Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis.
ABSTRACT Recent genetic and biochemical studies have revealed the existence in plants of a fourth RNA polymerase, RNAPIV, which mediates siRNA accumulation and DNA methylation-dependent silencing of endogenous repeated sequences. Here, we show that Arabidopsis expresses, in fact, two evolutionarily related forms of RNAPIV, hereafter referred to as RNAPIVa and RNAPIVb. These two forms contain the same second-largest subunit (NRPD2), but differ at least by their largest subunit, termed NRPD1a and NRPD1b. Unlike NRPD1a, NRPD1b possesses a reiterated CTD, a feature that also characterizes the largest subunit of RNAPII. Our data indicate that RNAPIVb is the most abundant form of RNAPIV in Arabidopsis. Selective disruption of either form of RNAPIV indicates that RNAPIVa-dependent siRNA accumulation is not sufficient per se to drive robust silencing at endogenous loci and that high levels of DNA methylation and silencing depend on siRNA that are accumulated through a pathway involving the concerted action of both RNAPIV forms. Taken together, our results imply the existence of a novel two-step mechanism in siRNA synthesis at highly methylated loci, with RNAPIVb being an essential component of a self-reinforcing loop coupling de novo DNA methylation to siRNA production.
- SourceAvailable from: skku.ac.kr[show abstract] [hide abstract]
ABSTRACT: Histone lysine methylation plays a key role in the organization of chromatin structure and the regulation of gene expression. Recent studies demonstrated that the yeast Set1 and Set2 histone methyltransferases are recruited to mRNA coding regions by the PAF transcription elongation complex in a manner dependent upon the phosphorylation state of the carboxy-terminal domain of RNA polymerase II. These studies define an unexpected link between transcription elongation and histone methylation.Cell 06/2003; 113(4):429-32. · 31.96 Impact Factor
- Genes & Development 07/2000; 14(12):1415-29. · 12.44 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: RNA polymerase II holoenzymes isolated from yeast and mammalian cells are large, preassembled complexes that contain some or all of the general transcription initiation factors and many other polypeptides. Recent experiments suggest that these holoenzymes may mediate alterations in chromatin structure and play a key role in regulatory mechanisms that influence transcriptional initiation, RNA chain elongation, RNA processing and transcription termination.Current Opinion in Cell Biology 07/1997; 9(3):310-9. · 11.41 Impact Factor
Reinforcement of silencing
at transposons and highly repeated
sequences requires the concerted action
of two distinct RNA polymerases IV
Dominique Pontier,1,5Galina Yahubyan,1,5,6Danielle Vega,1Agnès Bulski,2Julio Saez-Vasquez,1
Mohamed-Ali Hakimi,3Silva Lerbs-Mache,4Vincent Colot,2and Thierry Lagrange1,7
1LGDP, UMR 5096, Université de Perpignan, 66860 Perpignan Cedex, France;2Unité de Recherche en Génomique Végétale
(URGV), INRA/CNRS/UEVE, 91057 Evry Cedex, France;3Institut Jean Roget, 38000 Grenoble, France;4PDC, UMR 5575,
Université Joseph Fourier, 38000 Grenoble, France
Recent genetic and biochemical studies have revealed the existence in plants of a fourth RNA polymerase,
RNAPIV, which mediates siRNA accumulation and DNA methylation-dependent silencing of endogenous
repeated sequences. Here, we show that Arabidopsis expresses, in fact, two evolutionarily related forms of
RNAPIV, hereafter referred to as RNAPIVa and RNAPIVb. These two forms contain the same second-largest
subunit (NRPD2), but differ at least by their largest subunit, termed NRPD1a and NRPD1b. Unlike NRPD1a,
NRPD1b possesses a reiterated CTD, a feature that also characterizes the largest subunit of RNAPII. Our data
indicate that RNAPIVb is the most abundant form of RNAPIV in Arabidopsis. Selective disruption of either
form of RNAPIV indicates that RNAPIVa-dependent siRNA accumulation is not sufficient per se to drive
robust silencing at endogenous loci and that high levels of DNA methylation and silencing depend on siRNA
that are accumulated through a pathway involving the concerted action of both RNAPIV forms. Taken
together, our results imply the existence of a novel two-step mechanism in siRNA synthesis at highly
methylated loci, with RNAPIVb being an essential component of a self-reinforcing loop coupling de novo
DNA methylation to siRNA production.
[Keywords: RNAPIV; reiterated CTD; siRNA; DNA methylation; silencing]
Supplemental material is available at http://www.genesdev.org.
Received April 25, 2005; revised version accepted June 29, 2005.
A major evolutionary distinction separating prokaryotes
from eukaryotes is the passage from a unique multisub-
unit DNA-dependent RNA polymerase enzyme (RNAP)
to three complexes (Roeder and Rutter 1969), each re-
sponsible for the transcription of a subclass of nuclear
DNA sequences (Sentenac 1985). RNA polymerase I
(RNAPI) transcribes the repeated genes encoding the
large ribosomal RNAs, which represent up to four-fifths
of total RNA. RNA polymerase II (RNAPII) transcribes
all of the cell protein-coding messenger RNAs (mRNAs)
as well as some small nuclear RNAs (snRNAs). RNA
polymerase III (RNAPIII) is dedicated to the transcription
of a collection of genes whose main common feature is
that they encode structural or catalytic RNAs (tRNAs,
5S RNA, snRNA) that are components of protein syn-
thesis, splicing, and tRNA processing apparatuses. It is
believed that this triplication event provided the eukary-
otic cell with a greater flexibility toward energy-consum-
ing cellular functions such as ribosome synthesis, as
well as with more sophisticated means for the regulation
of gene expression.
Prokaryotic and eukaryotic RNA polymerases are
multisubunit enzymes that are evolutionarily related to
each other through their largest and second-largest sub-
units (Ebright 2000; Cramer 2002). The largest subunit
(≈160 kDa: ??; A; RPA1; RPB1; RPC1) contains eight re-
gions conserved in order and sequence (A to H), while the
second-largest subunit (≈150 kDa: ?; B; RPA2; RPB2;
RPC2), contains nine such regions (A to I) (Allison et al.
1985; Sweetser et al. 1987). Although the role of these
conserved domains is not yet fully understood, the struc-
ture determination of RNAPII suggests that they coop-
erate in the formation of a single fold cleft containing the
active site of the enzyme (Cramer et al. 2001).
5These authors contributed equally to this work.
6Present address: Department of Plant Physiology and Molecular Biol-
ogy, University of Plovdiv, Plovdiv-4000, Bulgaria.
E-MAIL email@example.com; FAX 0033-0033468668499.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/
2030GENES & DEVELOPMENT 19:2030–2040 © 2005 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/05; www.genesdev.org
A specific feature of RNAPII is the presence at the
C-terminal end of its largest subunit (RPB1) of variable
numbers of a conserved heptapeptide repeat motif (con-
sensus YSPTSPS), which form the so-called C-terminal
domain (CTD) (Corden 1990; Young 1991). In vivo and in
vitro studies indicated that this CTD is essential for cell
viability and transcriptional activation and that it under-
goes a cycle of extensive phosphorylation and dephos-
phorylation through each round of transcription (Dah-
mus 1996; Carlson 1997; Greenblatt 1997). Indeed, de-
pending on its phosphorylation state, the CTD recruits
essential proteins, which help regulate the many steps of
transcription from initiation to termination (Greenblatt
1997; Otero et al. 1999; Kim et al. 2004), as well as chro-
matin modification (Hampsey and Reinberg 2003), and
pre-mRNA processing (Bentley 1999; Hirose and Manley
2000). The acquisition of the CTD by RNAPII can there-
fore be considered as an important step in the evolution
of the multiple levels of transcriptional and post-tran-
scriptional regulation that are the hallmark of eukary-
An unexpected outcome of the Arabidopsis genome
sequence analysis was the identification of genetic infor-
mation encoding novel large subunits of a putative
fourth type of eukaryotic RNAP (Arabidopsis Genome
Initiative 2000). Specifically, this information consisted
in two pairs of related genes potentially coding for the
largest and second-largest subunits. Recent genetic data
have implicated the product of two of these genes
(NRPD1a and NRPD2a) in the silencing of transposable
elements and other repeats via short-interfering RNAs
(siRNAs) (Herr et al. 2005; Onodera et al. 2005). In the
present work, we have extended this analysis by charac-
terizing the full set of potential RNAPIV largest and sec-
ond-largest subunit genes. We show that Arabidopsis ex-
presses, in fact, two forms of RNAPIV, RNAPIVa and
RNAPIVb, which contain the same second-largest sub-
unit (NRPD2) but differ at least by the nature of their
largest subunits. Furthermore, we show that NRPD1b
possesses a reiterated CTD, unlike NRPD1a, and that
RNAPIVb is the most abundant form of RNAPIV in
Arabidopsis. Selective disruption of RNAPIVa and
RNAPIVb indicates that efficient silencing at trans-
posons and highly repeated sequences requires the con-
certed action of both RNAPIV forms, while a basal level
of silencing at low repetitive DNA is only dependent on
RNAPIVa. Taken together, our results indicate that a
self-reinforcing silencing pathway based on the nonre-
dundant action of two different RNAPIV forms, probably
via a novel two-step mechanism in siRNA synthesis, is
required to maintain a high level of methylation at trans-
posons and highly repeated sequences.
Arabidopsis expresses two class IV largest subunit
We have initiated the molecular characterization and the
functional analysis of the class IV largest and second-
largest subunit genes present in the Arabidopsis ge-
nome, namely, At1g63020, At2g40030, and At3g23780,
At3g18090. Using a combination of PCRs and cDNA li-
brary screens, full-length cDNA sequences were ob-
tained for these genes, indicating that they are all tran-
scribed in Arabidopsis. The corresponding cDNA se-
quences have been deposited in the GenBank database
and recently renamed according to the revised nomen-
clature (Herr et al. 2005; Onodera et al. 2005) as
AtNRPD1a (accession number AY826515), AtNRPD1b
(AY826516), and AtNRPD2a (AY935711), AtNRPD2b
(AY935712) (Fig. 1A; Supplementary Figs. S1A, S2A).
While producing a full-length RNA with the same exon
boundaries as NRPD2a, NRPD2b is unlikely to encode a
functional protein due to a premature stop codon in the
first coding exon of the full-length cDNA, and it is there-
fore likely to be an expressed pseudogene (Supplemen-
tary Fig. S2B). Accordingly, we refer to the 1172-amino-
acid product of the NRPD2a gene as NRPD2. NRPD1a
and NRPD2 correspond to the largest and second-largest
subunits of the recently identified RNAPIV enzyme
(Herr et al. 2005; Onodera et al. 2005). Comparison of the
full-length cDNA sequence of AtNRPD1b with that of
the genome sequence revealed that it spans, in fact, the
two misannotated genes At2g40030 and At2g40040 (Fig.
1A). Thus, AtNRPD1b contains 17 exons and encodes a
1976-amino-acid polypeptide with a molecular mass of
235 kDa (AtNRPD1b) (Fig. 1A). RT–PCR analysis indi-
cates that AtNRPD1b as well as AtNRPD1a are ex-
pressed in all tissues tested (Supplementary Fig. S1B).
As outlined in Figure 1A, AtNRPD1b presents se-
quence similarities to AtRPB1 and AtNRPD1a in the
evolutionary conserved regions A through H that are
conserved between all known largest RNAP subunits.
Except for the B and C regions, AtNRPD1a and
AtNRPD1b proteins are more similar to each other than
to AtRPB1, suggesting that they originate from a com-
mon ancestor (Fig. 1A; Supplementary Fig. S3A). Of note,
region G is located near the conserved region F in both
AtNRPD1a and b, which suggests that these two pro-
teins are missing the structural element known as the
foot domain in the RNAPII structure (Cramer et al.
2001). Library screening and database searches indicate
that NRPD1b genes are also present in other plants, in-
cluding rice (OsNRPD1b; OsAP004365.3) and spinach
(SoNRPD1b; accession number AY826517). As expected
for bona fide orthologs, these proteins share a significant
level of sequence identity throughout their RPB1-like
region, with values ranging from 55% to 84% (Fig. 1A). A
striking difference between NRPD1s and the other large
subunits of RNAP is the amino acid substitutions ob-
served at the first position (for the NRPD1b proteins) and
at the first two positions (for the NRPD1a proteins) of
the invariant NADFDGD motif found in the conserved
region D (Supplementary Fig. S3B). Although these two
positions are not directly engaged in the coordination of
the Mg2+ions that participate in catalysis and can toler-
ate conservative substitutions (Dieci et al. 1995), the
structure of the RNAPII elongation complex has recently
suggested a possible role for the first residue N in the
Reinforcement of silencing by two RNAPIVs
GENES & DEVELOPMENT 2031
specificity for ribo- rather than deoxyribonucleotide
(Gnatt et al. 2001). Whether the amino acid substitutions
found at this position in NRPD1a and NRPD1b are in-
dicative of a more relaxed specificity of the RNAPIV en-
zyme(s) toward the nucleotide substrate or reveal a more
specific adaptation to novel function remains to be de-
product. Predicted and reannotated exons are indicated with open and gray boxes, respectively. Vertical arrowheads indicate T-DNA
insertions. Evolutionarily conserved regions A to H are represented as gray boxes. The cysteine and histidine residues of the zinc-
binding domain in the conserved region A are indicated in red (cc). The catalytic aspartate residues present in the conserved region D
are indicated in blue. The hydrophilic S/G/A/D/E/K-rich region and the DCL-like domains that compose the CTD are red and green,
respectively. Reiterated motifs are underlined. Numbers refer to amino acid identities between AtNRPD1b conserved regions and the
corresponding domains in homologs corresponding to SoNRPD1b, OsNRPD1b, AtNRPD1a, and AtRPB1. (B) Conservation of an RNA
polymerase core structure in RNAPIVb. The active-site magnesium is indicated by a pink sphere. (C) Schematic structure of
AtNRPD1b and AtNRPD1a CTDs. The S/G/A/D/E/K-rich region is shown in red, and the DCL-like domains are shown in green.
Repeated motifs are indicated as vertical lines. (D) Amino acid alignment of the repeated motifs with the deduced consensus sequence.
Arabidopsis contains a second class IV largest subunit gene. (A) Diagram of AtNRPD1b gene and corresponding protein
Pontier et al.
2032 GENES & DEVELOPMENT
termined. Beside the overall sequence conservation,
AtNRPD1b presents several features that make it a
likely component of a functional multimeric RNAP, as
was previously shown for AtNRPD1a (Herr et al. 2005):
All the invariant aspartate residues known to be directly
involved in the catalytic activity are conserved (Fig. 1A),
and the zinc-binding motifs (cc) that are critical for
the assembly of the largest and second-largest subunits
are maintained (Fig. 1A). When compared with yeast
RNAPII, AtNRPD1b and AtNRPD2 (which compose
RNAPIVb [see below]) present blocks of sequence ho-
mology that cluster around the active center of the en-
zyme (Fig. 1B, green panel) in a way that is similar to
bacterial RNA polymerase (Fig. 1B, orange panel). This
suggests the conservation of an RNA polymerase core
structure in RNAPIVb.
Compared with NRPD1a, NRPD1b shows an addi-
tional feature: namely the presence of a long CTD that
extends beyond the DCL-like motif (defective chloro-
plast and leaves) and comprises a highly hydrophilic do-
main including 10 complete repeats of a 16-amino-acid
consensus sequence with multiple potential phosphory-
lation sites (Fig. 1C,D). This latter arrangement is remi-
niscent of the reiterations that characterize the CTD of
RPB1 (Corden 1990; Young 1991) and was also found in
rice and spinach NRPD1b, although the number of reit-
erations and the primary sequence of the repeat motifs
appear specific to each species (data not shown).
Arabidopsis harbors two forms of RNAPIV, RNAPIVa
The identification of two functional NRPD1 genes and
one functional NRPD2 gene raised the question of the
exact nature of RNAPIV(s) in Arabidopsis. To address
this question, we raised specific antibodies against
NRPD1a, NRPD1b, and NRPD2 proteins, and we char-
acterized several Arabidopsis lines (Alonso et al. 2003)
nrpd1b-2 (1b-2), nrpd2a-1 (2a-1), nrpd2a-2 (2a-2), and
nrpd2b-1 (2b-1) (Fig. 1A; Supplementary Figs. S1A, S2A).
Plants homozygous for these mutant alleles were iden-
tified by PCR analysis of segregating families and were
used as controls in Western blot experiments. In wild-
type extracts, proteins with an apparent mass identical
to the predicted molecular sizes of NRPD1b (≈250 kDa),
NRPD1a (≈150 kDa), and NRPD2 (≈135 kDa) were spe-
cifically detected as judged by the absence of signal in
corresponding KO insertion lines (Supplementary Figs.
S1C, S2C). This analysis also shows that the AtNRPD2b
gene does not contribute to NRPD2 protein accumula-
tion, in agreement with the cDNA sequence data
(Supplementary Figs. S2B, S2C). Finally, Western analy-
sis of fractions corresponding to total leaf extracts or
highly purified nuclei and chloroplasts indicated that, as
expected for eukaryotic-type RNAP subunits, NRPD1a
and NRPD1b proteins are specifically detected in nuclei
(Fig. 2A). NRPD2 was also found in the nucleus (Fig. 2A),
consistent with immunolocalization experiments (On-
odera et al. 2005). Analysis of the accumulation of these
proteins in various plant organs also reveals a very abun-
dant accumulation of the NRPD1a and NRPD1b pro-
teins in flowers compared with leaves (Fig. 2B).
Multisubunit RNAP holoenzymes are high-molecular-
weight protein complexes (Cramer et al. 2001). To test
whether NRPD1a and NRPD1b are also part of large
complexes in vivo, flower extracts were size-fractionated
by sephacryl S-300HR gel filtration chromatography, and
ern blot analysis of Arabidopsis proteins from total extracts (T),
percoll-purified nuclei (N), and chloroplasts (C) with NRPD1b,
NRPD1a, NRPD2, KARI, and RPB1 antibodies. KARI was used
as a plastid control protein, whereas RPB1 was used as a nuclear
control. (B) Western blot analysis and Coomassie blue staining
of leaf (L) and flower (F) extracts (∼20 µg) with NRPD1a and
NRPD1b antibodies. (C) Gel filtration chromatographic analy-
sis of the NRPD1-containing complexes. The eluted fractions
were analyzed by Western blots using the NRPD1b and
NRPD1a antibodies. Numbered lanes correspond to size-frac-
tionated protein fractions ranging from 250 kDa (lane 58) to 2
MDa (lane 38). The peak position of the ferritin protein marker
(440 kDa) is indicated. (In) Input material. (D) Mutual stability
of the class IV largest and second-largest subunits in various
null mutants. The steady state of NRPD1b and NRPD1a was
analyzed in various nrpd2 mutants (left panel). RPB1 protein
was used as a loading control. The steady state of NRPD2 was
analyzed in various nrpd1 mutants (right panel). A nonspecific
cross-reacting band indicated by an asterisk was used as a load-
Arabidopsis contains two RNAPIV forms. (A) West-
Reinforcement of silencing by two RNAPIVs
GENES & DEVELOPMENT 2033
the resulting fractions were analyzed for the presence of
NRPD1a and NRPD1b by Western blot. As shown in
Figure 2C, both proteins were eluted close to the void
volume, therefore indicating that they are present exclu-
sively as complexed forms in vivo. The average molecu-
lar masses of the NRPD1a/b-containing complexes are
close to 1 MDa, a typical size for nuclear RNAP holoen-
zymes (Greenblatt 1997).
To determine if NRPD1a, NRPD1b and NRPD2 are
part of one or several multisubunit RNAP complexes, we
analyzed the costability of these proteins in vivo using
NRPD1b was strongly reduced in nrpd2a-1 mutant ex-
tract, indicating that NRPD2 is required to stabilize
NRPD1b (Fig. 2D, left panel). In contrast, NRPD1a ac-
cumulation was unaffected by the absence of NRPD2,
which suggests that NRPD1a is either stable as a sub-
complex in the mutant extract or that it does not form a
tight complex with NRPD2 in vivo. In order to extend
this analysis, NRPD2 protein stability was assessed
in nrpd1a-1, nrpd1b-1, and nrpd1a-1/nrpd1b-1 mutant
plants. These experiments confirmed that NRPD1b sta-
bilizes NRPD2, as most of the NRPD2 signal disap-
peared in nrpd1b-1 mutant plants (Fig. 2D, right panel).
While a weak NRPD2 signal was reproducibly observed
in this background, no NRPD2 could be detected in
nrpd1a-1/nrpd1b-1 double-mutant plants (Fig. 2D, right
panel), thus suggesting that a minor fraction of NRPD2
is complexed with NRPD1a. Taken together, our results
are consistent with most of NRPD2 being engaged in a
complex with NRPD1b, therefore defining a bona fide
novel RNAPIV form in Arabidopsis thaliana. We refer to
this novel RNA polymerase complex as RNAPIVb and
renamed the RNAPIV form harboring NRPD1a as
RNAPIVa and RNAPIVb act in the same pathway
and direct silencing and DNA methylation
at endogenous repeated loci
To investigate the functional role of the RNAPIVa and
RNAPIVb complexes in vivo, two independent nrpd1a
and nrpd1b homozygous mutant lines were further char-
acterized. To rule out any possible functional redun-
dancy of NRPD1a and NRPD1b, we also generated
double homozygous lines for nrpd1a-1/nrpd1b-1 and
nrpd1a-1/nrpd1b-2 alleles. None of the mutant alleles
showed readily discernable morphological changes other
than a nonadditive delay in flowering that was more pro-
nounced when plants were grown under short days com-
pared with long days (Fig. 3A).
In order to understand the molecular basis of this phe-
notype, we analyzed several candidate genes whose ex-
pression is known to delay flowering time under both
light conditions. While the regulation of most genes ana-
lyzed was not affected, as exemplified by the floral gene
regulator FLC, all independent mutants showed signifi-
cant expression of the imprinted FWA gene, whose ec-
topic expression has been shown to induce a late flow-
ering phenotype in Arabidopsis (Fig. 3B, see panels FLC
and FWA; Soppe et al. 2000). Consistent with the ectopic
activation of FWA, we found that the long tandem repeat
that encompasses the FWA promoter and the first two
untranslated exons is transcriptionally active in all mu-
tants analyzed (Fig. 3B, panel FWA tr; Lippman et al.
2004). The promoter region of FWA in wild-type plants
has been shown to be methylated in all sequence con-
texts (CG, CNG, and CNN) within the two direct re-
peats region, causing FWA to be silenced (Soppe et al.
2000). In this regard, FWA activation in our mutants sug-
gested a possible loss of cytosine methylation and con-
notype of nrpd1 plants, grown under long days (top) and
short days (bottom, left). The picture represents one out
of nine plants per line. (Right) Comparison of the leaf
number at flowering of wild-type (WT) and nrpd1 single
and double mutants. Similar results were obtained for
1a-2 and 1b-2 mutants. (B) RT–PCR analysis of flower-
ing-related gene expression in homozygous single and
double mutants. The FWA panel corresponds to the am-
plification of the coding region spanning exons 3–5 of
the gene. The FWAtr corresponds to the tandem repeats
located upstream of FWA. The cartoon shows the loca-
tion of FWA-derived primer pairs used in panels B and C.
(C) Analysis of DNA methylation of the FWA tandem
repeats. DNA was digested with the methylation-sensi-
tive enzymes HhaI (top) and AvaII (bottom).
Phenotypes of nrpd1 mutant lines. (A) Phe-
Pontier et al.
2034 GENES & DEVELOPMENT
sequent heterochromatin disruption. To determine if
nrpd1a and nrpd1b mutant alleles affect FWA cytosine
methylation, DNA methylation assays using methyl-
sensitive restriction enzymes and PCR amplification
were performed on the FWA tandem repeats region in
genomic DNA extracted from wild type and nrpd1 mu-
tants. If the enzyme site is methylated, the DNA is not
cut and can therefore be amplified by PCR, whereas de-
methylation of the site will allow DNA digestion and
preclude PCR amplification due to restriction of the
DNA. While no difference in CG methylation level (as
judged by the use of the HhaI enzyme, which does not
recognize GCGC motifs specifically methylated on the
inner C) was observed in nrpd1 mutants, a strong reduc-
tion of CNN methylation (as judged by the use of AvaII
enzyme, which is blocked by methylation of the outer C
of the recognition motif GGTCC) was observed in all
mutants (Fig. 3C). Taken together, our results indicate
that while they retain pre-existing CG methylation at
the endogenous FWA locus, nrpd1 mutants are blocked
in the perpetuation of CNN methylation, an effect
that would probably account for the transcriptional
activation of this gene. Based on the effects of mutations
on flowering delay, FWA transcript accumulation,
and DNA cytosine methylation, our results are consis-
tent with a nonadditive effect of both mutations, sug-
gesting that both proteins act in the same regulatory
pathway leading to de novo methylation at the FWA
To determine if nrpd1 mutants have a more general
effect on the methylation of repeated endogenous DNA,
we tested the methylation status at retrotransposon
AtSN1 and the 5S rDNA cluster, two repeated loci that
are extensively methylated in all sequence contexts (CG,
CNG, and CNN) (Hamilton et al. 2002; Zilberman et al.
2003; Xie et al. 2004). In wild-type plants, the AtSN1
retroelement is resistant to cleavage by the methylation-
sensitive restriction enzyme HaeIII, whereas a strong re-
duction of CNN methylation is observed in the mutants
(Fig. 4A). This result was confirmed and extended by
quantitative PCR analysis of genomic DNA digested
with the methylation-dependent enzyme McrBC (Lipp-
man et al. 2003), which confirms a nonadditive effect of
the mutations (Fig. 4C; see Materials and Methods). As
for the 5S rDNA intergenic spacers, HpaII and MspI
digestions indicated again a loss of methylation in
both CG and CNG sequence contexts in mutant plants
(Fig. 4B). Comparison of the single to the double mu-
tant showed a similar sensitivity to HpaII and MspI.
This result was confirmed by quantitative PCR analy-
sis of genomic DNA digested with the methylation-
dependent enzyme McrBC with no apparent additive ef-
fect in this case (Fig. 4C). That both RNAPIV forms act
in a nonredundant manner to direct methylation at 5S
rDNA was further supported by the observation that the
extent of methylation loss in the nrpd2a-1 mutant
(which likely harbors no functional RNAPIV forms be-
cause of NRPD2 depletion) is similar to that seen in
nrpd1a and nrpd1b single mutants (Supplementary Fig.
Differential role of the two RNAPIV forms on siRNA
Silencing and DNA methylation at FWA, AtSN1, and 5S
gene clusters are associated with siRNAs (Hamilton et
al. 2002; Zilberman et al. 2003; Lippman et al. 2004; Xie
et al. 2004), which prompted us to examine the involve-
ment of RNAPIVa and RNAPIVb in their accumulation.
We found that the 24-nt siRNAs corresponding to FWA
and AtSN1 retrotransposons were absent in nrpd1 single-
and double-mutant alleles (Fig. 5A), which is consistent
with a nonredundant role of RNAPIVa and RNAPIVb
in the production of endogenous siRNAs. Likewise,
siRNAs corresponding to 5S rDNA (siRNA1003) were
also greatly reduced down to a level that reveals a 21-nt
nonspecific band also observed in the rdr2-1 mutant
control (Fig. 5A, left and right panels). This was in sharp
contrast to miR-159 accumulation, which remained
unchanged in the various mutants (Fig. 5A). Thus,
RNAPIVa and RNAPIVb appear to be nonredundant
components of a pathway that generates endogenous
siRNAs that guide DNA methylation at transposons and
highly repeated endogenous sequences.
same pathway controlling methylation at AtSN1 and 5s rDNA.
(A) Analysis of DNA methylation of AtSN1. HaeIII-digested
DNA was used as a template for PCR reactions using AtSN1 and
control primers. The cut3 primers would not amplify DNA if
the digestion were complete. Undigested corresponds to undi-
gested DNA. (B) Blot analysis of 5S rDNA digested with meth-
ylation-sensitive restriction enzymes HpaII (left) and MspI
(right) in nrpd1 mutants and hybridized to a 5S probe. (C) DNA
methylation at various repeated loci was assessed in different
mutant backgrounds by quantitative McrBC–PCR. Delta Ct
corresponds to the difference in Ct between digested and undi-
gested samples. Heavily methylated sequences give large Delta
Ct, whereas unmethylated sequences give a Delta Ct value of 0.
RNAPIVa and RNAPIVb act nonredundantly in the
Reinforcement of silencing by two RNAPIVs
GENES & DEVELOPMENT2035
To test if the requirement of both RNAPIV forms for
endogenous siRNA formation was general, we tested
three additional endogenous loci present in few copies in
the genome and that are associated with detectable lev-
els of siRNAs: siRNA02, cluster 2, and TR2558 loci
(Lippman et al. 2004; Xie et al. 2004). Whereas siRNAs
corresponding to these loci were eliminated in nrpd1a
and nrpd1a/nrpd1b mutants, they remained mostly
unaffected in nrpd1b (Fig. 5A). We conclude from this
result that RNAPIVb engagement in the siRNA synthe-
sis pathway is target-dependent. DNA methylation as-
says indicate that RNAPIVb-independent loci (TR2558,
siRNA02, and cluster 2) are less methylated than 5S
rDNA, FWA, and AtSN1 sequences (Fig. 4C; Lippman et
al. 2004; Zilberman et al. 2004).
The identification of genomic sequences that require
only RNAPIVa for the accumulation of corresponding
siRNAs gave us a functional way to confirm the associa-
tion between NRPD1a and NRPD2. The accumulation
of siRNAs was decreased at all loci tested in the nrpd2a
mutant (Fig. 5B; Herr et al. 2005; Onodera et al. 2005),
which is consistent with the presence of NRPD2 in both
The present work demonstrates that, besides the re-
cently characterized RNAPIVa (Herr et al. 2005; Onodera
et al. 2005), plants contain a novel form of RNAPIV,
RNAPIVb. Although the two forms contain the same
second-largest subunit (NRPD2), they differ by their larg-
est subunit, termed NRPD1a and NRPD1b. Unlike
NRPD1a, NRPD1b is characterized by the presence of a
long, repeat-containing CTD, and thus possesses a struc-
ture that is remarkably similar to RPB1, the largest sub-
unit of RNAPII. The conservation of NRPD1a and
NRPD1b genes in both monocot and dicot plants sug-
gests that the emergence of these two forms predated the
divergence between these two phyla.
Recent studies have implicated RNAPIVa in the si-
lencing of endogenous DNA in a siRNA-dependent regu-
latory pathway (Herr et al. 2005; Onodera et al. 2005).
Our work supports this conclusion but also reveals an
unexpected level of complexity by demonstrating an ad-
ditional requirement of RNAPIVb for siRNA accumula-
tion and silencing at specific endogenous repeated loci.
Indeed, our data indicate that RNAPIVa is necessary but
not sufficient for the accumulation of endogenous
siRNAs that guide de novo methylation at 5S rDNA and
retroelements. At loci that are characterized by a high
level of methylation, the synthesis of siRNAs and the
perpetuation of de novo methylation also necessitates
RNAPIVb. This is not the case at loci such as siRNA02,
cluster 2, and TR2558, which are not densely methylated
(Fig. 4C; Lippman et al. 2004; Zilberman et al. 2004).
These observations indicate a correlation between
RNAPIVb engagement and the capacity of the siRNA-
dependent regulatory pathway to drive robust DNA
methylation. This suggests, in turn, that RNAPIVb is an
essential component of a reinforcing silencing mecha-
nism acting downstream of RNAPIVa. Considering that
each of the two RNAPIV forms is likely to produce long
transcripts that can feed into the RNAi pathway involv-
ing RDR2 and DCL3 (Chan et al. 2004; Herr et al. 2005;
Onodera et al. 2005), their requirement for siRNA syn-
thesis at highly methylated loci implies the existence of
two rounds of siRNA production. Involvement of pri-
mary and secondary siRNAs has already been proposed
to explain heterochromatin formation at centromeric se-
quences in fission yeast (Sugiyama et al. 2005) and at
highly repeated loci in plants (Onodera et al. 2005;
Vaughn and Martienssen 2005). Our study suggests,
however, that in plants the primary siRNAs that initiate
silencing via de novo methylation derive from primary
transcripts produced by RNAPIVa and not by euchro-
matic RNAP as previously proposed (Onodera et al.
2005; Vaughn and Martienssen 2005). The secondary
siRNAs and the subsequent amplification of methyl-
ation would then result from the recognition, either di-
rect or indirect, of the methylated target by RNAPIVb.
This hypothesis is further supported by the observation
that siRNA accumulation at endogenous loci is differen-
tially affected in DNA methyltransferase mutants (Lip-
pman et al. 2003; Zilberman et al. 2004; Onodera et al.
The involvement of two forms of RNAPIV in siRNA-
dependent silencing pathway at specific targets implies
that their ability to direct silencing must be somehow
different. One major difference between them is the pres-
ence, in the CTD of RNAPIVb, of an additional region
mostly composed of tandemly repeated motifs. Taking
into account that the acquisition of a reiterated CTD has
cumulation. (A) Small RNA blot assays for miR-159 and various
endogenous siRNAs in various nrpd1 mutants. Blots in A were
stripped and reprobed multiple times as indicated on the right.
(Right panel) Small RNA blot assays for miR-159 and siRNA
1003 in the rdr2-1 mutant is shown. (B) Small RNA blot assays
for miR-159 and various endogenous siRNAs in nrpd2a-1 mu-
Differential role of the two RNAPIVs on siRNA ac-
Pontier et al.
2036GENES & DEVELOPMENT
dramatically enhanced the capacity of RNAPII to couple
transcription to post-transcriptional events by recruiting
essential transcription-related proteins (Hirose and Man-
ley 2000; Hampsey and Reinberg 2003), it is tempting to
propose, by analogy, that the RNAPIVb reiterated CTD
would facilitate the coupling between the transcription
and silencing pathways by providing a platform for
RNAi- and/or silencing-related proteins. The observa-
tion that ago4 and nrpd1b mutants show similar mo-
lecular phenotypes with respect to siRNA accumulation
at all loci tested (Zilberman et al. 2004) makes the AGO4
protein a good candidate for being a specific component
of the RNAPIVb-dependent pathway (Fig. 6). Based on
the above considerations, we propose a model for siRNA-
dependent silencing at repeated loci with two key steps
(Fig. 6). The first one (blue arrows) implicates RNAPIVa,
RDR2, and DCL3 in the synthesis of endogenous pri-
mary siRNA at all repeated loci. Their incorporation into
putative effector complexes and subsequent recruitment
of the DRM methyltransferase would trigger low level of
de novo methylation of the homologous target loci. This
would be the only operating step at loci such as TR2558,
siRNA02, and cluster 2, whose siRNA accumulation is
independent of RNAPIVb. The second step would sub-
sequently concern targets that are characterized by high
levels of methylation (red arrows). On such sequences
(5S rDNA, retroelements), DNA methylation directed by
primary siRNAs would create binding sites for the re-
cruitment of RNAPIVb that, together with RDR2 and
DCL3, will trigger production of secondary siRNA. Sub-
sequent methylation via a pathway involving both
AGO4 and DRMs would further recruit RNAPIVb: This
would constitute the first round of a self-reinforcing
RNAi loop linking siRNA synthesis to heterochromatin
What determines the engagement of RNAPIVb in the
siRNA-synthesizing pathway leading to silencing of a
subset of targets remains unclear. One can consider that
5S rDNA and retroelements are highly repeated se-
quences and consequently that the dependence on
RNAPIVb would rely on copy number. However, the
concerted action of both RNAPIVs is required for siRNA
synthesis at FWA tandem repeat, which is not apprecia-
bly repeated elsewhere in the genome. This suggests the
existence of another criterion discriminating RNAPIVb-
dependent elements that could be the transcriptional po-
tential of the endogenous locus. In this regard, it is in-
teresting to note that in ddm1 mutant plants, which lose
most DNA methylation, AtSN1 and FWA tandem repeat
are active loci while the TR2558 tandem repeat remains
transcriptionally silent (Lippman et al. 2004). One can
therefore imagine that, in the process of heterochroma-
tin formation, DNA methylation, together with another
chromatin mark left by the transcribing euchromatic
RNAP, could serve as specific binding sites for the re-
cruitment of RNAPIVb.
An alternative model for RNAPIV activity has been
recently proposed by Vaughn and Martienssen (2005),
who raises the possibility that RNAPIVa may, in fact,
synthetize primary transcripts from double-stranded
RNA templates. Similar template specificity could be
inferred to RNAPIVb since both polymerases present a
similar extent of divergence compared with RNAPII.
This would imply in our two-step model, that RNAPIVa
would synthesize primary transcripts in trans from mi-
totically transmissible double-stranded RNA templates
while RNAPIVb would probably be acting in cis. One
appealing aspect of this model is that it readily accounts
for the engagement of RNAPIVb at only a subset of loci
that lead to siRNA accumulation. In this model, diffus-
ible primary siRNA and associated effector complexes
would guide, via DRM-directed methylation, RNAPIVb
and the associated RNAi machinery to the DNA target
sequence. Once located at the transcriptionally active
target sites, RDR2 will convert nascent euchromatic
transcripts into dsRNA that then will be transcribed in
cis by RNAPIVb, therefore maintaining the sustained
production of secondary transcripts and subsequent sec-
ondary siRNAs. Future biochemical and functional stud-
ies will be necessary to understand precisely the inter-
play between both RNAPIV forms and the mechanism
by which RNAPIV triggers a high level of DNA methyl-
ation and heterochromatin assembly.
Materials and methods
Arabidopsis growth and genetic analysis
For plant growth, seeds were stratified at 4°C for 2 d before
growth at 23°C with 16 h light/8 h dark cycles on soil or under
continuous light on plates with MS medium. All mutants origi-
nated from the Salk Institute collection of T-DNA insertions
(http://signal.salk.edu/cgi-bin/tdnaexpress) obtained via the
Nottingham Arabidopsis Stock Center (http://nasc.life.nott
.ac.uk). The nrpd1a-1 (1a-1) and nrpd1a-2 (1a-2) alleles
correspond, respectively, to the lines SALK_583051 and
SALK_628428. The nrpd1b-1 (1b-1) and nrpd1b-2 (1b-2) alleles
correspond, respectively, to the lines SALK_029919 and
SALK_033852. Genomic DNA was amplified with the LBA1
and specific primers to detect the T-DNA (5?-CGCTGTCTC
GTTGCTGTAAACATGG-3? for 1b-1 and 1b-2 lines, 5?-AACC
TCGAAGCAACAAGAATCTCCG-3? for the 1a-1 line, and 5?-
methylation at endogenous repeated loci in plants. The blue
arrows correspond to the RNAPIVa-dependent pathway that
concerns siRNA02 and cluster 2. On targets such as AtSN1 and
5S repeats it would be followed by a self-reinforcing loop impli-
cating RNAPIVb (red arrows).
Hypothetical model for siRNA-dependent DNA
Reinforcement of silencing by two RNAPIVs
GENES & DEVELOPMENT 2037
GCGGATCCGCAATTATCAAAGCTTCTCC-3? for the 1a-2
line). To follow the presence of the endogenous copy of the
genes and identify homozygous lines, genomic DNA was PCR-
amplified with specific couples of primers (1b-1, 5?-CAAAGTG
GTGATGCATGGAGG-3? and 5?-ATGTAAATTTTGGAAGT
CGGC-3?; 1b-2, 5?-GCGCTCTAGACTTCAAATGGTCATAT
-3?; 1a-1, 5?-AACCTCGAAGCAACAAGAATCTCCG-3? and
5?-ATCACGGTGACTGTAGTTGAAGCC-3?; 1a-2, 5?-GCGG
ATCCGCAATTATCAAAGCTTCTCC-3? and 5?-CTCGTTTA
AAGATGAAACAACGG-3?). Homozygous lines were propa-
gated by repeated self-pollination.
For crosses, homozygous plants were grown on soil to matu-
rity and five to eight flowers were hand-pollinated after removal
of their immature stamens. The F2 generation was analyzed by
PCR to characterize double-homozygous plants from which
seeds were collected. The F3 generation was used for further
Bioinformatics and molecular visualization
Gene searching was done on the Arabidopsis genome sequence
database or the nonredundant databases by using the TBLASTN
program (Altschul et al. 1997) with the amino acid sequence of
the human RPB1 protein. The spinach NRPD1b ortholog gene
was isolated by screening of a ?Zap library (Clontech), using a
labeled DNA fragment corresponding to a part of the large exon
7 of AtNRPD1b as a probe. Phylogenetic studies were performed
with the conserved region comparison as described previously
(Lagrange et al. 2003). Molecular visualization of the RNAP
conserved regions was performed using the WebLab ViewerLite
software from Accelrys.
Protein isolation, cell fractionation, antibodies, and Western
Total plant protein extracts were obtained following the Tanaka
method (Hurkman and Tanaka 1986). Coomassie staining was
used to calibrate loadings. Intact Arabidopsis chloroplasts were
purified as described previously (Kunst 1998). Intact nuclei from
Arabidopsis leaves were purified as described previously onto
percoll gradients (Watson and Thompson 1986).
Proteins were separated onto SDS/PAGE gels and blotted
onto PVDF membrane (Immobilon-P, Millipore). Rabbit anti-
sera were raised against peptides designed in NRPD1a,
NRPD1b, and NRPD2 and affinity-purified. Dilutions of 1/1000
were usedto perform Western blot analysis of the indicated pro-
tein extracts. Dilutions of 1:1000 and 1:500 of KARI (Dumas et
al. 2001) and RPB1 monoclonal antibody 8WG16, respectively,
were used for plastid and nuclei controls.
Gel filtration chromatography
Total homogenates were prepared in BC500 buffer (Maldonado
et al. 1996) and centrifuged for 30 min at 20,000 rpm (rotor JA
25.50), and the supernatant was filtered through a 0.45-µm filter
(Gelman Sciences). About 400 µg of total soluble proteins were
fractionated through a HiLoad 16/60 Sephacryl S-300 HR FPLC
colum (Pharmacia), with BC500 buffer at a flow rate of 1 mL/
min. All fractionations were carried out in the 4°C cold room.
Fractions of 1 mL each were collected and concentrated by ac-
etone precipitation. An equal volume of individual fractions
was used for immunoblot analysis. The protein standards for
size estimation of the NRPD1-containing complex(es) were as
follows: blue dextran, 2 MDa; ferritin, 440 kDa.
RNA extraction and analysis
Total RNA was isolated from different organs from plants
grown in soil using the Invisorb Spin Plant RNA Mini Kit fol-
lowing the manufacturer’s instructions (Invitek) followed by a
DNase treatment using the RQ1 RNase-free DNase (Promega).
A PCR reaction was performed on RNA with primers for the
RPL21 gene (5?-TCCACTGCGTCGACGTCTCG-3? and 5?-CT
CCAACTGCAACATTGGGCG-3?) to check for the absence of
genomic DNA. Reverse transcription was performed on 3–5 µg
of total RNA using the ProSTAR First-Strand RT–PCR kit fol-
lowing the manufacturer’s instructions (Stratagene). Semiquan-
titative RT–PCR was calibrated by nonsaturating PCR reaction
(22 cycles) using primers designed to amplify the EF1-4? cDNAs
(5?-CTGCTAACTTCACCTCCCAG-3? and 5?-TGGTGGGTA
CTCAGAGAAGG-3?). A parallel set of reactions without addi-
tion of reverse transcriptase was run as a quality control. To
analyze NRPD1b and NRPD1a expression in wild-type plants,
semiquantitative RT–PCR was performed with the following
primers: 5?-CAAAGTGGTGATGCATGGAGG-3? and 5?-CG
GCTCATTATTTTCTGGGAC-3? for AtNRPD1b and 5?-ACT
TGCGCTGCTTGGAATGAC-3? and 5?-CAAAGACATTGTC
TGTTGTGC-3? for AtNRPD1a. Primers used to amplify FLC
and FWA cDNAs were, respectively, 5?-AAGATCCTTGATC
GATATGGG-3? and 5?-AAGTAGTGGGAGAGTCACCGG-3?
(27 cycles), and 5?-GCGGTTGGAAACATTCCAAAACC-3?
and 5?-AGTGTCTCCACAATTAGTTGCC-3? (40 cycles). The
primer pair 5?-TCCCATTCAACATTCATACGAGCGCCGC
-3? and 5?-TCTGATATTTGGCTGGAAAAAACAACAATAAT
C-3? was used to detect cDNAs corresponding to the long tan-
dem repeat that encompasses the FWA promoter and the first
two untranslated exons (40 cycles).
For siRNA analysis, total RNA samples were extracted from
inflorescences using TRIzol (Invitrogen). Thirty micrograms of
total RNA were separated on a 15% polyacrylamide gel contain-
ing 7 M urea and electroblotted onto Hybond N+membranes
(Amersham Pharmacia Biotech Inc.). Hybridization was per-
formed in PerfectHyb solution (Sigma) following the supplier’s
instruction. siRNA1003, miR159, and siRNA02 were detected
using end-labeled DNA oligonucleotides AS1003, AS159, and
AS-02, respectively (Xie et al. 2004). The probes for cluster2,
TR2558, and FWA siRNA detection were riboprobes synthe-
sized using the Riboprobe system T7 Kit (Promega) following
the supplier’s instruction. Primers used to amplify cluster2- and
TR2558-specific sequences were, respectively, 5?-CTTTTT
CAAACCATAAACCAGAAA-3? and 5?-TTGCTGATTTGTAT
TTTATGCAT-3?, and 5?-CAATATCTAAAATTATTTCTATG
AGTCGG-3? and 5?-GCATAACATAACTATGCAAAACATG
AGC-3?. Primers used to generate the FWA riboprobe template
were the ones used for amplifying the tandem repeat in RT–PCR
experiments. AtSN1 siRNA were detected using end-labeled
DNA oligonucleotides JP2107 (Zilberman et al. 2004).
Genomic DNA extraction and methylation detection assays
Genomic DNA was extracted from seedlings using the Wizard
Genomic DNA extraction kit (Promega). DNA was digested
with different restriction enzymes (HhaI, AvaII for FWA analy-
sis and HaeIII for AtSN1 analysis). HhaI recognizes the sequence
GCGC and is inhibited by methylation of either cytosine. AvaII
recognizes the sequence GGA/TCC and is inhibited by meth-
ylation at the outer cytosine, allowing detection of asymmetric
methylation. PCR amplification was subsequently done on 150
ng of digested DNA using the following primers. The 5?-GAAG
GTTCTCATCATATACCG-3? and 5?-GGTTTTGGAATGTTT
CCAACCGC-3? primer pair (FWA SINE) was used to identify
the methylation status of the tandem repeats that encompass
Pontier et al.
2038GENES & DEVELOPMENT
the FWA promoter and the first two untranslated of FWA. Prim-
ers designed for RT–PCR amplification of the FWA coding re-
gion flanked an unmethylated HhaI site and were used to check
the completion of genomic DNA digestion (cut1). Since the am-
plified DNA sequence is devoid of AvaII, this pair was used to
control equal template concentration in the AvaII experiment
(control panel). Internal PCR control primers (5?-GGAGAGAG
GCTTGTTGGATACTGC-3? and 5?-GAACACGCATGACA
GTGGGTGGAG-3?) span a region lacking HhaI as well as
HaeIII sites and were used to control equal template concentra-
tion in both HhaI and HaeIII experiments (control panels). In
contrast, this control region contains an unmethylated AvaII
site, and, therefore, those primers were used to check the
completion of genomic DNA digestion by this enzyme (cut2).
For AtSN1 methylation status, the following primers were
T-3? and 5?-TTTAAACATAARAARAARTTCCTTTTTCATC
TAC-3? (Hamilton et al. 2002). The primers FWA SINE de-
scribed above flank a DNA sequence containing unmethylated
HaeIII sites and are used to check the completion of HaeIII
digestion (cut3 panel).
The Southern experiment was performed on 5 µg of genomic
DNA digested by the methylation-sensitive enzymes HpaII or
MspI and separated on 0.9% agarose gel. After blotting on Hy-
bond N+membranes (Amersham Pharmacia Biotech Inc.), hy-
bridization was performed in PerfectHyb solution (Sigma) fol-
lowing the supplier’s instructions, with the transcribed region
of the 5S rRNA gene.
McrBC-based methylation analysis was performed as follows,
based on a previously published protocol (Lippman et al. 2003).
Genomic DNA (500 ng) was digested for 2 h at 37°C with 30 U
of McrBC enzyme (NEB). A mock digestion was done in parallel,
with no enzyme. Quantitative PCR (ABI Prism 79000 HT) was
directly performed on 1/50th of the digested and mock samples,
using the ABI SybrGreen PCR master mix. Reactions were per-
formed on two independent digests, and were usually dupli-
cated. Differences in Ct values between digested and undigested
samples were averaged over three or four sets of PCR reactions.
Primers used for quantitative PCR were as follows: 5?-AACGT
GCTGTTGGCCCAGT-3? and 5?-CTGGAAGTTCAAGCCC
AAAG-3? for AtSN1; 5?-CTTTTCGGGCNTTTTNGTG-3? and
5?-CGAAAAGGTATCACATGCC-3? for the 5S spacer; 5?-TTC
CGAAACAGTAAACCATCG-3? and 5?-TCAAAGTGAAAGT
GGTTCTTGG-3? for siRNA02up; 5?-GCCATCCCAAGCTG
TTCTCTC-3? and 5?-CCCTCGTAGATTGGCACAGT-3? for
We thank E. Lam, A.M. Henry, and our lab colleagues for fruit-
ful discussions. We thank O. Voinnet for providing mutant seed
stocks. We thank M. Block for providing KARI antibodies. G.Y.
was the recipient of a post-doctoral fellowship from CNRS. This
work was supported by a grant from CNRS (Programme ATIP to
T.L.) as well as by a grant from the European Union (Epigenome
Network of Excellence to V.C.).
Note added in proof
A genetic screen for mutants defective in RNA-directed DNA
methylation and silencing of a transgene promoter in Arabidop-
sis identified nrpd1b and nrpd2a as drd3 and drd2 (Kanno et al.
2005). These findings are in agreement with those reported here
that RNA-mediated DNA methylation at endogenous repeated
loci requires the action of two forms of RNAPIV in Arabidopsis.
Allison, L.A., Moyle, M., Shales, M., and Ingles, C.J. 1985. Ex-
tensive homology among the largest subunits of eukaryotic
and prokaryotic RNA polymerases. Cell 42: 599–610.
Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H.,
Shinn, P., Stevenson, D.K., Zimmerman, D.K., Barajas, P.,
Cheuk, R., et al. 2003. Genome-wide insertional mutagen-
esis of Arabidopsis thaliana. Science 301: 653–657.
Miller, W., and Lipmann, D.J. 1997. Gapped BLAST and PSI-
BLAST: A new generation of protein database search pro-
grams. Nucleic Acids Res. 25: 3389–3402.
Arabidopsis Genome Initiative 2000. Analysis of the genome
sequence of the flowering plant Arabidopsis thaliana. Na-
ture 408: 796–815.
Bentley, D. 1999. Coupling RNA polymerase II transcription
with pre-mRNA processing. Curr. Opin. Cell Biol. 11: 347–
Carlson, M. 1997. Genetics of transcriptional regulation in
yeast: Connections to the RNA polymerase CTD. Annu.
Rev. Cell. Dev. Biol. 13: 1–23.
Chan, S.W.-L., Zilberman, D., Xie, Z., Johansen, L.K., Carring-
ton, J.C., and Jacobsen, S.E. 2004. RNA silencing genes con-
trol de novo DNA methylation. Nature 303: 1336.
Corden, J.L. 1990. Tails of RNA polymerase II. Trends Biochem.
Sci. 15: 383–387.
Cramer, P. 2002. Multisubunit RNA polymerases. Curr. Opin.
Struct. Biol. 12: 89–97.
Cramer, P., Bushnell, D.A., and Kornberg, R.D. 2001. Structural
basis of transcription: RNA polymerase II at 2.8 Å resolu-
tion. Science 292: 1863–1876.
Dahmus, M.E. 1996. Reversible phosphorylation of the C-
terminal domain of RNA polymerase II. J. Biol. Chem.
Dieci, G., Hermann-Le Denmat, S., Lukhtanov, E., Thuriaux, P.,
Werner, M., and Sentenac, A. 1995. A universally conserved
region of the largest subunit participates in the active site of
RNA polymerase III. EMBO J. 14: 3766–3776.
Dumas, R., Biou, V., Halgand, F., Douce, R., and Duggleby, R.
2001. Enzymology, structure, and dynamics of acetohydroxy
acid isomeroreductase. Accounts Chem. Res. 34: 399–408.
Ebright, R.H. 2000. RNA polymerase: Structural similarities be-
tween bacterial RNA polymerase and eukaryotic RNA poly-
merase II. J. Mol. Biol. 304: 687–698.
Gnatt, A.L., Cramer, P., Fu, J., Bushnell, D.A., and Kornberg,
R.D. 2001. Structural basis of transcription: An RNA poly-
merase II elongation complex at 3.3 Å resolution. Science
Greenblatt, J. 1997. RNA polymerase II holoenzyme and tran-
scriptional regulation. Curr. Opin. Cell Biol. 9: 310–319.
Hamilton, A.J., Voinnet, O., Chappell, L., and Baulcombe, D.
2002. Two classes of short interfering RNA in RNA silenc-
ing. EMBO J. 21: 4671–4679.
Hampsey, M. and Reinberg, D. 2003. Tails of intrigue: Phos-
phorylation of RNA polymerase II mediates histone meth-
ylation. Cell 113: 429–432.
Herr, A.J., Jensen, M.B., Dalmay, T., and Baulcombe, D.C. 2005.
RNA polymerase IV directs silencing of endogenous DNA.
Science 308: 118–120.
Hirose, Y. and Manley, J.L. 2000. RNA polymerase II and the
integration of nuclear events. Genes & Dev. 14: 1415–1429.
Hurkman, W.J. and Tanaka, C.K. 1986. Solubilization of plant
membrane proteins for analysis by two-dimensional gel elec-
trophoresis. Plant Physiol. 116: 1209–1218.
Kanno, T., Huettel, B., Mette, M.F., Aufsatz, W., Jaligot, E.,
Reinforcement of silencing by two RNAPIVs
GENES & DEVELOPMENT2039
Daxinger, L., Kreil, D.P., Matzke, M., and Matzke, A.J. 2005.
Atypical RNA polymerase subunits required for RNA-di-
rected DNA methylation. Nat. Genet. 37: 761–765.
Kim, M., Krogan, N.J., Vasiljeva, L., Rando, O.J., Nedea, E.,
Greenblatt, J.F., and Buratowski, S. 2004. The yeast Rat1
exonuclease promotes transcription termination by RNA
polymerase II. Nature 432: 517–522.
Kunst, L. 1998. Preparation of physiologically active chloro-
plasts from Arabidopsis. Methods Mol. Biol. 82: 43–48.
Lagrange, T., Hakimi, M.-A., Pontier, D., Courtois, F., Alcaraz,
J.P., Grunwald, D., Lam, E., and Lerbs-Mache, S. 2003. Tran-
scription factor IIB (TFIIB)-related protein (pBrp), a plant-spe-
cific member of the TFIIB-related protein family. Mol. Cell.
Biol. 23: 3274–3286.
Lippman, Z., May, B., Yordan, C., Singer, T., and Martienssen,
R. 2003. Distinct mechanisms determine transposon inher-
itance and methylation via small interfering RNAs and his-
tone modification. PLoS Biol. 1: E67.
Lippman, Z., Gendrel, A.-V., Black, M., Vaughn, M.W., Dedhia,
N., McCombie, W.R., Lavine, K., Mittal, V., May, B., Kass-
chau, K.D., et al. 2004. Role of transposable elements in
heterochromatin and epigenetic control. Nature 430: 471–
Maldonado, E., Drapkin, R., and Reinberg, D. 1996. Purification
of human RNA polymerase II and general transcription fac-
tors. Methods Enzymol. 274: 72–100.
Onodera, Y., Haag, J.R., Ream, T., Nunes, P.C., Pontes, O., and
Pikaard, C.S. 2005. Plant nuclear RNA polymerase IV medi-
ates siRNA and DNA methylation-dependent heterochro-
matin formation. Cell 120: 513–522.
Otero, G., Fellows, J., Li, Y., de Bizemont, T., Dirac, A.M., Gus-
tafsson, C.M., Erdjument-Bromage, H., Tempst, P., and Sve-
jstrup, J.Q. 1999. Elongator, a multisubunit component of a
novel RNA polymerase II holoenzyme for transcriptional
elongation. Mol. Cell 3: 109–118.
Roeder, R.G. and Rutter, W.J. 1969. Multiple forms of DNA-
dependent RNA polymerase in eukaryotic organisms. Na-
ture 224: 234–237.
Sentenac, A. 1985. Eukaryotic RNA polymerases. CRC Crit.
Rev. Biochem. 18: 31–90.
Soppe, W.J.J., Jacobsen, S.E., Alonso-Blanco, C., Jackson, J.P.,
Kakutani, T., Koornneef, M., and Peeters, A.J.M. 2000. The
late flowering phenotype of fwa mutants is caused by gain-
of-function epigenetic alleles of a homeodomain gene. Mol.
Cell 6: 791–802.
Sugiyama, T., Cam, H., Verdel, A., Moazed, D., and Grewal, S.I.
2005. RNA-dependent RNA polymerase is an essential com-
ponent of a self-enforcing loop coupling heterochromatin
assembly to siRNA production. Proc. Natl. Acad. Sci.
Sweetser, D., Nonet, M., and Young, R.A. 1987. Prokaryotic and
eukaryotic RNA polymerases have homolgous core sub-
units. Proc. Natl. Acad. Sci. 84: 1192–1196.
Vaughn, M.W. and Martienssen, R.A. 2005. Finding the right
template: RNA Pol IV, a plant-specific RNA polymerase.
Mol. Cell 17: 754–756.
Watson, J.C. and Thompson, W.F. 1986. Purification and restric-
tion endonuclease analysis of plant nuclear DNA. Methods
Enzymol. 118: 57–75.
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 functional diversification of small RNA
pathways in plants. Plos Biol. 2: E104.
Young, R.A. 1991. RNA polymerase II. Annu. Rev. Biochem.
Zilberman, D., Cao, X., and Jacobsen, S.E. 2003. Argonaute 4
control of locus-specific siRNA accumulation and DNA and
histone methylation. Science 299: 716–719.
Zilberman, D., Cao, X., Johansen, L.K., Xie, Z., Carrington, J.C.,
and Jacobsen, S.E. 2004. Role of Arabidopsis Argonaute4 in
RNA-directed DNA methylation triggered by inverted re-
peats. Curr. Biol. 14: 1214–1220.
Pontier et al.
2040 GENES & DEVELOPMENT