Identification of an RNA-dependent RNA polymerase
in Drosophila involved in RNAi and
transposon suppression
Concetta Lipardi and Bruce M. Paterson1
Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Building 37, Room 6118, 9000 Rockville Pike,
Bethesda, MD 20892
Here, we show that recombinant Drosophila elp1 (D-elp1) produced
in Sf9 cells or Escherichia coli, corresponding to the largest of the three
subunits in the RNA polymerase II core elongator complex, has
RNA-dependent RNA polymerase (RdRP) activity. D-elp1 is a noncanonical RdRP that can synthesize dsRNA from different ssRNA templates using either a primer-dependent or primer-independent initiation mechanism. Of the three core subunits, only D-elp1 depletion
inhibits RNAi in S2 cells but does not affect micro RNA function.
Furthermore, D-elp1 depletion results in increased steady state levels
of representative transposon RNAs and a decrease in the corresponding transposon antisense transcripts and endo siRNAs. In contrast,
although Dcr-2 depletion results in increased transposon RNA levels
and a reduction in the corresponding endo siRNAs, there is no change
in the transposon antisense RNA levels. In D-elp1 null third instar
larvae transposon RNA levels are also increased and the corresponding transposon antisense RNAs are reduced. D-elp1 associates tightly
with Dcr-2, similar to the Dicer-RdRP interaction observed in lower
eukaryotes. These results identify an aspect of the RNAi pathway in
Drosophila that suggest transposon derived endo siRNAs, critical for
transposon suppression, are produced, in part, in a D-elp1 dependent
step that converts transposon RNA into dsRNA that is subsequently
processed by Dcr-2. The generality of this mechanism in genome
defense and RNA silencing in higher eukaryotes is suggested.
elongator 兩 genome defence 兩 cellular stress
I
n Arabidopsis Thaliana, Neurospora crassa, Schizosaccharomyces
pombe and Caenorhabditis elegans, RNA-dependent RNA polymerases (RdRPs) are required for RNA silencing (1, 2). However,
even though robust RNAi occurs in response to exogenous dsRNAs
and siRNAs in Drosophila, no RdRP homologs have been identified
in flies or higher eukaryotes. Endogenous siRNAs in Drosophila
and vertebrates, called endo siRNAs, have been shown to arise
primarily from specific regions in the genome transcribed by pol II
that produce double stranded RNAs that are subsequently processed by Dcr-2 (3, 4). A large body of evidence representing a wide
range of organisms has shown that siRNAs target the cognate RNA
through an Ago-mediated endonucleolytic cleavage event in the
RISC (5). In flies and mammals, siRNAs have not been shown to
participate in a further amplification step involving an RdRP and
the synthesis of dsRNA, as has been observed in S. pombe, C.
elegans and various plant species. However, independent studies
demonstrating RNAi spreading in Drosophila in response to very
low doses of dsRNA suggests an amplification process is present in
flies (6). Mathematical models of RNAi also indicate that a primed
amplification step involving dsRNA synthesis from siRNAs and the
target RNA is required to explain dose-dependent responses,
transient and sustained responses, transgene or transposon-induced
silencing, and the avoidance of self-directed reactions that would
occur unabated in the catalytic Ago-2 based mechanism proposed
for RNAi in higher eukaryotes (2, 7). Therefore, on theoretical
grounds, questions concerning the role of an RdRP in RNA
silencing in higher eukaryotes are still unresolved.
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0904984106
Indirect evidence for the existence of an RdRP in Drosophila
comes from (i) observation of siRNA-primed dsRNA synthesis in
embryo extracts (8, 9) and (ii) copy number-dependent post transcriptional silencing of an Adh transgene that is correlated with the
appearance of Adh siRNAs, derived from Adh dsRNA generated
in response to increased levels of Adh mRNA (10). Deep sequence
analysis of endogenous Drosophila siRNAs (endo siRNAs) from
embryos, larval discs, and adult structures, as well as from S2 cells
and Kc cells, indicates almost twenty percent of the endo siRNAs
are derived from transposable elements in a Dcr-2-dependent process
and represent both the sense (41%) and antisense strand (59%) of the
transposon RNAs along the entire body of the transposable element,
consistent with the formation of transposon dsRNA (11, 12). In line
with these observations, significant amounts of cytoplasmic, polyadenylated dsRNA, representing the entirety of the transposons
mdg1 and mdg3, was identified in the Drosophila cell line 67J25D
by the Georgiev laboratory nearly 30 years ago (13). At that time,
the authors proposed either symmetric transcription or RNAdependent RNA synthesis gave rise to the mdg1/mdg3 dsRNAs. In
this study we show that elongator subunit 1 of the Drosophila pol II
core elongator complex, D-elp1, has RdRP activity and plays a role
in both RNAi and the Dcr-2 dependent formation of transposon
specific endo-siRNAs correlated with transposon silencing.
Results and Discussion
Identification of D-elp1 as an RdRP. To characterize the proteins
involved in siRNA-primed dsRNA synthesis identified previously
(8), Drosophila embryo extract was fractionated by ion exchange
chromatography (DEAE and Heparin Sepharose), gel filtration
(Superose 6), and a final velocity sedimentation step (15–45%
glycerol gradient) (Fig. S1 A). Fractions were dialyzed against
reaction buffer and tested for primed fill in synthesis using full
length GFP dsRNA with approximately 40 bp of single-stranded 5⬘
overhang on each end as the template, and 0.1 to 2.0 ␮g of protein
under reaction conditions outlined in SI Text and described previously, with minor buffer modifications (8) (Fig. S1B). Reaction
products were analyzed on 1.5% agarose-formaldehyde denaturing
gels due to the size of the labeled RNA. Fill-in specific activity
(defined as cpm (PSL)/mm2/mg protein and measured on the
FujiFilm FLA-5100) was enriched approximately nine hundredfold in the final gradient fraction (Fig. S1 A). Comparative mass
spec analysis (W. Lane, Harvard Microchemistry Laboratory) of
closely spaced gradient fractions that were either active or inactive
in the primed fill-in reaction (fraction 4 and 6, respectively,
Author contributions: C.L. and B.M.P. designed research, performed research, analyzed
data, and wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To
whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/
0904984106/DCSupplemental.
PNAS Early Edition 兩 1 of 6
BIOCHEMISTRY
Edited by Maxine F. Singer, Carnegie Institution Washington, DC, and approved July 31, 2009 (received for review May 8, 2009)
B
D-elp1
125
ssRNA
Template :
(~ 142 kD)
250bp
350bp cap
380bp cap-An
α-Flag
kD M
225
CB
A
RNaseA/T1
MW bp
Hsp83
75
350 bp
D-elp1
dsRNA
350 bp
Template
ssRNA
500
51
BSA
E
Dcr-2
bp
MW
D
250 bp
C
380 bp cap-An
125
350 bp
Cp
Ap
+ + + + - - - + - +
bp MW - - + -
500
125
50
Up
125
Gp
25
21-25 bp
origin
n.n.a.
Fig. S1B) resulted in the identification of approximately 25 proteins
highly enriched or unique to the active fraction (Table S1). These
included members of the COP9 signalosome complex, initiation
factors, DNA polymerase sigma and epsilon, heat shock proteins,
an ATP-dependent helicase, mcm4 and mcm6, and eight peptides
for CG10535, corresponding to the Drosophila homolog of the
RNA polymerase II core elongator complex subunit, elp1 (D-elp1),
also known as IKAP in mammals (Fig. S1C). The other two subunit
proteins of the core elongator complex usually associated in a
stochiometric ratio with D-elp1, the Drosophila elp-2 (CG11887)
and elp-3 (CG15433) homologs, were not detected in either fraction
at the single peptide level.
Since Drosophila does not contain a canonical RdRP gene, we
screened selected proteins in the active fraction for their ability to
inhibit siRNA-mediated silencing of a GFP reporter in S2 cells, by
soaking the cells with double stranded RNA for the various
candidate proteins. Using a mixture of dsRNAs previously screened
for the absence of off target effects against D-elp1 (D-elp1.A and
D-elp1.B) (14, 15), the knockdown of D-elp1 strongly inhibited RNAi
(see below). After two rounds of dsRNA treatment the cells were
healthy and did not take up trypan blue. Taken together with the
evidence indicating elp-1 has multiple roles in transcription, cytoplasmic
kinase signaling, exocytosis, tRNA modification and disease (16) we felt
further analysis of the Drosophila elp1 (D-elp1) was warranted.
Baculovirus Recombinant D-elp1 Has Primed Fill-In Activity. Flagtagged D-elp1protein was expressed in Sf9 cells using a modified
baculovirus vector containing the heat shock inducible HSP70
promoter (17). High level recombinant D-elp1 (rD-elp1) expression from the polh promoter in the standard baculovirus vector was
toxic to Sf9 cells. Acrylamide/SDS gel analysis of Flag-tag purified
rD-elp1 showed a single major band of the expected molecular
weight (⬃142 kD) that reacted with anti-Flag antibody (Fig. 1A).
Western blot analysis demonstrated a small amount of Hsp83
protein remained tightly bound to the affinity isolated rD-elp1. Sf9
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sense strand template
sense primer
antisense primer
antisense 3ʼP04primer
~ 600 bp
Fig. 1. D-elp1protein expressed in Sf9 cells has RdRP
activity. (A) Flag tag affinity purified D-elp1 stained with
colloidal blue (CB) is shown. The presence of the Flag tag
was verified by Western blot using M2 HRP Flag antibody
(␣-Flag). M, the SeeBlue plus2 marker (Invitrogen) (B)
D-elp1 directs unprimed synthesis from different templates (Left). Newly synthesized dsRNA is resistant to
RNase A/T1 digestion (Right). (C) Nearest neighbor analysis (n.n.a.) confirms template directed synthesis. Black
arrowheads mark the ribonucleotide-3⬘monophosphates. (D) Dcr-2 digests the 350-bp dsRNA to yield 21–25
bp siRNAs. (E) D-elp1 directs primed dsRNA synthesis to
give the expected ⬃600 bp extension product. MW,
25-bp DNA ladder end-labeled with 32P dCTP.
cells infected with a GFP control baculovirus yielded no high molecular
weight proteins when processed similarly (Fig. S2B, Center).
rD-elp1 protein directed the labeling of the primed fill-in substrate with ␣32P-UTP in a concentration and time dependent
manner (PSL/mm2, FujiFilm FLA-5100) using 30–250 ng protein
per reaction incubated for 2 h, or 100 ng of protein in reactions
incubated for various times up to 2 h, respectively (Fig. S2 A).
Labeling depended upon the 5⬘ overhangs since pretreatment of the
substrate with RNase One to generate blunt-ended dsRNA eliminated incorporation entirely but nuclease treatment after the
reaction had no effect on the labeled RNA (8) (Fig. S2 A). Nearest
neighbor analysis had previously confirmed internal labeling of the
filled in 5⬘ overhang (8). Nearest neighbor analysis measures the
distribution of the nucleosides that are 5⬘-adjacent to the ␣-labeled
position in the RNA product after RNase One digestion and TLC
of the resultant nucleoside-3⬘-monophosphates. Actinomycin-D
and ␣-amanitin at concentrations that inhibited uridine incorporation ⬎95% in S2 cells and inhibit the activity of all three
DNA-dependent RNA polymerases, respectively, did not affect
rD-elp1 activity (Fig. S2 A) (8). Replacement of CTP and GTP with
deoxynucleotides or 3⬘-O-methyl ribonucleotides prevented ␣32PUTP incorporation (Fig. S2 A) (18). When synthetic 30-bp dsRNA
or dsDNA templates with a single 18-bp 5⬘ overhang were tested
with rD-elp1 or the GFP control proteins eluted from the flag
beads, only the dsRNA template incubated with rD-elp1protein
produced the expected 48-bp fill-in product resistant to RNase One
digestion (Fig. S2C, Left). Furthermore, neither single- or doublestranded DNA templates served as substrate for rD-elp1 directed
labeling (Fig. S2C, Right).
Baculovirus rD-elp1 Has RdRP Activity. Baculovirus rD-elp1 was
tested with different single-stranded RNA templates, including
unmodified, capped, or capped and polyadenylated RNAs of
250–380 bp. Template length labeled RNA was produced that was
resistant to RNaseA/T1 concentrations that completely degraded
the input RNA, as shown by analysis on 6% polyacrylamide-8M
Lipardi and Paterson
empty vector
46 kD
75
500
350 bp
125
142 kD
96 kD
142 kD
96 kD
125
bp MW
aa
empty vector
D-elp1
∆C
∆N
aa
D-elp1
∆C
∆N
empty vector
∆N
kD M
225
1253
96 kD
1253
1
∆C
∆C
B
aa
∆N
142 kD
D-elp1
1
843
D-elp1
844
A
50
25
51
46 kD
C
D
RNaseA/T1
+ +
+ +
bp MW 350 bp
D-elp1
dsRNA
500
sense strand template
sense primer
antisense primer
E bp MW
BSA
Dcr-2
α-T7
Coomassie
350 bp
~ 600 bp
125
50
350 bp
Template
ssRNA
125
25
urea gels (Fig. 1B). To independently confirm the RNaseA/T1
digestion results and to rule out possible nuclease titration artifacts,
rD-elp1 reaction products were also digested with Dcr-2 (recombinant
Dcr-2 prepared in Sf9 cells, SI Text), the ribonuclease III-related
enzyme that digests dsRNA to produce siRNAs (19). Dcr-2 cleaved the
labeled RNAs into siRNA length fragments of ⬃22–25 bp, as shown
here for the 380-bp labeled RNA derived from a polyadenylated
template, confirming independently that the rD-elp1 reaction products
are dsRNAs (Fig. 1D). Material prepared similarly from Sf9 cells
infected with a GFP control virus did not produce labeled RNA in
response to any of the template RNAs, and rD-elp1-dependent
dsRNA synthesis required the presence of both the ssRNA template and rD-elp1 protein in the reaction (Fig. S2B, Right).
Nearest neighbor analysis of the 250-bp and 380-bp labeled
RNAs confirmed that ␣-UTP was incorporated internally into the
newly synthesized RNA strand and gave the expected nucleoside3⬘-monophosphate ratios predicted by the template sequence for
each template normalized to 3⬘-CMP (FujiFilm FLA 5100, PSL/
mm2): 250-bp RNA with Cp, Ap, Up and Gp ratios distributed as
1.0 to 1.3 to 1.5 to 1.0, and 380-bp RNA (380-bp polyadenylated
template) ratios distributed as 1.0 to 1.1 to 2.2 to 1.0 (Fig. 1C). Thus,
baculovirus rD-elp1 is similar to N. crassa Qde-1 and can initiate
primer-independent, template directed dsRNA synthesis on a variety of
ssRNA templates (2). But unlike Qde-1, we did not observe significant
back-primed or self-primed synthesis that generated product larger
than the input template using template RNAs from 50–700 bp.
To ascertain if baculovirus rD-elp1 was also able to initiate
primer-dependent RNA synthesis, as describe for N. crassa Qde-1
and S. pombe Rdp-1 (20, 21), GFP ssRNA (716 bp) was annealed
either with the 5⬘-32P- labeled sense or anti-sense strand of a GFP
siRNA and tested as substrate in reactions with rD-elp1 protein and
unlabeled ribonucleotide triphosphates. First, only the antisense primer
directed dsRNA synthesis to gave the expected ⬃600 bp full-length
primer extended product when analyzed on 6% polyacrylamide-8M
urea gels (Fig. 1E). Second, chemical addition of a 3⬘-phosphate
Lipardi and Paterson
21-25 bp
Fig. 2. D-elp1 protein expressed in E. coli has RdRP
activity. (A) D-elp1 protein (1–1252 aa) and the ⌬C (1– 843
aa) and ⌬N (844 –1252 aa) deletions are schematized.
Proteins stained with Coomassie blue (Lower Left) react
with anti-T7-tag antibody to identify D-elp1 (Lower
Right). M, SeeBlue Plus2 marker (Invitrogen) (B) Full
length D-elp1 and the ⌬C deletion are active in unprimed
dsRNA synthesis while the ⌬N deletion and the empty
vector are inactive. MW is the 25-bp ladder (Invitrogen)
(C) The 350-bp dsRNA is resistant to RNase A/T1 digestion. (D) E. coli D-elp1 directs primed synthesis with a 5⬘
32P-labeled siRNA antisense strand but not with the labeled sense strand. MW, 25-bp ladder (Invitrogen) (E)
The 350-bp dsRNA produced by E. coli D-elp1 is cleaved
by Dcr-2.
group on the antisense primer prevented primer extension (Fig.
1E). Together the data confirmed that baculovirus rD-elp1 can
initiate dsRNA synthesis using either primer-dependent or primerindependent mechanisms, similar to other known cellular RdRPs.
Using the specific activity of the ␣UTP or the primer to estimate
the relative moles of product produced per mole of template {cpm
[PSL units]/mm2/mg protein (AU units): 32P incorporation measured on the FujiFilm FLA 5100; protein amount determined by
Western blot analysis using a flag tagged protein standard measured on the FujiFilm LAS-3000}, unprimed dsRNA synthesis
produced at least five times more RNA than the primed reaction.
In both instances the relative ratio indicated less than one mole of
product was produced per mole of template, consistent with a single
transcription initiation event. The difference in product yield
between the unprimed and primed synthesis reactions could reflect
the efficiency of the primer-template annealing step, i.e., unusable
primer-template complexes are formed. However, we cannot exclude the possibility that the differences also arise at the transcription initiation step.
An rD-elp1 Deletion Made in E. coli Has RdRP Activity. To strengthen
the argument that baculovirus rD-elp1 RdRP activity was not due
to an Sf9-asociated viral RdRP or an unidentified protein contaminant, and to initiate studies to identify domains in the protein
responsible for RdRP activity, we prepared full length soluble
rD-elp1 protein in E. coli. E. coli lacks the RNA silencing machinery
and has not been shown to have an RdRP related gene or RdRP
activity. N-terminal His6-T7-tagged rD-elp1, isolated on nickel
agarose resin, was of the expected size on Coomassie blue-stained
gels, ⬃142 kDa, and reacted with antibody to the T7 peptide
sequence fused to amino terminus of D-elp1 (Fig. 2A, D-elp1 lanes)
(22). Similar to rD-elp1 produced in baculovirus, E. coli rD-elp1
(Fig. 2 A) was active in both primer-independent (Fig. 2 B and C)
and primer-dependent dsRNA synthesis, as shown by the analysis
of the reaction products on 6% polyacrylamide-8M urea gels (Fig.
PNAS Early Edition 兩 3 of 6
BIOCHEMISTRY
46 kD
B 110
100
90
80
70
60
50
40
30
20
10
0
dsRNA:
control: w/o F-Luc dsRNA
exp:
with F-Luc dsRNA
Firefly Luc./Renilla Luc (%)
normalized qRT-PCR RNA levels
+
+
-
+
+
IgG-hc
IgG-hc
IgG-hc
IgG-lc
IgG-lc
IgG-lc
IgG-lc
IP FLAG
Blot V5
IP V5
Blot Flag
2D). The labeled dsRNA from the unprimed synthesis reaction was
also cleaved into siRNA length fragments when incubated with
recombinant Dcr-2 (Fig. 2E). Again, using the specific activity of
the ␣UTP to measure primer-independent RNA synthesis levels
and calibrated Western blots to measure D-elp1 protein amounts
per reaction, as described above, the baculovirus and E. coli
recombinant D-elp1 proteins produce similar amounts of dsRNA
under comparable reaction conditions. Just like the baculovirus
rD-elp1, the primer-independent dsRNA yield with the E. coli
protein was greater than its primer-dependent yield. The product to
template ratio was also less than one in both the primed and
unprimed RNA synthesis reactions.
Deletion analysis indicated that RdRP activity was associated
with the amino terminal 96-kD fragment (⌬C, CDS 1–2,528) since
the carboxy terminal 46-kD polypeptide (⌬N, CDS 2,528–3,759)
was inactive in dsRNA synthesis. The negative control, prepared
identically to rD-elp1 using E. coli transfected with the empty
vector, produced background proteins that had no RdRP activity
(Fig. 2 A and B). Unfortunately, due to the aggregation and
nonspecific binding properties of recombinant D-elp1 protein,
prepared either in Sf9 cells or E. coli, we have so far been unable
to obtain highly purified material. We cannot formally exclude the
possibility that D-elp1 is not acting as a cofactor to stimulate RdRP
activity in a minor protein contaminant. However, given the fact
that D-elp1 prepared either in Sf9 cells or E.coli has RdRP activity,
and that activity is associated with the amino terminal 96-kD
fragment of the protein, the results support the conclusion that
D-elp1 protein, and not a minor contaminant, is responsible for the
RdRP activity identified here.
D-elp1 Interacts with Dcr-2 and Is Involved in RNAi. Knockdown of
D-elp1 inhibited dsRNA-mediated gene silencing to the same
extent as Dcr-2 depletion (Fig. 3 A and B). As mentioned above,
inhibition of siRNA-mediated silencing of a GFP reporter was similarly
affected by the knockdown of D-elp1 (Fig. S3B). Although we do not
have antibody to endogenous D-elp1, S2 cells expressing V5-tagged
D-elp1 and a GFP marker selectively lost D-elp1 expression when
knocked down in parallel cultures, suggesting the endogenous
protein was also depleted under the treatment conditions used.
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D-elp3
Dcr-1
Ago-2
D-elp1
IgG-hc
IP V5
Blot FLAG
V5 Dcr-1
-
D-elp2
V5 Ago-2
Flag-D-elp1
Dcr-2
D-elp1
D-elp1
V5 vector
+
Dcr-2
V5 Dcr-1
-
D
GFP
V5 Ago-2
+
D-elp3
V5 vector
Flag-D-elp1 -
D-elp2
V5 Dcr-2
C
D-elp1
V5 vector
Dcr-2
V5 Dcr-2
RNA:
V5 vector
A
IP Flag
Blot V5
Fig. 3. D-elp1 is required for RNAi and interacts with
Dcr-2. (A) Quantitative RT-PCR analysis for the expression
levels of Dcr-2, D-elp1, D-elp2, and D-elp3 RNAs in S2 cells
after depletion. (B) S2 cells were soaked with dsRNA
either to GFP, Dcr-2, D-elp2, D-elp3, and D-elp1 (a mixture of D-elp1.A and D-elp1.B) and were then cotransfected with Renilla and firefly luciferase reporters, with
or without dsRNA to firefly luciferase, to determine
changes in firefly luciferase activity due to the inhibition
of RNAi. Three separate experiments are shown. (C)
D-elp1 interacts with V5 tagged Dcr-2 in S2 cells. Western
blot analysis was performed using either antiFlag or V5
antibody in the initial pulldown followed by blot analysis
with the counter antibody. The Ig heavy and light chains
are indicated by IgG-hc and IgG-lc, respectively. (D) Ectopically expressed D-elp1 and either Ago-2 or Dcr-1
show weak but reproducible interactions in S2 cells.
D-elp1 is the largest of the three subunits in the core elongator
complex, and D-elp3 has been shown to have histone acetyl
transferase activity that facilitates pol II transcription through
chromatinized templates in vitro (16, 23, 24). We wanted to know
if D-elp1 depletion alone was responsible for the effects on RNA
silencing or if the three subunits of the core elongator complex were
equally involved, suggesting a role for the elongator complex itself.
Unlike the results with D-elp1, the knockdown of either D-elp2 or
D-elp3 RNA had no affect on RNAi, indicating the role of D-elp1
is relatively specific and outside the context of the elongator
complex (Fig. 3 A and B). In support of this interpretation, the
Elp1-related proteins are found predominately in the cytoplasmic
compartment in cells examined from yeast to man (23, 25, 26) and
ectopically expressed D-elp1 in S2 cells was localized mainly in the
cytoplasmic compartment, in agreement with previous observations (Fig. S3A, Upper). However, the RNA pol II CTD, which has
been shown to bind the core elongator complex, was observed only
in the nuclear compartment in S2 cells (Fig. S3A, Lower).
The cytoplasmic location of D-elp1 and its role in RNAi suggested it might be interacting with components of the RISC. RdRP
has been shown to form a complex with Dicer in S. pombe and
Tetrahymena thermophilia (27, 28). To test this, V5-tagged Ago-2, Dcr-2
and Dcr-1 were individually coexpressed with Flag-tagged D-elp1 or the
empty vector in S2 cells and subjected to either V5 or Flag antibody
pulldown and blot analysis with the counter tag antibody. Pulldowns
were performed in the presence of Ribonuclease A to eliminate
possible RNA-mediated interactions. Dcr-2 showed extremely robust interaction with D-elp1, using either V5 or Flag antibody in the
pulldown reaction (Fig. 3C), while the interactions observed with
Ago2 and Dcr-1 were evident but not as intense (Fig. 3D).
Given that D-elp1 interacts strongly with Dcr-2, it was formally
possible that loss of D-elp1 was affecting the stability of Dcr-2
resulting in the inhibition of RNAi. However, depletion of D-elp1
protein in transfected S2 cells did not reduce Dcr-2 protein expression or affect Dcr-2 RNA levels (Fig. 3A). Ago2 RNA levels were
also unaffected by D-elp1 depletion. In contrast to the RNAi
pathway, micro RNA regulation was unaffected since targeting of
the nautilus gene transcript by miR-3 was normal in D-elp1 depleted
Lipardi and Paterson
21-23 bp
297 antisense
21-23 bp
mdg1 sense
21-23 bp
mdg1 antisense
endo-siRNA
D
mdg1
Canton S
D-elp1 null
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
transposon :
RNA
297
mdg1
antisense
transposon:
RNA
Rrp49
Het-A
Het-A
E
Canton S
antisense
transposon:
RNA
297
S2 cells [Ravulapalli et al. (2008) 49th Annual Drosophila Research
Conference Abst. no. 111] (Fig. S3C).
D-elp1 Has a Role in Transposon Suppression. The Drosophila genome
harbors a variety of retrotransposons and mobile elements that are
silenced post transcriptionally through the RNAi pathway (11, 12,
29–31). We looked at the impact of D-elp1 depletion on transposon
suppression and the production of the corresponding endo siRNA
for representative transposons highly expressed in S2 cells. Previous
studies have shown that a reduction in the RISC components Ago2
and Dcr-2, but not Dcr-1, result in the increased steady state levels
of several transposon RNAs, including 297, mdg1, and the nonLTR transposon TART-B (11, 12, 32) accompanied by a decrease
in the corresponding transposon endo-siRNAs. We measured the
RNA levels for 297, mdg1, and the telomeric non-LTR transposon,
HetA, after treating S2 cells with dsRNA to D-elp1, using Dcr-2
depletion as the positive control (32). We also determined the endo
siRNA levels for 297 and mdg1. Just as with Dcr-2 depletion,
knockdown of D-elp1 resulted in a similar increase in RNA levels
for the mdg1, 297, and HetA transposons and a dramatic decrease
in the corresponding endo-siRNAs for mdg1 and 297 (Fig. 4 A and
B) (33). By contrast knockdown of D-elp2 or D-elp3 had no effect
on transposon RNA levels (Fig. 4A). This demonstrates that D-elp1
is specifically involved in the post transcriptional regulation of
transposon RNAs and their regulatory endo siRNAs.
The fact that D-elp1 knockdown and transposon siRNA depletion were correlated, and that D-elp1 has RdRP activity, suggested
a pathway in which transposon RNAs are converted into dsRNA by
D-elp1 and processed into endo siRNAs by Dcr-2. This interpretation was strengthened with the observation that transposon
antisense RNA levels for mdg1 and 297 decreased dramatically in
S2 cells treated with D-elp1 dsRNA (Fig. 4C). This result establishes a direct link among D-elp1 depletion, a decrease in transposon antisense RNA levels, and a decline in transposon endo siRNA
abundance, presumably due to a decrease in the levels of transposon
dsRNA available for Dcr-2 cleavage. Importantly, Dcr-2 depletion,
which also reduces endo siRNA production and increases transposon RNA levels, did not result in a reduction in the transposon
antisense RNA transcripts (Fig. 4C).
Two different P-element insertions in the D-elp1 gene are both
late larval lethals, indicative of maternally loaded D-elp1 protein:
Lipardi and Paterson
297
D-elp1 null
normalized qRT-PCR RNA levels
297
normalized qRT-PCR RNA levels
transposon :
RNA
mdg1
mdg1
Fig. 4.
D-elp1 plays a role in transposon
suppression. Transposon sense and antisense
RNA abundance levels were determined in S2
cells and in Canton S and D-elp1 null larvae
using quantitative RT-PCR (12, 32). (A) S2 cells
were treated with dsRNA against Dcr-2, Delp1 (D-elp1.A; D-elp1.B), D-elp2, D-elp3. RTPCR assays were performed three times independently with the error bars representing
the average value. (B) Endogenous siRNAs
(endo-siRNA) from transposons 297 and mdg1
were detected with sense and antisense
probes (SI Text). Rrp49 was used as loading
control. (C) Quantitative RT-PCR was perfomed to determine the abundance of antisense transcripts for transposons 297 and
mdg1 as in (A). (D) RNA abundance levels for
transposons 297, mdg1, and Het-A were determined in Canton S wild-type and D-elp1
null larvae using quantitative RT-PCR as described. (E) Quantitative RT-PCR was used to
determine that the loss of D-elp1 gene function in the fly larvae reduced antisense RNA
transcripts for transposon 297 and mdg1.
P-element insertion [l (3) 4,629] (34), and a Gypsy P-element
insertion targeted to the coding region, obtained from the Harvard
Exelixis collection (pBac [PB] aCG10350 c00296). When either
P-element insertion was placed over a deficiency that removes
D-elp1 [Df (3R) Exel6276, Bloomington 7743] or over one another,
the phenotype was identical i.e., late larval lethal. In the D-elp1
null, the third instar larvae never form pupae but wander in the
food for several days. The null larvae are viable and therefore can
be analyzed for transposon RNA levels. Similar to the D-elp1
knockdown in S2 cells, RNAs for mdg1, 297 and HetA transposons
were increased substantially in D-elp1 null larvae while the corresponding transposon antisense RNA transcripts were reduced (Fig.
4 D and E). Based upon these results with representative transposon RNAs, we conclude D-elp1 plays an important role in transposon regulation in S2 cells and during Drosophila development.
Elp-1-Related Proteins Are Conserved in All Eukaryotes. Amino acid
sequence comparisons (Fig. S4) show both S. pombe iki-3 and
human elp-1 proteins have ⬃45–50% similarity to D-elp1 across the
entire protein, while the S. cerevisae and C. elegans proteins have
significant regions of nonhomology. There are no conserved amino
acid domains between the elp1 family members and the canonical
cellular RdRPs, particularly the highly conserved DXDGD amino
acid sequence required for activity of the cellular RdRPs (35). We
have recently produced recombinant S. pombe iki-3, C. elegans
elpc-1, and human IKAP and all three have RdRP activity, suggesting there is a conservation of an alternative RdRP pathway in
most eukaryotes involving the elp1-related proteins. Recombinant
S. cerevisiae elp-1 was not active in our assay, consistent with the lack
of the RNAi machinery in budding yeast. However, we cannot
exclude the possibility that other factors may explain the inactivity
of S. cerevisiae elp-1. Both S. pombe and C. elegans have classical
cellular RdRPs, Rdp-1 and rrf1, respectively, as well as elp-1
homologs with RdRP activity indicating that the two enzyme
activities are not mutually exclusive and may have nonoverlapping
functions. A recently published genetic interaction screen in S.
pombe indicates that the double null for Dicer and iki3, the elp-1
homolog in S. pombe, is synthetically sick/lethal indicating there is
genetic cross talk between the RNAi and elongator functional
modules, in support of our findings (36). We speculate that the
PNAS Early Edition 兩 5 of 6
BIOCHEMISTRY
297 sense
21-23 bp
normalized qRT-PCR RNA levels
C
Dcr-2
D-elp1.A
normalized qRT-PCR RNA levels
dsRNA:
GFP
B
A
functional conservation among these proteins points to a common
role in transposon regulation and genome defense.
Elongator genes are essential in metazoans and the D-elp1
(CG10350) loss-of-function is a late larval lethal, as discussed above
(16, 34). In C. elegans, RNAi knockdown of elpc-1 is embryonic
lethal (wormbase.org) and a recent study using IKBKAP ⫺/⫺
mouse embryos reports loss of IKBKAP is an embryonic lethal as
well (11). In the mouse IKBKAP ⫺/⫺, the phenotype could be
rescued by the human gene. Just D-elp1 itself has been reported to
be involved in Drosophila immunodefence (flybase.org) and is
upregulated along with Dcr-2, Ago-2 and Ars-2 in S2 cells depleted
for the UPF proteins that are involved in nonsense-mediated decay
(32). This reinforces the interpretation that D-elp1 has separate
functions outside the elongator complex and is involved with
components of the RNAi machinery.
Mutations in the human elp-1 gene, also known as the IKBKAP
gene that encodes the IKAP protein, are correlated with a recessive
hereditary neuropathy common in the Ashkenazi Jewish population, called familial dysautonomia (FD), or the Riley-Day syndrome
(16). The most prominent mutation, an intronic noncoding point
mutation (T to C) in intron 20 that disrupts splicing and results in
the variable skipping of exon 20, is found in all FD patients.
However, the molecular basis for the disease has not been determined and has been attributed to defects in tRNA modification,
defects in transcription, and disruption of the cytoskeleton and cell
motility (16, 37). If truncated IKAP lacks or has reduced RdRP
activity, or fails to interact with components of the RISC, particularly Dcr-2, our findings suggest this could affect the RNA
silencing pathway and the normal level of post transcriptional gene
regulation, thus providing a possible alternative explanation for
neuronal cell death. Deletion studies are in progress to determine
if either the D-elp1 or human IKAP FD deletions have RdRP
activity and can interact with Dcr-2.
In summary, we have shown that Drosophila RNA polymerase II
elongator complex Subunit I, D-elp1, is noncanonical RdRP that
plays a role in both dsRNA and siRNA mediated RNAi, as well as
transposon suppression by modulating the levels of transposon
dsRNA and the corresponding endo siRNAs in the later instance.
This outcome highlights the multifunctional role of elongator in
RNAi, genome defense and transcription. Although earlier reports
concluded transitive and systemic RNAi did not occur in Drosophila, a recent study has clearly shown that antiviral immunity in flies
requires systemic RNAi spreading that involves a dsRNA uptake
pathway. Furthermore, protection against viral infection could be
established by pretreatment with very low doses of viral dsRNA,
similar to immunity in plants and C. elegans (6, 38). This means
dsRNA has to spread in the absence of viral infection. Earlier
studies have also shown that low levels of injected dsRNA induced
RNAi in adult flies (39, 40). The role identified here for D-elp1 as
an RdRP involved in transposon regulation suggests spreading via
the production of either unprimed or siRNA primed dsRNA
synthesis could mediate this process. Production of dsRNA from
aberrant RNAs through a similar mechanism may also complement
and facilitate Ago-2 mediated RNAi in higher eukaryotes under
certain conditions of cellular stress.
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6 of 6 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0904984106
Materials and Methods
Flag tagged recombinant D-elp1 and Dcr-2 were produced in Sf9 cells using the
Bac-to-Bac baculovirus system according to the manufacturers (Invitrogen). Delp1 containing an amino terminal 6-His and a carboxyl terminal Flag tag in Pet
28 was expressed in E. coli using either Rosetta BL21-LysS cells (Novagen) or BL21
CodonPlus (DE3)-RIPL (Stratagene) (22). The recombinant D-elp1 proteins were purified as described in the SI Text and stored in 10% glycerol at ⫺80 °C. The enzymatic
assays and nearest neighbor analysis were performed as described (8, 20).
S2 cell transfection, Western analysis protocols, and the luciferase assay are
described in SI Text. Flag tagged D-elp1 and V5 tagged Ago2, Dicer-1 and Dcr-2
cDNAs were cloned into pIZT/V5-His (invitrogen) for expression in S2 cells. RNA
expression levels for transposons 297, mdg1, and Het-A were measured by quantitative RT-PCR in S2 cells using the primers and protocol described previously (12, 32).
SI Text including full methods, Tables S1–S3, and any associated references are
available in the online version of the paper.
ACKNOWLEDGMENTS. We thank S. Grewal and M. Lichten for comments and
the National Cancer Institute confocal facility.
Lipardi and Paterson