNA methylation pathway C Co-immunoprecipitation of Pol IV and RDR2
Non
-tra
nsge
nic
Non
-tra
nsge
nic
RD
R2-
HA
NR
PD
1-F
LAG
NR
PE
1-F
LAG
DC
L3-F
LAG
RD
R6-
FLA
GN
RP
B2-
FLA
G
α-FLAG
α-RDR2
α-NRPD1
α-NRPE1
α-NRP(D/E)2
α-DCL3
α-RDR6
α-Pol I, II, III
Lane: 1 2 3 4 5 6 7 8
kDa260
160
110
α-HA α-FLAGIP antibody:
immunoblot antibody:
WT
(C
ol-
0)
WT
(C
ol-
0)
no
RN
A
nrp
d1-
3
nrp
e1-1
1
NR
PD
1-F
LA
G n
rpd
1-3
NR
PD
1(A
SM
)-F
LA
G n
rpd
1-3
NR
PE
1(A
SM
)-F
LA
G n
rpe1
-11
NR
PE
1-F
LA
G n
rpe1
-11
kDa190
120
190
120
190
120
RD
R2-
HA
WT
(C
ol-
0)
NR
PD
1-F
LA
G
NR
PD
1(A
SM
)-F
LA
G
IP: α-HA α-FLAG
Lane: 1 2 3 4 5
soloLTR (+RT)
actin (+RT)
actin (-RT)
soloLTR (-RT)
Lane: 1 2 3 4 5 6 7 8
α-FLAG
α-NRPD2
α-RDR2
F Test of RDR2 associationwith Pol IV active site mutant
D Metal A sites and mutagenesis E soloLTR silencing
B RDR2 peptides (highlighted) detected in affinity purified Pol IV
G RNase insensitivity of Pol IV - RDR2 interaction
Lane: 1 2
kDa190
120
190
120
Un
trea
ted
RN
ase
A
NRPD1-FLAGIP Samples
α-NRPD1
α-RDR2
Figure 1. Pol IV and RDR2 Interact in an RNA-Independent Fashion
(A) Model for the RNA-directed DNA methylation pathway in Arabidopsis thaliana.
(B) RDR2 tryptic peptides detected upon LC-MS/MS analysis of affinity-purified Pol IV are highlighted within the RDR2 sequence. The peptide shaded in green
was sequenced individually and as part of a larger peptide.
(C) RDR2 and Pol IV coimmunoprecipitate (see also Tables S1 and S2 and Figure S1). HA-taggedRDR2 or FLAG-tagged NRPD1, NRPE1, DCL3, RDR6, or NRPB2
was immunoprecipitated (IPed) using anti-HA or anti-FLAG antibodies. Immunoblots were then probed with anti-FLAG, anti-RDR2, anti-NRPD1, anti-NRPE1,
anti-NRP(D/E)2, anti-DCL3, or anti-RDR6. The anti-Pol I, anti-Pol II, anti-Pol III antibody recognizes the second subunits of Pols I, II or III but not Pols IV or V.
Molecular Cell
In Vitro Transcription by Pol IV, Pol V, and RDR2
812 Molecular Cell 48, 811–818, December 14, 2012 ª2012 Elsevier Inc.
Molecular Cell
In Vitro Transcription by Pol IV, Pol V, and RDR2
RESULTS
Pol IV and RDR2 Associate In VivoWe rescued an nrpd1-3 null mutant lacking the Pol IV largest
subunit with a FLAG epitope-tagged NRPD1 transgene
(NRPD1-FLAG), allowing Pol IV affinity purification using anti-
FLAG resin. Trypsin digestion and LC-MS/MS mass spectrom-
etry identified peptides of Pol IV’s 12 core subunits (Ream
et al., 2009) as well as 10 peptides corresponding to RDR2 (Fig-
ure 1B), confirming a recent report (Law et al., 2011).
As an independent test of Pol IV- RDR2 interaction, we
rescued an rdr2-1 null mutant with a RDR2 transgene (see
Figures S1A–S1C online) that includes the RDR2 promoter, all
exons and introns, and a C-terminal HA epitope tag. Following
anti-HA immunoprecipitation (IP) and immunoblotting, RDR2-
HA is readily detected using anti-RDR2 antisera (Figure 1C,
lane 2, row 2), as are the catalytic subunits of Pol IV, NRPD1
and NRPD2 (Figure 1C, lane 2, rows 3 and 5). LC-MS/MS anal-
ysis of affinity-purified RDR2-HA identified 9 of the 12 Pol IV
subunits, including major (3a) and alternative (3b) forms of the
third subunit (Tables S1 and S2). No Pol I-, Pol II-, Pol III-, or
Pol V-specific subunits were detected.
Consistent with the RDR2-HA IP and mass spectrometry
results, RDR2 coIPs with FLAG-tagged NRPD1 (Figure 1C,
lane 3), but not with Pol V (NRPE1-FLAG, lane 4), Pol II
(NRPB2-FLAG; lane 7), or Pols I or III (lane 2, row 8). NRPD1
does not coIP with RNA-DEPENDENT RNA POLYMERASE 6
(Figure 1C, lane 6 and lane 3), involved in 21 nt siRNA biogenesis
(Figure S1D), indicative of Pol IV’s specificity for RDR2. No asso-
ciation between RDR2 and DCL3 was detected by immunoblot
(Figure 1C, lanes 2 and 5) or LC-MS/MS analyses.
To test if Pol IV and RDR2 might associate via RNA, we
made use of Pol IV rendered catalytically inactive (Haag et al.,
2009) by changing to alanines the three invariant aspartates of
the NRPD1 Metal A site (Figure 1D). Whereas nrpd1-3 null
mutants are rescued by a wild-type NRPD1-FLAG transgene,
NRPD1 bearing the active site mutations (ASM) fails to restore
siRNA biogenesis, RdDM, or transposon silencing (Haag et al.,
2009). For example, a soloLTR element silenced in wild-type
cells (Figure 1E, lane 1) but derepressed in nrpd1-3 (Pol IV) or
nrpe1-11 (Pol V) null mutants (lanes 3 and 4) is resilenced by
NRPD1-FLAG or NRPE1-FLAG transgenes (lanes 5 and 6), but
not by NRPD1(ASM)-FLAG (active site mutant) or NRPE1
(ASM)-FLAG transgenes (Figure 1E, lanes 7 and 8).
NRPD1-FLAG or NRPD1(ASM)-FLAG associates with equiva-
lent amounts of NRPD2 (the Pol IV second subunit) and RDR2
(Figure 1F, compare lanes 4 and 5), suggesting that active site
mutations do not disrupt Pol IV assembly or RDR2 associa-
tion. Pol IV-RDR2 association is also unaffected by RNase A
(Figure 1G, lane 2). Collectively, these results suggest that Pol
(D) Amino acids at the Metal A sites of Pol I–Pol V largest subunits. Invariant aspa
shaded.
(E) RT-PCR analysis of solo LTR expression in wild-type, nrpd1 or nrpe1mutants,
Actin and no reverse transcriptase (�RT) controls are included.
(F) Test of RDR2 interaction with wild-type or ASM forms of Pol IV. RDR2-HA,
antisera. Immunoblots were probed using anti-FLAG, anti-RDR2, or anti-NRPD2
(G) NRPD1-FLAG was immunoprecipitated from control or RNaseA-treated cell e
Molec
IV-RDR2 interaction does not require Pol IV transcripts or
other RNAs.
Affinity-Purified Pol IV-RDR2 Complexes GenerateTranscripts In VitroIn the search for templates for Pols IV or V (e.g., see Figure S2),
we found that Pol IV, like Pol II, will transcribe a tripartite oligonu-
cleotide template that mimics a transcription bubble (Figure 2A).
Features of this template include an 8 bp RNA-DNA hybrid,
single-stranded DNA and RNA upstream of the hybrid region,
and double-stranded DNA downstream of the DNA-RNA hybrid
(Figure 2B). Pols I and II transcribe such tripartite templates, ex-
tending the RNA in a DNA-templated manner (Kuhn et al., 2007;
Lehmann et al., 2007).
Using the tripartite template, Arabidopsis Pol II and Pol IV-
RDR2 complexes catalyze a-32P-CTP incorporation into RNA
extension products that can be resolved on sequencing gels
and visualized by autoradiography (Figure 2C). The RNA of the
tripartite template is 16 nt; its DNA-templated extension can
yield a full-length product of 32 nt. Consistent with previous
studies using yeast Pol II, Arabidopsis Pol II catalyzes the
synthesis of RNA products up to 32 nt (lane 5) and is inhibited
by 5 mg/ml a-amanitin (lane 6). The Pol IV-RDR2 complex gener-
ates abundant 12–16 nt transcripts and longer transcripts up
to 32 nt (lane 2). Pol IV-RDR2 transcripts are insensitive to
a-amanitin (Figure 2C, lane 3), consistent with the divergence
in Pols IV and V of the a-amanitin binding pocket of Pol II (Fig-
ure S3) and the expected a-amanitin insensitivity of RNA-depen-
dent RNA polymerases, such as RDR2. Cloning and sequencing
of RNA-primed extension products confirmed that all Pol II, IV,
and V transcripts are DNA templated (Figure S4).
Catalytically crippled (ASM) and wild-type Pol IV both asso-
ciate with RDR2 (Figure 1F). Affinity-purified Pol IV(ASM)-RDR2
generates 12–16 nt RNA products (Figure 2C, lane 4) as effi-
ciently as wild-type Pol IV-RDR2 (lanes 2 and 3), but most long
transcription products are absent. Long RNAs dependent on
the Pol IV active site are interpreted to be DNA-templated Pol
IV transcripts. Transcripts unaffected by mutating the Pol IV
active site are presumably generated by RDR2; these are mostly
smaller than the 16 nt RNA oligonucleotide in the reactions (a 50
end-labeled aliquot of this RNA is present in lane 8), consistent
with RDR2 transcribing the 16 nt RNA. A transcript of �26 nt
generated by the Pol IV(ASM)-RDR2 complex (lane 4) is also
RDR2 dependent based on subsequent experiments using Pol
IV isolated from an rdr2 null mutant background (see below).
We next deconstructed the tripartite template, testing its
component oligonucleotides as templates (Figure 2D). Pol IV-
RDR2 or Pol II transcription reactions performed using a bipartite
template, consisting of the 16 nt RNA hybridized to the 31 nt DNA
template, yielded products similar to those obtained using the
rtates changed to alanines in NRPD1 and NRPE1 active site mutants (ASM) are
or mutants expressing wild-type or ASMmutantNRPD1 orNRPE1 transgenes.
NRPD1-FLAG, or NRPD1(ASM)-FLAG were IPed using anti-HA or anti-FLAG
antibodies.
xtracts. Immunoblots were probed with anti-NRPD1 or anti-RDR2 antibodies.
ular Cell 48, 811–818, December 14, 2012 ª2012 Elsevier Inc. 813
Figure 2. Pol IV Displays DNA-Dependent
RNA Polymerase Activity
(A) Model of the nucleic acids within a Pol II tran-
scription bubble, adapted from Gnatt et al. (2001).
(B) A tripartite oligonucleotide template that
mimics essential aspects of a transcription
bubble, modeled after Kuhn et al. (2007).
(C) Autoradiogram of 32P-CTP-labeled transcrip-
tion products catalyzed by Pol II or Pol IV-RDR2
using the tripartite template. Unmutated or active
site mutant (ASM) forms of Pol IV were tested in
parallel (lanes 2–4). Anti-FLAG IP of nontransgenic
tissue extract served as a control (lane 1),
revealing two background bands present in all
reactions. Reactions conducted in the presence of
a-amanitin are shown in lanes 3 and 6. Lane 8
shows a 32P end-labeled aliquot of the 16 nt RNA
oligonucleotide used in the tripartite template.
(D) In vitro reactions using dissected components
of the tripartite template. (Lanes 1–4) Affinity-
purified Pol II, Pol IV-RDR2, or Pol IV(ASM)-RDR2
complexes incubated with the tripartite template.
(Lanes 5–8) Transcription using a bipartite tem-
plate consisting of the RNA and template DNA.
(Lanes 9–12) Transcription using the template
DNA oligonucleotide only. (Lanes 13–16) Tran-
scription using the RNA oligonucleotide only. An
aliquot of 50 end-labeled 16 nt RNA was loaded in
lane 17. Figures S2–S4 show results for other
templates tested, the basis for Pol IV/V a-amanitin
insensitivity, and sequences of Pol II, IV, and V
transcripts generated using the bipartite template.
Molecular Cell
In Vitro Transcription by Pol IV, Pol V, and RDR2
tripartite template (Figure 2D, lanes 6–8; compare to lanes 2–4),
indicating that nontemplate DNA downstream of the DNA-RNA
hybrid is dispensable. In fact, transcription was more robust
without the need to displace the nontemplate DNA oligonucleo-
tide, allowing more full-length transcription by Pols II and IV.
Using only the 31 nt DNA oligonucleotide as the template,
a ladder of transcription products were generated by both Pol
IV-RDR2 and Pol II (Figure 2D, lanes 9–12). Many of these prod-
ucts were less abundant using the Pol IV active site mutant
form of the Pol IV-RDR2 complex, indicating that Pol IV (like Pol
II) is able to transcribe single-stranded DNA to some extent.
The 12–16 nt RNA products obtained using the tripartite or bipar-
814 Molecular Cell 48, 811–818, December 14, 2012 ª2012 Elsevier Inc.
tite templates are absent in reactions con-
taining only the 31 nt DNA template (lanes
9–12). Conversely, transcription reactions
using the 16 nt RNA oligonucleotide alone
support 12–16 nt RNA production in Pol
IV-RDR2 and Pol IV(ASM)-RDR2 reac-
tions (Figure 2D, lanes 14 and 15), but
not in Pol II reactions (lane 16), consistent
with these being RDR2 transcripts tem-
plated by the 16 nt RNA oligonucleotide.
Genetic and Biochemical Testing ofPol IV-RDR2 InterdependenceBecause Pol IV and RDR2 copurify,
we disentangled them by introgressing
NRPD1-FLAG or NRPD1(ASM)-FLAG transgenes into an rdr2-
1 null mutant background and by introgressing an RDR2-HA
transgene into a Pol IV null mutant (nrpd1-3). In the rdr2-1mutant
background, affinity-purified NRPD1 or NRPD1(ASM) lack asso-
ciated RDR2, as expected (Figure 3A, lanes 4 and 5). Likewise,
RDR2-HA normally associates with Pol IV (Figure 3B lane 2),
but not in an nrpd1-3 null mutant background (Figure 3B, lane 3).
As shown previously, Pol IV-RDR2 generates both long (>16
nt) and short (<16 nt) transcripts using the bipartite template (Fig-
ure 3C, lane 2), with most long transcripts dependent on the Pol
IV active site (lane 3). Importantly, 12–16 nt transcripts are no
longer produced in reactions utilizing Pol IV isolated from an
Non
-tra
nsge
nic
Pol
IV-R
DR
2P
ol IV
(AS
M)-
RD
R2
Pol
IV (
no R
DR
2)P
ol IV
(AS
M)
(no
RD
R2)
Non
-tra
nsge
nic
RD
R2-
Pol
IVR
DR
2 (n
o P
ol IV
)
RN
A s
tran
d
Non
-tra
nsge
nic
Pol
IV-R
DR
2P
ol IV
(AS
M)-
RD
R2
Pol
IV (
no R
DR
2)P
ol IV
(AS
M)
(no
RD
R2)
Non
-tra
nsge
nic
RD
R2-
Pol
IVR
DR
2 (n
o P
ol IV
)
α-NRPD1
α-RDR2
α-NRPD2
α-RDR2
α-NRPD2
α-NRPD1
kDa190
120
190
120
85
190
120
85
kDa120
250
130
130
30nt
20nt
10nt
16nt
1 2 3 4 5 6 7 8 9
1 3 52 4
1 32
A Pol IV isolated from WT or rdr2-1
B RDR2 from WT or nrpd1-3
C Test of Pol IV - RDR2 co-dependence
5' C A G T C T G A C T G T G T A C G C C T G G T CCGACTCG3'
CGGACCAG A A A U A C G U 5'
Figure 3. Pol IV Transcription Is Indepen-
dent of RDR2, but RDR2 Requires Pol IV
(A) FLAG-tagged Pol IV or Pol IV(ASM) (active site
mutant) complexes were immunoprecipitated
from wild-type (RDR2; lanes 2 and 3) or rdr2-1
mutant (no RDR2; lanes 4 and 5) backgrounds.
Anti-FLAG IP of nontransgenic plant extract
served as a negative control. Immunoblots were
probed using anti-NRPD1, anti-RDR2, or anti-
NRPD2 antibodies.
(B) RDR2-HA was IPed from wild-type Pol IV (lane
2) or nrpd1-3 mutant (no Pol IV, lane 3) back-
grounds, and resulting immunoblots were probed
using anti-RDR2, anti-NRPD1, or anti-NRPD2
antibodies. Anti-HA IP of nontransgenic plant
extract served as a negative control.
(C) In vitro transcription of bipartite templates by
Pol IV-RDR2 or by Pol IV or Pol IV(ASM) isolated in
an rdr2 mutant background (lanes 2–5). Lanes 7
and 8 compare Pol IV-RDR2 or RDR2 isolated in
a Pol IV mutant (nrpd1-3) background. In lanes
2–5, complexes were isolated via anti-FLAG IP of
FLAG-tagged NRPD1. In lanes 7 and 8, RDR2-
containing complexes were isolated by anti-HA
IP of HA-tagged RDR2. Anti-FLAG or anti-HA
immunoprecipitations of nontransgenic plant ex-
tracts serve as negative controls (lanes 1 and 6).
End-labeled 16 nt RNA was loaded in lane 9.
Molecular Cell
In Vitro Transcription by Pol IV, Pol V, and RDR2
rdr2mutant (Figure 3C, lane 4), consistent with their synthesis by
RDR2. Products of �16 and 31 nt observed in anti-FLAG and
anti-HA IP controls from nontransgenic plants (Figure 3C, lanes
1 and 6, respectively) are due to end-labeling activities that are
neither Pol IV nor RDR2 dependent.
The Pol IV-RDR2 complex or complexes isolated upon IP of
RDR2 or NRPD1 have similar activities (Figure 3C, compare
lanes 7 and 2). However, RDR2 isolated from the Pol IV null
mutant background (nrpd1-3) no longer synthesizes 12–16 nt
transcripts (Figure 3C, lane 8). We conclude that RDR2 requires
association with Pol IV, or a Pol IV-associated factor, for activity
in vitro. In contrast, Pol IV activity is not dependent on RDR2
association.
Affinity-Purified Pol V Is Transcriptionally Active In VitroWe tested the activity of Pol V affinity purified from an nrpe1-11
null mutant rescued with wild-type or active site mutant (ASM)
forms of FLAG-tagged NRPE1 (see Figure 1D). NRPE1-FLAG
complements the nrpe1-11 mutant, but NRPE1(ASM)-FLAG
does not (Figure 1E, compare lanes 6 and 8) (Haag et al., 2009).
Like Pol IV, no significant Pol V activity was detectable using
sheared genomic DNA, chromatin, or ssDNA templates (Fig-
Molecular Cell 48, 811–818, D
ure S2). Unlike Pol IV, no Pol V activity
was detected using the tripartite template
(data not shown). However, using the
bipartite template, which lacks a non-
template DNA strand in need of dis-
placement, Pol V generates transcription
products that are similar to those of Pol
II (Figure 4A, compare lanes 6 and 7 to
lanes 9 and 10). Pol V transcripts are abol-
ished upon mutation of the Pol V active site (lane 8), but their
synthesis is insensitive to a-amanitin (lanes 4 and 7).
The ability of both Pols IV and V to transcribe bipartite DNA-
RNA templates prompted us to test their ability to transcribe
bipartite RNA-RNA templates (Figure 4B). Interestingly, Pol IV
is able to generate transcripts up to �27 nt in length (Figure 4B,
lanes 3 and 4), but Pol V lacks significant activity using the all-
RNA template.
DISCUSSION
Our results provide biochemical demonstrations of RNA poly-
merase activity for Pol IV, Pol V, and RDR2. Pol IV and RDR2
physically associate, and we find that RDR2 activity is depen-
dent on this association, suggesting that Pol IV and RDR2 activ-
ities are coupled, thereby channeling RNA intermediates early in
the 24 nt siRNA-directed DNA methylation pathway.
Compared to Pol II, the transcriptional activity of Pol IV and Pol
V is relatively weak. One possibility is that cofactors that increase
Pol IV and Pol V activity do not copurify with the polymerases.
Pols IV and V also have numerous amino acid substitutions or
deletions at positions that are invariant, or highly conserved, in
ecember 14, 2012 ª2012 Elsevier Inc. 815
Figure 4. Comparison of Pol II, IV, and V
Transcripts Generated In Vitro
(A) In vitro transcription products catalyzed by
Pol II, Pol IV, Pol IV active site mutant (ASM);
Pol V; or the Pol V active site mutant (ASM)
using the bipartite DNA-RNA template. Reac-
tions were conducted in the absence or presence
of a-amanitin, as indicated. No added protein
and nontransgenic controls are shown in lanes
1 and 2.
(B) In vitro transcription products catalyzed by
immunoprecipitated Pol IV, Pol IV active site
mutant (ASM), Pol V, or Pol V active site mutant
(ASM) using an RNA-RNA bipartite template. See
also Figure S4.
Molecular Cell
In Vitro Transcription by Pol IV, Pol V, and RDR2
Pols I, II, and III, which might compromise their activities.
Compared to Pol IV, associated RDR2 activity is strong. Low-
abundance Pol IV transcripts might be amplified significantly
by RDR2 in vivo, particularly if RDR2 can use its own transcripts
as templates. If so, Pol IV’s RNA polymerase activity may not
need to be particularly robust.
Thus far, we have been unable to detect significant Pol IV
or Pol V transcription in vitro in the absence of an RNA primer.
A trivial explanation could be that Pols IV and V require uniden-
tified cofactors that are not needed by Pols I, II, or III to initiate
transcription using sheared genomic DNA or other DNA tem-
plates. However, the mislocalization of Pols IV and V, but not
Pol II, in nuclei treatedwith RNase A is consistent with an involve-
ment of RNA as a template or primer (Pontes et al., 2006).
Abortive transcripts or persistent RNA-DNA hybrids resulting
from Pol II transcription, or small RNAs that invade duplex DNA
to generate R loops, are potential sources of DNA-RNA hybrid
templates.
Pol V’s ability to carry out transcription using the bipartite
oligonucleotide template, but not the tripartite template, sug-
gests an inability to disrupt downstream dsDNA during tran-
scription. The potential ramifications of this observation in vivo
are intriguing. We’ve shown that DRD1, a putative chromatin
remodeler in the SWI2/SNF2 protein family, and DMS3, a
protein that shares homology with the hinge domain region
of cohesin and condensin ATPases, enable Pol V transcrip-
tion in vivo (Wierzbicki et al., 2008, 2009). These proteins
interact with RDM1 (Law et al., 2010), a single-stranded DNA
binding protein (Gao et al., 2010), suggesting that the com-
plex may play a role in generating, and stabilizing, melted
816 Molecular Cell 48, 811–818, December 14, 2012 ª2012 Elsevier Inc.
duplex DNA regions to provide Pol V
with a single-stranded template. Further
development of Pol V in vitro transcrip-
tion assays should allow tests of this
hypothesis.
EXPERIMENTAL PROCEDURES
Plant Materials
A. thaliana mutants studied were nrpd1-3, nrpe1-
11, and rdr2-1 (Onodera et al., 2005; Pontier
et al., 2005; Xie et al., 2004). Transgenic lines
were NRPD1-FLAG nrpd1-3, NRPE1-FLAG
nrpe1-11, DCL3-FLAG dcl3-1, NRPB2-FLAG nrpb2-1, NRPD1(ASM)-FLAG
nrpd1-3, and NRPE1(ASM)-FLAG nrpe1-11 (Haag et al., 2009; Pontes et al.,
2006; Ream et al., 2009).
Generation of Transgenic Lines
Full-length RDR2 sequences were PCR amplified, captured in pENTR-TOPO
(Invitrogen), and recombined into the pEarleyGate301 plant transformation
vector (Earley et al., 2006). Cloning details are provided in the Supplemental
Information. A. thaliana transformation was by the floral dip method (Clough
and Bent, 1998).
Antibodies
Affinity-purified anti-NRPD1, anti-NRPE1, anti-NRP(D/E)2, anti-NRPA2/
NRPB2/NRPC2 (anti-Pol I, anti-Pol II, anti-Pol III), and anti-RDR6 were
described previously (Hoffer et al., 2011; Onodera et al., 2005; Ream et al.,
2009). Anti-FLAGM2-HRP and anti-HAwere purchased fromSigma (St. Louis,
USA). Antibodies against bacterially expressed 6xHis-RDR2-C (amino acids
786–1133) and 6xHis-DCL3-N (amino acids 1–393) were raised in rabbits
and affinity purified. Additional details are provided in the Supplemental
Information.
Immunoprecipitation
Frozen leaf tissue (4.0 g) ground in liquid nitrogen using a mortar and pestle
was homogenized in extraction buffer, subjected to centrifugation to pellet
cell debris, and the supernatant incubated with 25 ml anti-FLAG-M2 or anti-
HA resin (Sigma) for >2 hr on a rotating mixer. Resin was recovered and
washed twice with extraction buffer supplemented with 0.5% NP-40. For
immunoblot experiments, proteins were eluted from the resin by boiling
5 min in two bed volumes of 23 SDS sample buffer. Proteins resolved on
7.5% Tris-glycine SDS-PAGE gels were transferred to nitrocellulose or PVDF
and probed with rabbit antibodies (see the Supplemental Information for
antibody dilutions) and anti-rabbit-HRP (Amersham) secondary antibody.
Immunoblots were visualized using ECL or ECL Plus (GE Healthcare) chemilu-
minescent detection.
Molecular Cell
In Vitro Transcription by Pol IV, Pol V, and RDR2
Mass Spectrometry
Tryptic digests of affinity-purified Pol IV (Ream et al., 2009) and RDR2 were
subjected to LC-MS/MS analysis. Details are provided in the Supplemental
Information.
In Vitro Transcription of Oligonucleotide Templates
Pol IV or Pol V transcription reactions used the total IP fraction from 4.0 g leaf
tissue and were performed according to Kuhn et al. (2007). Polymerase-bound
resin was mixed with transcription reaction buffer containing 2 mM each of
ATP, UTP, and GTP; 0.08 mM unlabeled CTP; 0.2 miC/mL a-32P-CTP; and
4 pmol template nucleic acid(s). As appropriate, a-amanitin was added
5 min prior to the reaction buffer. Labeled transcripts were resolved on 15%
polyacrylamide and 7 M urea gels, transferred to Whatman 3MM filter paper,
and dried under vacuum prior to phosphorimaging or film exposure. Additional
details are provided in the Supplemental Information.
To prepare tripartite and bipartite templates, oligonucleotides at a concen-
tration of 10 mM each in T4 Polynucleotide Kinase buffer (New England Bio-
labs), 50 mM NaCl, were annealed by incubating for 2 min in a 95�C water
bath, then allowing the water bath to cool to room temperature.
SUPPLEMENTAL INFORMATION
Supplemental Information includes four figures, two tables, Supplemental
Experimental Procedures, and Supplemental References and can be found
with this article at http://dx.doi.org/10.1016/j.molcel.2012.09.027.
ACKNOWLEDGMENTS
J.R.H. and C.S.P. designed the study and wrote the paper. T.S.R. generated
the RDR6-FLAG line and the DCL3 and RDR6 antibodies. Pol IV samples for
LC-MS/MS were prepared by T.S.R. and analyzed by C.D.N., A.D.N., and
L.P.-T. Transcript sequences were determined by M.M. All other experiments
were performed by J.R.H. Portions of this research were supported by the NIH
National Center for Research Resources (RR18522) and the W.R. Wiley Envi-
ronmental Molecular Science Laboratory, a national scientific user facility
sponsored by the U.S. Department of Energy, located at PNNL and operated
by Battelle Memorial Institute under DOE contract DE-AC05-76RL01830. Pi-
kaard lab research was supported by National Institutes of Health grant
GM077590. C.S.P. is an Investigator of the Howard Hughes Medical Institute
and the Gordon and Betty Moore Foundation. Opinions are those of the
authors and do not necessarily reflect the views of our sponsors.
Received: April 2, 2012
Revised: August 13, 2012
Accepted: September 19, 2012
Published online: November 8, 2012
REFERENCES
Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,
735–743.
Cramer, P., Armache, K.J., Baumli, S., Benkert, S., Brueckner, F., Buchen, C.,
Damsma, G.E., Dengl, S., Geiger, S.R., Jasiak, A.J., et al. (2008). Structure of
eukaryotic RNA polymerases. Annu. Rev. Biophys. 37, 337–352.
Earley, K.W., Haag, J.R., Pontes, O., Opper, K., Juehne, T., Song, K., and
Pikaard, C.S. (2006). Gateway-compatible vectors for plant functional geno-
mics and proteomics. Plant J. 45, 616–629.
Erhard, K.F., Jr., Stonaker, J.L., Parkinson, S.E., Lim, J.P., Hale, C.J., and
Hollick, J.B. (2009). RNA polymerase IV functions in paramutation in Zea
mays. Science 323, 1201–1205.
Gao, Z., Liu, H.L., Daxinger, L., Pontes, O., He, X., Qian, W., Lin, H., Xie, M.,
Lorkovic, Z.J., Zhang, S., et al. (2010). An RNA polymerase II- and AGO4-asso-
ciated protein acts in RNA-directed DNA methylation. Nature 465, 106–109.
Molec
Gnatt, A.L., Cramer, P., Fu, J., Bushnell, D.A., and Kornberg, R.D. (2001).
Structural basis of transcription: an RNA polymerase II elongation complex
at 3.3 A resolution. Science 292, 1876–1882.
Haag, J.R., and Pikaard, C.S. (2011). Multisubunit RNA polymerases IV and V:
purveyors of non-coding RNA for plant gene silencing. Nat. Rev. Mol. Cell Biol.
12, 483–492.
Haag, J.R., Pontes, O., and Pikaard, C.S. (2009). Metal A and metal B sites of
nuclear RNA polymerases Pol IV and Pol V are required for siRNA-dependent
DNA methylation and gene silencing. PLoS ONE 4, e4110. http://dx.doi.org/
10.1371/journal.pone.0004110.
Havecker, E.R., Wallbridge, L.M., Hardcastle, T.J., Bush, M.S., Kelly, K.A.,
Dunn, R.M., Schwach, F., Doonan, J.H., and Baulcombe, D.C. (2010). The
Arabidopsis RNA-directed DNA methylation argonautes functionally diverge
based on their expression and interaction with target loci. Plant Cell 22,
321–334.
Herr, A.J. (2005). Pathways through the small RNA world of plants. FEBS Lett.
579, 5879–5888.
Herr, A.J., Jensen, M.B., Dalmay, T., and Baulcombe, D.C. (2005). RNA poly-
merase IV directs silencing of endogenous DNA. Science 308, 118–120.
Hoffer, P., Ivashuta, S., Pontes, O., Vitins, A., Pikaard, C., Mroczka, A.,
Wagner, N., and Voelker, T. (2011). Posttranscriptional gene silencing in nuclei.
Proc. Natl. Acad. Sci. USA 108, 409–414.
Huang, L., Jones, A.M., Searle, I., Patel, K., Vogler, H., Hubner, N.C., and
Baulcombe, D.C. (2009). An atypical RNA polymerase involved in RNA
silencing shares small subunits with RNA polymerase II. Nat. Struct. Mol.
Biol. 16, 91–93.
Kanno, T., Huettel, B., Mette, M.F., Aufsatz, W., Jaligot, E., Daxinger, L., Kreil,
D.P., Matzke, M., and Matzke, A.J. (2005). Atypical RNA polymerase subunits
required for RNA-directed DNA methylation. Nat. Genet. 37, 761–765.
Kuhn, C.D., Geiger, S.R., Baumli, S., Gartmann, M., Gerber, J., Jennebach, S.,
Mielke, T., Tschochner, H., Beckmann, R., and Cramer, P. (2007). Functional
architecture of RNA polymerase I. Cell 131, 1260–1272.
Lahmy, S., Pontier, D., Cavel, E., Vega, D., El-Shami, M., Kanno, T., and
Lagrange, T. (2009). PolV(PolIVb) function in RNA-directed DNA methylation
requires the conserved active site and an additional plant-specific subunit.
Proc. Natl. Acad. Sci. USA 106, 941–946.
Landick, R. (2009). Functional divergence in the growing family of RNA poly-
merases. Structure 17, 323–325.
Law, J.A., and Jacobsen, S.E. (2010). Establishing, maintaining and modifying
DNAmethylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220.
Law, J.A., Ausin, I., Johnson, L.M., Vashisht, A.A., Zhu, J.K., Wohlschlegel,
J.A., and Jacobsen, S.E. (2010). A protein complex required for polymerase
V transcripts and RNA- directed DNA methylation in Arabidopsis. Curr. Biol.
20, 951–956.
Law, J.A., Vashisht, A.A., Wohlschlegel, J.A., and Jacobsen, S.E. (2011).
SHH1, a homeodomain protein required for DNA methylation, as well as
RDR2, RDM4, and chromatin remodeling factors, associate with RNA
polymerase IV. PLoS Genet. 7, e1002195. http://dx.doi.org/10.1371/journal.
pgen.1002195.
Lehmann, E., Brueckner, F., and Cramer, P. (2007). Molecular basis of RNA-
dependent RNA polymerase II activity. Nature 450, 445–449.
Li, J., Yang, Z., Yu, B., Liu, J., and Chen, X. (2005). Methylation protects
miRNAs and siRNAs from a 30-end uridylation activity in Arabidopsis. Curr.
Biol. 15, 1501–1507.
Onodera, Y., Haag, J.R., Ream, T., Costa Nunes, P., Pontes, O., and Pikaard,
C.S. (2005). Plant nuclear RNA polymerase IV mediates siRNA and DNAmeth-
ylation-dependent heterochromatin formation. Cell 120, 613–622.
Pontes, O., Li, C.F., Costa Nunes, P., Haag, J., Ream, T., Vitins, A., Jacobsen,
S.E., and Pikaard, C.S. (2006). The Arabidopsis chromatin-modifying nuclear
siRNA pathway involves a nucleolar RNA processing center. Cell 126, 79–92.
Pontier, D., Yahubyan, G., Vega, D., Bulski, A., Saez-Vasquez, J., Hakimi,
M.A., Lerbs-Mache, S., Colot, V., and Lagrange, T. (2005). Reinforcement
of silencing at transposons and highly repeated sequences requires the
ular Cell 48, 811–818, December 14, 2012 ª2012 Elsevier Inc. 817
Molecular Cell
In Vitro Transcription by Pol IV, Pol V, and RDR2
concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes
Dev. 19, 2030–2040.
Qi, Y., He, X., Wang, X.J., Kohany, O., Jurka, J., and Hannon, G.J. (2006).
Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed
DNA methylation. Nature 443, 1008–1012.
Ream, T.S., Haag, J.R., Wierzbicki, A.T., Nicora, C.D., Norbeck, A.D., Zhu,
J.K., Hagen, G., Guilfoyle, T.J., Pasa-Toli�c, L., and Pikaard, C.S. (2009).
Subunit compositions of the RNA-silencing enzymes Pol IV and Pol V reveal
their origins as specialized forms of RNA polymerase II. Mol. Cell 33, 192–203.
Werner, F., and Grohmann, D. (2011). Evolution of multisubunit RNA polymer-
ases in the three domains of life. Nat. Rev. Microbiol. 9, 85–98.
818 Molecular Cell 48, 811–818, December 14, 2012 ª2012 Elsevier
Wierzbicki, A.T., Haag, J.R., and Pikaard, C.S. (2008). Noncoding transcription
by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of over-
lapping and adjacent genes. Cell 135, 635–648.
Wierzbicki, A.T., Ream, T.S., Haag, J.R., and Pikaard, C.S. (2009). RNA poly-
merase V transcription guides ARGONAUTE4 to chromatin. Nat. Genet. 41,
630–634.
Xie, Z., Johansen, L.K., Gustafson, A.M., Kasschau, K.D., Lellis, A.D.,
Zilberman, D., Jacobsen, S.E., and Carrington, J.C. (2004). Genetic and func-
tional diversification of small RNA pathways in plants. PLoS Biol. 2, E104.
http://dx.doi.org/10.1371/journal.pbio.0020104.
Zhang, H., and Zhu, J.K. (2011). RNA-directed DNA methylation. Curr. Opin.
Plant Biol. 14, 142–147.
Inc.