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A potential link between transgene silencing and poly(A) tails
MIKIKO C. SIOMI, HIROKO TSUKUMO, AKIRA ISHIZUKA, TOMOKO NAGAMI and HARUHIKO SIOMIRNA 2005 11: 1004-1011
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REPORT

A potential link between transgene silencing
and poly(A) tails
MIKIKO C. SIOMI, HIROKO TSUKUMO, AKIRA ISHIZUKA, TOMOKO NAGAMI, and HARUHIKO SIOMI
Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan

ABSTRACT
Argonaute proteins function in gene silencing induced by double-stranded RNA (dsRNA) in various organisms. In Drosophila,
the Argonaute proteins AGO1 and AGO2 have been implicated in post-transcriptional gene-silencing (PTGS)/RNA interference(RNAi). In this study, we found that AGO1 and AGO2 depletion caused the accumulation of multicopied enhanced green
fluorescence protein (EGFP) transgene transcripts in Drosophila S2 cells. Depletion of AGO1, the essential factor for miRNAbiogenesis, led to an increased transcriptional rate of the transgenes. In contrast, depletion of AGO2, the essential factor for
siRNA-directed RNAi, resulted in EGFP mRNA stabilization with concomitant shortening of the EGFP mRNA poly(A) tail. Ourfindings suggest that AGO1 and AGO2 mediate multicopied transgene silencing by different mechanisms. Intriguingly, Dicer2
depletion phenocopies AGO2 depletion, with an increase in EGFP protein levels and shortening of the EGFP mRNA poly(A) tail.
The possibility that AGO2 and Dicer2 involve, at least in part, poly(A) length maintenance of transgene mRNA suggests a
potentially important link between transgene silencing and poly(A) tails.
Keywords: argonautes; TGS; PTGS; poly(A); Drosophila

INTRODUCTION
Double-stranded (ds) RNA induces sequence-specific posttranscriptional gene silencing (PTGS) of cognate genes in awide range of organisms (Meister and Tuschl 2004; Melloand Conte 2004; Sontheimer 2005). The ribonuclease III
enzyme Dicer converts long dsRNA to short interfering
RNAs (siRNAs) of 21–23 nucleotides (nt) in length (Bernstein
et al. 2001; Ketting et al. 2001; Knight and Bass 2001), whichare, in turn, unwound and loaded onto an RNA-induced
silencing complex (RISC), an enzymatic complex thatmediates cleavage of the mRNA targets depending on thesequence of the siRNA molecule within the complex.
This process is known as RNA interference (RNAi) (Fireet al. 1998). RNAi is not the only PTGS triggered by dsRNAs
in vivo. MicroRNAs (miRNAs) are a class of endogenously
encoded small noncoding regulatory RNAs that are processed from hairpin-type precursor RNA transcripts by

Reprint requests to: Mikiko C. Siomi, Institute for Genome Research,
University of Tokushima, Tokushima 770-8503, Japan; e-mail: siomim@
genome.tokushima-u.ac.jp.
Article and publication are at http://www.rnajournal.org/cgi/doi/
10.1261/rna.2280105.

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a22801

Dicer (Ambros 2004; Bartel 2004). miRNAs negatively control translation of mRNA targets containing sequences thatare imperfectly complementary to them (Ambros 2004;
Bartel 2004). Many factors that function in small RNAmediated PTGS have been identified in various organisms.
Among these, members of the Argonaute family of proteins
are the most extensively studied (Carmell et al. 2002). In
Caenorhabditis elegans, genetic analyses suggest that RDE-1,
a member of the Argonaute family, is required for the
initiation of RNAi, whereas Alg-1 and Alg-2, other membersof the family, are required for the accumulation of stablemature miRNAs, but not for RNAi driven by dsRNA(Grishok et al. 2001). In cultured Drosophila S2 cells,
AGO2 was identified as a core component of biochemicallypurified RISC (Hammond et al. 2001). Further, it has beenshown that embryos lacking AGO2 are siRNA-directed
RNAi defective, but are still capable of miRNA-directed
target RNA cleavage (Okamura et al. 2004). In contrast,
AGO1 is dispensable for siRNA-directed RNA cleavage, but
is necessary for the accumulation of stable mature miRNAs,
thus impacting on miRNA-directed target RNA cleavage.
Recently, human AGO2 was shown to be the small RNAguided endonuclease that cleaves the mRNA targets in RNAi
(Liu et al. 2004; Song et al. 2004). In contrast, human AGO1,

RNA (2005), 11:1004–1011. Published by Cold Spring Harbor Laboratory Press. Copyright ª 2005 RNA Society.

Siomi et al.

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Transgene silencing and poly(A) tails

AGO3, and AGO4 did not show such target cleavage activity(Liu et al. 2004; Meister et al. 2004). These findings indicatethat the Argonautes are not functionally redundant.
It has been also shown that some of the Argonaute family ofproteins direct transcriptional gene silencing (TGS) inducedby dsRNAs complementary to DNA sequences in the nucleus.
For instance, Ago1, the only member of the Argonaute infission yeast, is required for silencing transcription at centromeres (Volpe et al. 2002) and initiation of silent heterochromatin at the mating-type locus (Hall et al. 2002), whichsuggests that the RNAi mechanism is closely related to thatof TGS. Very recently, Ago1 was shown to be necessary not
only for TGS, but also for the RNAi pathway in fission yeast(Sigova et al. 2004). Genetic studies in plants have likewisedemonstrated that the RNAi mechanism
is closely related to that of TGS (Finnegan
and Matzke 2003; Baulcombe 2004). In
Arabidopsis thaliana, Ago1 is involved in
RNAi and miRNA-directed gene silencing
pathways (Vaucheret et al. 2004), whereas
Ago4 is implicated in guiding the chromatin silencing pathway (Zilberman et al.
2003; Chan et al. 2004).
Although for many years transgene
silencing was thought to be a phenomenon unique to plants, recent genetic
studies have shown that it also occurs
in Drosophila (Birchler et al. 2003).
piwi, a member of the Argonaute family,is necessary for post-transcriptionaltransgene silencing and some aspectsof transcriptional transgene silencing(Pal-Bhadra et al. 2002, 2004). aubergine(a.k.a. sting), another member of thefamily, is responsible for acting post-transcriptionally to maintain silencing of the
X-linked repetitive Stellate locus that isnecessary for male fertility (Livak et al.
1990, Schmidt et al. 1999; Aravin et al.
2001, 2004) and is also necessary for some
aspects of post-transcriptional transgene
silencing (Pal-Bhadra et al. 2002). However, it is also known that transgene
silencing in Drosophila differs frommany cases in plants, in that the degreeof silencing is not as strong and seldom
reaches complete silencing (Birchler
et al. 2003). To investigate the possible
involvement of AGO1 and AGO2 intransgene silencing in Drosophila-cultured S2 cells, we have sought to determine the effects of these Argonaute
proteins on the expression of multicopied enhanced green fluorescence protein (EGFP) transgenes in S2 cells.

RESULTS AND DISCUSSION
Depletion of AGO1 or AGO2 by RNAi results inthe accumulation of multicopied EGFP transgene
transcripts in S2 cells
We produced an S2 cell line stably expressing EGFP (S2EGFP) and verified by Southern blot analysis that EGFP
transgenes are present in 20 copies per genome in thiscell line (data not shown). Although little is known abouttransgene silencing in cultured cells, the presence of multiple
copies of EGFP was expected to trigger PTGS of the EGFP
transgenes. Therefore, we asked whether AGO1 or AGO2, or
both, was involved in silencing of the EGFP transgenes.

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RNAi-based reverse-genetic methods have been widely
applied to study gene function in S2 cell cultures (Boutros
et al. 2004). AGO2 depletion from S2-EGFP cells by RNAi
caused an approximately twofold accumulation of EGFP
protein compared with control (Fig. 1A). In contrast,
AGO1 depletion did not cause significant accumulationof EGFP protein. These experiments were repeated fourtimes and the quantitative data are shown in Figure 1A.
We then determined whether the levels of EGFP mRNA
were affected upon depletion of AGO1 or AGO2 in S2
cells. We carried out Northern blot analysis on total RNAs
from S2-EGFP cells and found that the steady-state concentration of EGFP mRNA was increased approximately
threefold upon depletion of AGO1 or AGO2 (Fig. 1B). In
the case of EGFP mRNA transiently expressed by transfection, depletion of AGO1 or AGO2 did not cause any
significant change in EGFP transcript levels (Fig. 1C).
These findings suggest that AGO1 and AGO2 functionin the silencing of multicopied EGFP transgenes.
AGO1 and AGO2 depletion both caused the accumulation of EGFP mRNA (Fig. 1B). On the other hand, EGFP
protein accumulation was only observed in AGO2-depleted
cells (Fig. 1A). From these results, it could be speculated
that the depletion of AGO1 in some way affects the nuclear
and cytoplasmic distribution of EGFP mRNA. Thus, we
performed Northern blot analysis on total RNAs isolated
separately from the nucleus and the cytoplasm of AGO1and AGO2-depleted cells. The relative amounts of the
EGFP transcripts in the cytoplasmic fraction to those in
the nuclear fraction were calculated; they were approximately one and a half fold in both cases (Fig. 1D), indicating that EGFP mRNA distribution in the cellular com-

partments is the same in both cases. Why increased EGFPmRNA does not result in increased production of EGFP
protein in AGO1-depleted cells is unclear.
AGO2 depletion by RNAi leads to poly(A)shortening of the EGFP transcripts

Intriguingly, we noticed that when AGO2 was depleted, EGFP
transcripts were shorter by 100 nucleotides (nt) (Fig. 1B). In
contrast, AGO1 depletion did not affect the size of mRNA. In
the case of an endogenous gene, ribosomal protein P1(RpP1), the size of the transcripts was not altered upon
depletion of AGO1 or AGO2 (Fig. 1B). The size of EGFPmRNA transiently expressed in S2 cells was also not affectedby either AGO1 or AGO2 depletion (Fig. 1C), suggesting thatmRNA shortening is specific for transgenes. We hypothesizedthat poly(A) tails of EGFP transcripts might be shortened in
the AGO2-depleted cells. To test this, we isolated total RNAs
from cells depleted of AGO1 or AGO2 and examined thepoly(A) length of EGFP transcripts by RNaseH cleavage assay(Fig. 1E). When AGO2 was depleted, the poly(A) tails of
EGFP transcripts were indeed shorter by 100 bases thanthose present in control and AGO1-depleted cells. In contrast,
the poly(A) length of tubulin mRNA were not altered in
AGO2-depleted cells (Fig. 1E, right). In most of the cases,
the length of poly(A) tails determines the translational statusof a regulated transcript (for review, see Parker and Song2004), such that translational silencing correlates withpoly(A) shortening. In the case of EGFP transgene transcripts
in AGO2-depleted cells, however, the poly(A) shortening did
not down-regulate the translation; rather, EGFP protein
expression level was observed to be higher than that in controls (Fig. 1A). This is, admittedly, a rareexample of poly(A) shortening, leading
FIGURE 1. Depletion of members of the Argonaute family of proteins by RNAi causes an increase to increased protein expression, although
in expression of multicopied EGFP transgenes. (A) S2-EGFP cells were transfected with double- there are precedents; for example, in
stranded RNAs (dsRNAs) corresponding to the indicated cDNAs (AGO1 and AGO2). Four days mice, the activation of translation oflater, cells were harvested and the expression of EGFP protein analyzed. Values shown represent theexpression of EGFP protein in AGO1 or AGO2 dsRNA-transfected cells relative to EGFP protein some mRNA during spermiogenesis has
expression from cells transfected with control dsRNA. Four independent experiments were per- been shown to be accompanied byformed. Expression levels of ribosomal protein P1 (RpP1) were also checked as a control. (B) poly(A) shortening (Kleene 1989).
Northern blots show that depletion of AGO1 or AGO2 causes the accumulation of EGFP transcripts. Values shown represent the expression of EGFP mRNA in AGO1 or AGO2 dsRNAtransfected cells relative to EGFP mRNA expression from cells transfected with control dsRNA.
Three independent experiments were performed. EGFP mRNAs are 100 nt shorter, specificallywhen AGO2 is depleted. The levels of the RpP1 transcripts are also shown as a control, indicatingthat both amount and length of RpP1 mRNA are not altered upon depletion of AGO1 or AGO2.
(C) Northern blots show that the levels of transiently expressed EGFP mRNA do not significantly
change upon depletion of AGO1 or AGO2. The levels of RpP1 mRNA are shown as a control. (D)Northern blots show the nuclear and cytoplasmic distribution of EGFP mRNA. EGFP mRNA levelsin the cytoplasmic fractions relative to those in the nuclear fractions are 1.52- and 1.53-fold in
AGO1- and AGO2-depleted cells, respectively. Total RNAs (5 mg) prepared either from the nuclear
or the cytoplasmic fraction were applied per lane. 5S rRNA is shown as an internal control. Western
blots (bottom ) show that lamin is only observed in the cytoplasmic fraction and cytochrome c onlyin the nuclear fraction, indicating that both fractions were well separated from each other. (E) AGO2
depletion leads to poly(A) shortening of the EGFP transcripts. The length of the EGFP mRNApoly(A) was compared under conditions with or without AGO2 expression. Northern blots after
treatment of total RNAs with RNaseH and a specific oligo DNA hybridizing to EGFP 30 UTR
sequence show that the poly(A) tails are shorter by an 100 nt, but only in AGO2-depleted cells(left). The length of tubulin mRNA poly(A) tail was not altered upon AGO2 depletion (right).

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AGO1 and AGO2 mediate transgene
silencing by different mechanisms
There are at least two possibilities
explaining the observed accumulation of
EGFP mRNA upon depletion of AGO1
or AGO2, namely, an increase in thetranscriptional rate or an increase inmRNA stability. To distinguish these,
we first examined the levels of EGFPmRNA by quantitative Northern blotting
in AGO1- or AGO2-depleted cells after
treatment with Actinomycin D (to inhibit synthesis of nascent transcripts). Only

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Transgene silencing and poly(A) tails

AGO2 depletion led to the stabilization of EGFP mRNA
(Fig. 2A). Tubulin mRNA stability was, on the other hand,
not affected by AGO2 depletion (Fig. 2A). These results
indicate that AGO2 is involved in destabilizing EGFP transgene transcripts in the stable cell line. The stabilization of
EGFP mRNA upon AGO2 depletion might explain the mild
increase in EGFP protein observed in AGO2-depleted cells.
We also measured the nuclear transcriptional rate of EGFP
transgenes by nuclear run-on experiments in cells where
AGO1 or AGO2 was depleted. AGO1 depletion, but notthat of AGO2, led to an increase in EGFP transgene transcription levels (Fig. 2B), suggesting that AGO1 is involved in TGSof the EGFP transgenes in S2 cells.
It has been shown that siRNAs guide heterochromatin
formation or DNA methylation of cognate DNA sequences,result-ing in transcriptional silencing in plants, fission
yeast, and human (Baulcombe 2004; Lippman andMartienssen 2004). In plants, heterochromatin formation
has been shown to be strongly related to DNA methylation
at the promoter regions (Soppe et al. 2000; Matzke et al.
2003; Chan et al. 2004). The clear difference between plants
and S2 cells in transgene silencing is the degree of thesilencing; in plants, the expression is completely silenced,
whereas in S2-EGFP cells, the silencing degree is so low that
the EGFP expression is still partly apparent. This could be

due to the low amount of EGFP antisense transcripts. In
agreement with this, we could not detect EGFP antisensetranscripts by Northern blot analysis in S2-EGFP cells (datanot shown). However, we were able to detect the presence of
EGFP antisense transcripts in S2-EGFP cells by RT–PCR(Fig. 2C), which suggests that duplex forms of EGFP transcripts could exist in S2-EGFP cells. We found that methylation of the EGFP promoters in S2-EGFP cells was quite scarceby bisulfite DNA sequencing analyses (data not shown).
Therefore, unlike in plants, DNA methylation does notappear to participate in TGS of the EGFP transgenes in
S2 cells. It is known that heterochromatin negatively effects
gene expression (Richards and Elgin 2002), and that the formation and maintenance of heterochromatin is characterized byaddition of specific modifying groups of histones, which areconsequently bound by heterochromatin protein 1 (HP1)(Maison and Almouzni 2004). We found that EGFP transgenes are heterochromatinized at the promoter areas as
judged by heterochromatin immunoprecipitation assay
using anti-HP1 antibody (Fig. 2D). However, we were unable
to detect any significant change in heterochromatin for-FIGURE 2. AGO1 and AGO2 are involved in silencing of EGFP transgenes in two ways. (A) AGO2 functions in PTGS of the transgenes. S2EGFP cells were first treated with dsRNAs corresponding to AGO1 or
AGO2 cDNAs. Four days later, transfected cells were treated with ActinomycinD. At the times indicated, total RNAs in each case were isolated
and EGFP transcripts analyzed by Northern blot analyses. Ribosomal
RNAs (rRNA) stained with ethidium bromide are shown as a loading
control. A rapid decrease of the EGFP mRNA was observed in AGO1depleted cells, as in the control. In contrast, the rate of decrease wasmuch slower in AGO2-depleted cells, indicating that the EGFP transcripts are stabilized upon AGO2 depletion. Quantitation of the RNA
bands by a PhosphorImager from three independent experiments isshown below. Tubulin mRNA levels were not altered upon AGO2
depletion in contrast to EGFP mRNA levels. (B) Nuclear run-on showsthat an increase in transcription of the EGFP transgenes was observed
only upon AGO1 depletion. Four days after dsRNA treatment, cells were
harvested and the nuclei isolated. Transcription was then performed innuclei in the presence of [32P]UTP. The data shown below are theaverage 6 standard deviation for three trials. No increase in transcriptionof tubulin was observed when AGO1 was silenced by RNAi. Tubulin
mRNA levels are shown as an internal control. (C) Detection of EGFP
antisense transcripts in S2-EGFP cells. EGFP antisense transcripts were
not detected in S2-EGFP cells by Northern blotting (data not shown).
RT–PCR analysis, however, reveals that EGFP antisense transcripts areindeed present in S2-EGFP cells, suggesting that EGFP dsRNAs may exist
in S2-EGFP cells. RT–PCR was also performed for tubulin as an internal
control. (D) Heterochromatin formation at the EGFP promoter region.
Dot-blot hybridization was performed on DNA fragments isolated fromthe immunoprecipitated complexes with anti-HP1 antibody (right). The
probe used contains the sequence corresponding to the EGFP promoter.
(n.i.) nonimmune IgG shows the background level of this experiment.
Western blot using anti-HP1 (left) shows that anti-HP1, but not n.i.,
specifically immunoprecipitates HP1 protein. (h.c. and l.c.) Protein
bands corresponding to IgG heavy and light chains, respectively.

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mation at the EGFP promoter region upon AGO1 depletion(data not shown).

Dicer2 depletion phenocopies AGO2 depletion
Drosophila Dicer2 and AGO2 act in concert in the RNAi
pathway, while Dicer1, together with AGO1, is primarily
involved in miRNA processing, and consequently, inmiRNA function (Liu et al. 2003; Lee et al. 2004; Okamura
et al. 2004). We sought to determine whether depletion of
Dicer1 or Dicer2 by RNAi could also affect expression of the
EGFP transgenes. Depletion of Dicer2, but not Dicer1,
resulted in accumulation of EGFP protein (Fig. 3A), as was
the case for depletion of AGO2 (Fig. 1A). Both Dicer1 andDicer2 depletion caused the accumulation of EGFP mRNA
(Fig. 3B), but poly(A) tail shortening was observed only inDicer2-depleted cells (Fig. 3B).
The CCR4–NOT complex is known to be the major
enzyme catalyzing mRNA deadenylation in Saccharomyces
cerevisiae (Denis and Chen 2003). The fly homologs of thecatalytic subunits of the complex, CCR4 and CAF1, wereshown to be associated with a cytoplasmic poly(A)-specific30 exonuclease activity, and RNAi knockdown of each ofthese genes led to a lengthening of bulk mRNA poly(A)tails (Temme et al. 2004). Thus, we performed double depletion of AGO2 and CAF1, or Dicer2 and CAF1 in S2-EGFP
cells, and examined the poly(A) length of EGFP transcripts.
Depletion of AGO2 and CAF1, or Dicer2 and CAF1, led to
the restoration of the length of the EGFP transcript poly(A)tails (Fig. 3C). These findings imply that AGO2 and Dicer2
protect poly(A) tails of EGFP mRNA from being shortened
by deadenylase activity in vivo.

EGFP mRNA stabilization upon depletion of AGO2 isaccompanied by shortening of the EGFP poly(A) tail (100
nt shorter). Increased EGFP mRNA does not appear toresult in increased production of EGFP protein in AGO1depleted S2-EGFP cells. However, in AGO2-depleted cells,
EGFP protein levels were increased as EGFP mRNA. The
nuclear and cytoplasmic distributions of EGFP mRNA in
both cases are the same. It is speculated that the length ofthe poly(A) tail may directly correlate with the expression
levels of EGFP protein. Interestingly, Dicer2 depletion phe-Conclusion
Transgene silencing has been observed in a wide variety oforganisms, including plants, fungi, nematodes, and Drosophila melanogaster (Birchler et al. 2003). Our findings clearlyshow that the presence of multiple copies of EGFP triggers
PTGS and probably TGS of the EGFP transgenes in cultured
S2 cells. Both AGO1 and AGO2 were observed to functionin the silencing of the EGFP transgenes, but their mechanisms of action clearly differ from each other. A generalmodel has been proposed for heterochromatin formationand epigenetic gene silencing in different species (Lippman
and Martienssen 2004). We found that EGFP transgenes areheterochromatinized at the promoter areas, indicating thatthe model also fits well to S2 cultured cells. Nuclear run-on
assays show an increase in EGFP mRNA only in AGO1depleted cells, but no differences in EGFP mRNA turnover.
Our failure to detect any significant change of heterochromatin formation at the EGFP promoter areas upon AGO1
depletion could reflect a loss of heterochromatinization at
only a few of the multicopied EGFP promoters.
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FIGURE 3. Dicer2 depletion phenocopies AGO2 depletion. (A) Western blots show that depletion of Dicer2, but not Dicer1, causes theaccumulation of EGFP protein. S2-EGFP cells were transfected withdsRNAs corresponding to the indicated cDNAs. Three days later, cells
were harvested and the expression of EGFP protein analyzed byWestern blotting. Values shown represent the expression of EGFP
protein in Dicer1 or Dicer2 dsRNA-transfected cells relative to
EGFP protein expression from cells transfected with control dsRNA.
Three independent experiments were performed. (B) Dicer2 depletion
phenocopies AGO2 depletion, with an increase in EGFP mRNA levels
and poly(A) shortening. Of note, is that an increase in EGFP mRNAlevels was observed in both Dicer1-depleted and Dicer2-depleted cells.
Tubulin mRNA levels are also shown as an internal control. (C)Depletion of CAF1, a component of the mRNA deadenylation complex, together with depletion of AGO2 or Dicer2 caused the restoration of the poly(A) length of the EGFP transcripts.

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Transgene silencing and poly(A) tails

nocopies AGO2 depletion, with an increase in EGFP protein levels and shortening of EGFP mRNA poly(A) tails. In
flies, Dicer2 and AGO2 are part of a holo-RISC and act inconcert in the processing and function of siRNAs derived
from dsRNA (Liu et al. 2003; Lee et al. 2004; Okamura
et al. 2004). Thus, our findings raise the possibility that inaddition to their role in RNAi, AGO2 and Dicer2 alsofunction in PTGS of multicopied transgenes and in protection of the poly(A) tails from being shortened. The precise
mechanisms involved in the shortening of the poly(A) tails
of EGFP transcripts upon depletion of AGO2 or Dicer2 areunclear. Whatever the mechanisms might be, our findings
provide a functional connection between RNAi complexes
and enzymes that mediate poly(A) tail modifications.
Recent findings in fission yeast and C. elegans also appear
to point to a potentially important link between an RNAimediated process and poly(A) tails. RNAi-mediatedheterochromatin assembly in fission yeast requires theRNA-induced transcriptional silencing (RITS) complex
that interacts with a complex termed RNA-directed RNA
polymerase complex (RDRC) containing Cid12, a memberof the poly(A) polymerase family, in a manner that requires
Dicer (Motamedi et al. 2004). The C. elegans gene rde-3,
which is required for siRNA accumulation and for efficient
RNAi in all tissues, also encodes a member of the poly(A)polymerase family (Chen et al. 2005).

MATERIALS AND METHODS
Antibodies and Western blot analyses
Anti-EGFP antibody was described previously (Okamura et al.
2004). Anti-RpP1 antibody was a kind gift from T. Uchiumi(Niigata University). Anti-HP1 antibody, C1A9, was purchasedfrom the Developmental Studies Hybridoma Bank of the University of Iowa. Antibody against cytochrome c was purchased fromBD Biosciences Pharmingen. Ant-lamin (lamin C) was a kind giftfrom K. Furukawa (Niigata University). Western blot analyses
were performed as described in Ishizuka et al. (2002).

Northern blot analyses and RT–PCR
Northern blotting was performed as described in Ishizuka et al.
(2002). For EGFP, the full-length open reading flame (ORF) wasused as a probe. In Figure 1B, dsRNA of AGO1 or AGO2 wasintroduced into S2-EGFP cells by soaking for depletion, andNorthern blotting was performed using 1.5% agarose gels. Total
RNAs used was 5 mg/lane in each case. In Figure 2A, RNAi for
AGO1 or AGO2 was performed for 4 d, and the cells were treatedwith ActinomycinD at 5 mg/mL. At each of the indicated times,
total RNAs were isolated and Northern blotting performed. Total
RNAs used were 5 mg/lane in each case. RT–PCR for detecting
EGFP antisense and tubulin was performed using 3 mg of total
RNAs isolated from S2-EGFP or parental S2 cells and treated with
DNase. The RT–PCR kit (ProSTAR Ultra-HF RT-PCR system)

was purchased from Stratagene. The primer used for RT for
detecting EGFP antisense was 50 -GCTGTTCACCGGGGTGGTGC
CC-30 . For tubulin, oligo d(T) was utilized for RT reaction. For
fractionating the nuclear and the cytoplasmic fractions, S2-EGFP
cells were suspended into Lysis buffer (30 mM HEPES at pH 7.3,
2 mM MgOAc, 5 mM DTT, 2 mg/mL Leupeptin, 2 mg/mL Pepstatin, and 0.5% Aprotinin) and lysed by passing through a 30G
needle. After centrifugation, the supernatant (cytoplasmic fraction) and the precipitate were separated. The pellet was washedtwice with Lysis buffer and lysed by sonication in Lysis buffer
containing 100 mM KOAc and 20% glycerol. After centrifugation,
the supernatant was collected and used as the nuclear fraction.
RNAs were isolated from both fractions using ISOGEN LS (Nippon
Gene).

HP1 immunoprecipitation and dot-blot hybridization
For cross-linking, S2-EGFP cells were first incubated for 10 min in
medium containing 1% formaldehyde. The cells were then washedtwice with PBS, resuspended in a buffer containing 50 mM Tris-HCl(pH 8.0), 10 mM EDTA, 1% SDS, 2 mg/mL leupeptin, 2 mg/mL
pepstatin, and 0.5% aprotinin, and then sonicated on ice. Aftercentrifugation at 4 C, the supernatant was subjected to immunoprecipitation with anti-HP1 or nonimmune IgG on Gamma Bind
beads (Amersham Bioscience). Prior to immunoprecipitation, thesupernatant was diluted with a buffer containing 20 mM Tris-HCl(pH 8.0), 1 mM EDTA, 1% Triton X-100, 170 mM NaCl, 2 mg/mL
leupeptin, 2 mg/mL pepstatin, 0.5% aprotinin and DNA carriers.
After extensive washing, the beads were treated with a buffer
containing 0.1 M NaHCO3 and 1% SDS. The eluates were thentreated with NaCl to reverse the cross-link. All of the contained
protein was digested with proteinase K; DNAs were then precipitated after being extracted with phenol/CHCl3. A portion of the
DNA solution obtained was immobilized on Hybond-N+ nylon
membranes and probed with a specific DNA fragment recognizing
the EGFP promoter. Western blotting analysis was performed
using anti-HP1 antibody to show that anti-HP1, but not nonimmune IgG precipitated HP1 protein as expected.

Nuclear run-on assays
Nuclear run-on assay was performed as described (Hirayoshi and Lis
1999). Briefly, 4 d after dsRNA treatment, the cells were harvested
and the nuclei isolated. Transcription was then performed in the
nuclei in the presence of [32P]UTP. After reaction, all transcripts
were isolated from nuclei and probed with both EGFP and tubulin
cDNA fragments (both being full-length ORF) immobilized on
Hybond-N+ nylon membranes (Amersham Bioscience).

Poly(A) length determinations
Four days after dsRNA treatment, total RNAs were isolated fromthe cells and 15 mg of each sample were incubated with an oligo
DNA hybridizing to EGFP 30 UTR (130 nt upstream of polyadenylation signal). After incubation, samples were treated with
RNaseH enzyme for 30 min at 37 C. The resultant RNAs wereseparated on 2% agarose gels and subjected to Northern blotting
using a specific probe recognizing just downstream of the cleaved
site by RNaseH. Total RNAs used in the lanes without RNaseH

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treatment were 5 mg/lane in each case. To determine the poly(A)length of tubulin mRNA, an oligo DNA hybridizing to the transcript just upstream of the stop codon was used.

ACKNOWLEDGMENTS
We thank Dr. Z. Mourelatos and Dr. J. Nakayama for many
helpful discussions, as well as members of the Siomi laboratoryfor discussions and comments on the manuscript. We acknowledge Dr. S. Kashiwabara for his valuable comments and suggestions on mRNA poly(A) length analysis, Dr. K. Hirayoshi for the
protocol of nuclear run-on, and Dr. T. Uchiumi for anti-P1 antibody. A.I. was a predoctoral fellow of the 21st century COE
program of the Ministry of Education, Culture, Sports, Science,
and Technology of Japan (MEXT) and currently is a predoctoral
fellow (DC1) of the Japan Society for the Promotion of Science(JSPS). This work was supported in part by grants to M.C.S and
H.S from MEXT and JSPS.
Received February 9, 2005; accepted April 20, 2005.

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