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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

mailto:[email protected]


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


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.


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


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-


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


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


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


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


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

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