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Pimet, the Drosophila homolog of HEN1, mediates 2'-O-methylationof Piwi- interacting RNAs at their 3' ends
Kuniaki Saito, Yuriko Sakaguchi, Takeo Suzuki, Tsutomu Suzuki, Haruhiko Siomi and Mikiko C. Siomi
Genes & Dev. 2007 21: 1603-1608
Access the most recent version at doi:10.1101/gad.1563607

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RESEARCH COMMUNICATION

Pimet, the Drosophila
homolog of HEN1, mediates
2ⴕ-O-methylation of Piwiinteracting RNAs at their3ⴕ ends
Kuniaki Saito,1 Yuriko Sakaguchi,2Takeo Suzuki,2 Tsutomu Suzuki,2Haruhiko Siomi,1 and Mikiko C. Siomi1,3
1

Institute for Genome Research, University of Tokushima,
Tokushima 770-8503, Japan; 2Department of Chemistry
and Biotechnology, Graduate School of Engineering,
The University of Tokyo, Tokyo 113-8656, Japan

Piwi-interacting RNAs (piRNAs) consist of a germlinespecific group of small RNAs derived from distinct intergenic loci in the genome. piRNAs function in silencing selfish transposable elements through binding with
the PIWI subfamily proteins of Argonautes. Here weshow that piRNAs in Drosophila are 2ⴕ-O-methylated attheir 3ⴕ ends. Loss of Pimet (piRNA methyltransferase),
the Drosophila homolog of Arabidopsis HEN1 methyltransferase for microRNAs (miRNAs), results in loss of2ⴕ-O-methylation of fly piRNAs. Recombinant Pimetshows single-stranded small RNA methylation activity
in vitro and interacts with the PIWI proteins within
Pimet mutant ovary. These results show that Pimet mediates piRNA 2ⴕ-O-methylation in Drosophila.
Supplemental material is available at http://www.genesdev.org.
Received April 20, 2007; revised version accepted May 24,
2007.

Small, 20- to 30-nucleotide (nt)-long, RNAs trigger genesilencing mechanisms, collectively called RNA silencing, which include cleavage-dependent and -independent
mRNA decay, translational repression, DNA methylation, and heterochromatin formation in organisms
ranging from fission yeast to humans (Tomari andZamore 2005; Zaratiegui et al. 2007). RNA silencingtriggering small RNAs are processed from long precursors and are then loaded onto Argonaute proteins (Parker
and Barford 2006), guiding the Argonautes to specific sequences within the target RNAs (Tomari and Zamore
2005; Pillai et al. 2007).
Numbers of the Argonaute protein family members,
defined by the PAZ and PIWI domains, differ among species (Carmell et al. 2002); for instance, fly has five members (Williams and Rubin 2002) whereas mouse has
seven. The Argonautes can be divided into two subgroups, the AGO and PIWI subfamilies, according to sequence similarity. In Drosophila, AGO1 and AGO2 of[Keywords: piRNA; 2⬘-O-methylation; HEN1; Pimet; Drosophila]
3Corresponding author.
E-MAIL siomim@genome.tokushima-u.ac.jp; FAX 81 88 6339451.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1563607.

the AGO subfamily function in the microRNA (miRNA)
pathway and RNA interference (RNAi) by binding with
miRNAs and short interfering RNAs (siRNAs), respectively (Okamura et al. 2004; Tomari et al. 2004; Miyoshi
et al. 2005). Recent studies have revealed that PIWI proteins are specifically associated with a subset of endogenous small RNAs, termed Piwi-interacting RNAs
(piRNAs; in flies, these are also called repeat-associatedsiRNAs, or rasiRNAs), which are distinct from miRNAs
in many respects such as size, expression pattern, andeven function (O’Donnell and Boeke 2007). Unlike
miRNAs, piRNAs are 24–30 nt long, are expressed only
in gonads, and function in genome surveillance throughassociation with the PIWI proteins (for review, see Kim
2006; Saito et al. 2006; Vagin et al. 2006; Brennecke et al.
2007; Gunawardane et al. 2007; Houwing et al. 2007) bysilencing transposable elements that have sufficient potency to invade the genome by inserting themselves into
DNA elements.
Recently, a model for piRNA biogenesis in Drosophila
was proposed (Brennecke et al. 2007; Gunawardane et al.
2007). In this model, PIWI proteins determine and form5⬘ ends of piRNAs, which will then be loaded onto theother PIWI members, by cleaving their target transcripts
in a manner depending on the associated piRNA sequence. This process not only continuously generates
piRNAs but also keeps destroying target RNAs transcribed from selfish genes. Fish and mammals may have
similar mechanisms (Aravin et al. 2007; Houwing et al.
2007). However, many aspects of piRNA biogenesis in
Drosophila remain unclear; for example, protein factor(s)determining and forming the 3⬘ ends of piRNAs are not
identified. How nascent piRNAs are loaded onto theother PIWI members also has not been elucidated.
In plants, miRNAs are methylated at the 2⬘-terminalhydroxyl group on the ribose at the last nucleotide (Yu et
al. 2005). A factor responsible for such modification in
Arabidopsis is HEN1 (Yu et al. 2005). This is a protein
containing a double-stranded RNA (dsRNA)-binding domain at the N terminus and a catalytic domain at the C
terminus that transfers methyl groups from S-adenosyl
methionine (SAM) to miRNA/miRNA* duplexes. Fiveconsecutive residues (Asp–Phe–Gly–Cys–Gly) residingin the catalytic domain of Arabidopsis HEN1 correspondto the SAM-binding motif. Mutations introduced in the
SAM-binding motif abolish the methylation activity. In
the hen1 mutant, miRNA are reduced in abundance andwhen detectable are uridylated, a cause of miRNA destabilization (Li et al. 2005). It can be concluded that
HEN1 is the stabilizer of miRNAs in plants. Arabidopsis
HEN1 methylates even siRNA duplexes with perfect sequence complementarity (Yang et al. 2006), but singlestranded small RNAs and pre-miRNAs forming a hairpin
structure fail to be methylated in vitro by recombinant
HEN1 (Yu et al. 2005). It is thus postulated that miRNA
methylation by HEN1 occurs between two steps during
the miRNA maturation process; namely, after Dicer-mediated excision of miRNA/miRNA* duplexes from theprecursors and before the unwinding process of the duplexes.
piRNAs in Drosophila are modified at their 3⬘ ends, as
judged by their resistance to periodate oxidation/␤-elimination (Vagin et al. 2006; Gunawardane et al. 2007). In

GENES & DEVELOPMENT 21:1603–1608 © 2007 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/07; www.genesdev.org

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this study, we determined that piRNAs in Drosophila
are 2⬘-O-methylated at their 3⬘ ends, as in mouse and rat(Houwing et al. 2007; Kirino and Mourelatos 2007;
Ohara et al. 2007). In Drosophila, CG12367 encodes aprotein showing similarity at the peptide levels with themethyltransferase catalytic domain of Arabidopsis
HEN1. Loss of CG12367 results in loss of piRNA methylation in vivo; thus, we refer to this as Pimet (for piRNA
methyltransferase). Indeed, Aub- and Piwi-associatedpiRNAs isolated from Pimet mutant ovary showed sensitivity to oxidation/␤-elimination as oppose to those inwild-type ovary. Recombinant Pimet showed activity invitro for transferring methyl groups from SAM to singlestranded small RNAs bearing a 2⬘, 3⬘-terminal-cis-diolgroup, but not to 2⬘-O-methylated small RNAs. Aubassociated piRNAs from Pimet mutant ovary were methylated by recombinant Pimet in vitro; by contrast,
AGO1-associated miRNAs did not serve as a substrate
for the methylation. Taken together, these results showthat Pimet is the factor responsible for piRNA methylation in Drosophila germlines.
Results and Discussion
piRNAs in fly ovary are 2⬘-O-methylated
at their 3⬘ ends
piRNAs in fly ovary are resistant to periodate oxidation/
␤-elimination treatment, indicating that these piRNAs
are modified at their 3⬘ ends (Vagin et al. 2006; Gunawardane et al. 2007). In this study, similar experiments were
performed for piRNAs associated with Aub and Piwi inovary. In both cases piRNAs did not gain mobility evenafter periodate oxidation/␤-elimination treatment (Fig.
1A), indicating that they are modified at their 3⬘ ends.
Under the same conditions, miRNAs associated with

Figure 1. piRNAs associated with Aub and Piwi in fly ovary are
methylated at their 3⬘ ends. (A) piRNA associated with Aub andPiwi in fly ovary were subjected to periodate oxidation/␤-elimination chemical reactions. In both cases, piRNAs did not gain mobility, indicating that these piRNAs with Aub and Piwi were modified
at their 3⬘ ends. Under these conditions, a synthetic 21-nt small
RNA (luc guide siRNA) (Okamura et al. 2004) gained mobility bynearly 2 nt, a feature of RNAs bearing a 2⬘-, 3⬘-terminal-cis-diolgroup at the last nucleotide (Hutvagner et al. 2001). (B) miRNAs
associated with AGO1 in fly ovary show that they are not modified
at their 3⬘ ends, in contrast to plant miRNAs (Yu et al. 2005).

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AGO1 increased mobility by nearly 2 nt (Fig. 1B), a feature of RNAs bearing a 2⬘-, 3⬘-terminal-cis-diol group(Hutvagner et al. 2001). This is consistent with the factthat animal miRNAs are not modified at their 3⬘ ends
(Elbashir et al. 2001; Hutvagner et al. 2001). piRNAs expressed in mouse and rat have recently been shown to be2⬘-O-methylated at their 3⬘ ends (Houwing et al. 2007;
Kirino and Mourelatos 2007; Ohara et al. 2007). To identify the terminal modification of piRNA in Drosophila,
Aub-associated piRNAs were gel-purified and digested
by RNase T2. The digest was analyzed by capillary liquid
chromatography (LC) and mass spectrometry (MS) (Fig.
2). In this treatment, only 3⬘-terminal residues are converted to nucleosides (Ohara et al. 2007). As shown in
Figure 2B, four kinds of 2⬘-O-methyl nucleosides wereclearly observed as major products. Each proton adductwas further analyzed by MS/MS using collision-induced
dissociation (CID). Selected reaction monitoring (SRM)
for base-related product ions (Fig. 2C) and mass spectrum(Supplementary Fig. S1) of each 2⬘-O-methyl nucleosidealso confirmed that a methyl group is attached to eachribose portion. To eliminate the possibility that they
were 3⬘-O-methyl nucleosides, RNase T2-digested
piRNAs were coinjected with a series of synthetic 3⬘-Omethyl nucleosides. Each 3⬘-O-methyl nucleoside eluted
at a distinct retention time from each 3⬘-terminal
nucleoside of piRNAs (Fig. 2D). On the other hand, eachsynthetic 2⬘-O-methyl nucleoside eluted at the same retention time with its corresponding 3⬘-terminal nucleoside of piRNAs (data not shown). These results indicatethat piRNAs in flies are almost entirely methylated atthe 2⬘-hydroxyl group on the ribose of the last nucleotide.
CG12367 is involved in piRNA methylationin the ovary
In Arabidopsis, miRNAs are methylated at their 3⬘ ends
by HEN1 while still in a duplex form with miRNA*
molecules (Yu et al. 2005). HEN1 is a methyltransferasethat contains two characteristic motifs, a dsRNA-binding domain and a catalytic domain necessary for the
methyltransferase activity. The Drosophila genome possesses a single gene (CG12367) that encodes a protein
showing a strong similarity to the methyltransferase
catalytic domain of Arabidopsis HEN1 (Supplementary
Fig. S2A). CG12367 lacks the dsRNA-binding domain
found in HEN1 (Supplementary Fig. S2A). In fact, manyother HEN1 homologs in divergent organisms including
nematodes, fish, and mammals also lack dsRNA-binding
domains (Supplementary Fig. S2B; Park et al. 2002). To
assess possible roles in piRNA methylation, a CG12367
mutant fly (piggyBacf00810) containing a piggyBac insertion within the first intron of CG12367 (Supplementary
Fig. S3A) was obtained from the EXELIXIS-Drosophila
Stock Collection. The expression levels of CG12367
were checked by RT–PCR. We found that the homozygous piggyBacf00810 mutant does not express CG12367
mRNAs, but expresses transcripts of CG8878, a gene encoded in a region corresponding to the first exon of
CG12367 in the reverse orientation (Fig. 3A). piRNAs
associated with Aub and Piwi were isolated from
CG12367 mutant ovary, and oxidation/␤-elimination
was performed. Western blotting analysis of the PIWI
proteins Aub, Piwi, and AGO3 revealed that their expression levels did not seem to be changed by loss ofDownloaded from www.genesdev.org on July 3, 2007

Pimet methylates fly piRNAs

transfer methyl groups from SAM to both 3⬘ ends ofmiRNA/miRNA* duplex in vitro (Yu et al. 2005; Yang etal. 2006). We produced GST-tagged full-length Pimet recombinant protein in E. coli (Supplementary Fig. S4A)
and performed in vitro small RNA methylation assays.
GST-HEN1 was employed as a control in the assays. We
found that miR-1/miR-1* duplex (0.04 nmol/50 µL) was
methylated by GST-HEN1 in the presence of 14C-SAM(Fig. 4A), as reported. By contrast, under the same conditions, GST-Pimet did not methylate the substrate (Fig.
4A). When a 26-nt single-stranded RNA (ssRNA) was thesubstrate (0.5 nmol/50 µL), even GST-HEN1, which has
been reported to fail to methylate ssRNAs (Yu et al.
2005), showed weak activity for ssRNA methylation(Fig. 4B). GST-Pimet methylated ssRNA more efficiently
than did GST-HEN1 (Fig. 4B). GST-Pimet mutant (GSTPimet-mt) (see Supplementary Fig. S4A), in which theresidues in the SAM-binding motif were mutated,showed no methylation activity (Fig. 4B). GST-Pimet
was able to methylate small ssRNAs ranging from 22 to
38 nt in length (Fig. 4C). It thus seems that Pimet isindifferent to the length of substrates, unlike HEN1 that
hardly recognizes 25 nt or longer RNA duplexes as substrates (Yang et al. 2006). 2⬘-O-methyl modification of
26-nt ssRNAs prevented the methylation (Fig. 4D), suggesting that GST-Pimet transfers methyl groups only
onto the 2⬘-hydroxyl group on the ribose of the far 3⬘
nucleotide. Similarly, 26-nt ssRNAs phosphorylated atthe 3⬘-hydroxyl group did not serve as substrates (Supplementary Fig. S4B). Pimet seems to require the two hydroxyl groups on the 3⬘-most nucleotides of substrates,
Figure 2. piRNAs associated with Aub in fly ovary are 2⬘-O-methylated at their 3⬘ ends. (A) Total ion chromatogram (TIC) of thepiRNAs digested by RNase T2. The retention times of the observed
3⬘-phospho nucleotides (Cp, Up, Gp, and Ap) are indicated. The
expected retention times of unmodified nucleosides (C, U, G, and A)
are shown in gray. The considerable amount of adenosine observed
is due to contamination (data not shown), as in the case of mouse
piRNA MS analysis (Ohara et al. 2007). (B) Mass chromatograms for
proton adducts of piRNA 3⬘-terminal nucleosides, 2⬘-O-Me C (m/z258; black line), 2⬘-O-Me U (m/z 259; green line), 2⬘-O-Me G (m/z298; red line), and 2⬘-O-Me A (m/z 282; blue line). (C) Mass chromatograms of SRM for base-related product ions of 2⬘-O-Me C (m/z112; black line), 2⬘-O-Me U (m/z 113; green line), 2⬘-O-Me G (m/z152; red line), and 2⬘-O-Me A (m/z 136; blue line). (D) piRNAs digested by RNase T2 were coinjected with a series of synthetic 3⬘O-methyl nucleosides. Mass chromatograms for proton adducts ofpiRNA 3⬘-terminal nucleosides and a series of synthetic 3⬘-Omethyl nucleosides, 2⬘ or 3⬘-O-Me C (m/z 258; black line), 2⬘ or
3⬘-O-Me U (m/z 259; green line), 2⬘ or 3⬘-O-Me G (m/z 298; red line),and 2⬘ or 3⬘-O-Me A (m/z 282; blue line). Peaks for 3⬘-O-methyl
nucleosides are indicated by arrowheads.

CG12367 expression (Supplementary Fig. S3B). piRNAs
isolated from the mutant ovary were sensitive to␤-elimination (Fig. 3B) in contrast to those from wildtype ovary (Fig. 1A). piRNAs from PiggyBacf00810/
Df(2R)BSC40 transheterozygote ovaries also showed thesensitivity to the chemical treatment (Supplementary
Fig. S3C). These imply that CG12367 is involved inpiRNA methylation; thus, being Pimet.
Recombinant Pimet shows activity to methylate
single-stranded small RNAs in vitro
It has been demonstrated that recombinant GST-HEN1
protein produced in Escherichia coli shows activity to

Figure 3. Involvement of CG12367 in piRNA methylation in fly
ovary. (A) RT–PCR shows that the homozygous piggyBacf00810 mutant does not express CG12367 mRNAs, but does express transcripts of CG8878. A schematic drawing of CG12367 (blue) andCG8878 (red) genes in Drosophila is shown in Supplementary Figure
S3A. RT–PCR reaction was performed using primers that are indicated in Supplementary Figure S3A. (−/+) PiggyBacf00810/CyO; (−/−)
PiggyBacf00810/PiggyBacf00810; (wild type) yellow-white. (B) piRNAs
associated with Aub and Piwi in the CG12367 mutant ovary (shownin A) were subjected to oxidation/␤-elimination chemical reactions.
In both cases, piRNAs gain mobility, indicating that piRNAs associated with Aub and Piwi in the CG12367 mutant are not modified
at their 3⬘ ends.

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5A, top panel). piRNA methylated in the assay showed
resistance to oxidation and ␤-elimination treatment (Fig.
5B). Interestingly, miRNAs associated with AGO1 were
not methylated, although these miRNAs are singlestranded in a complex form with AGO1 (Miyoshi et al.
2005). Confirmation that the miRNA levels in Figure 5A
were several-fold higher than those of piRNAs was provided by phosphorylation of these small RNAs (Fig. 5A,
bottom panel). It seems that small RNA methylation by
Pimet is largely influenced by the accessibility of the 3⬘
ends of the substrates to Pimet itself. Structural analysis
of Argonaute proteins suggests that the 5⬘ end of thesmall guide RNA is anchored in a highly conserved
pocket in the PIWI domain, whereas the 3⬘ end of thesmall RNA is embedded in the PAZ domain (Ma et al.
2004; Parker and Barford 2006). Taken together, our results suggest that the 3⬘ ends of Aub-associated piRNAs
are not tightly bound to the PAZ domain, but are exposed to the surface of the protein. On the other hand,the 3⬘ ends of AGO1-associated miRNAs are likely to beembedded in the PAZ domain and therefore are not exFigure 4. GST-Pimet methylates single-stranded small RNAs in
vitro. (A) miR-1/miR-1* duplexes (0.04 nmol/50 µL) were incubatedwith GST, GST-Pimet, and GST-HEN1 in the presence of 14C-labeled SAM. Resultant small RNAs were run on a denaturing acrylamide gel. The top panel (14C; autoradiograph of 14C-labeled RNAs)
shows that only GST-HEN1 is able to transfer methyl groups from
SAM to miRNA/miRNA* duplexes. The bottom panel shows astaining image (TBO, Toluidine Blue O) of the top gel, which indicates that the same amounts of miRNA/miRNA* duplexes wereused in all the lanes. (B, top panel) When single-stranded small
RNAs (26 nt) are the substrate for the methylation assay (0.5 nmol/
50 µL), GST-Pimet shows stronger activity compared with that of
GST-HEN1. The bottom panel shows a staining image of the top gel.
(C) ssRNAs (22 nt, 26 nt, and 38 nt) were methylated by GST-Pimetin vitro. GST-Pimet seems to be indifferent to the length of substrates, unlike Arabidopsis HEN1 that hardly recognize 25-nt or
longer RNAs as substrates in an in vitro methylation assay (Yang etal. 2006). (D) 2⬘-O-methyl modification of 26-nt ssRNAs prevents
methylation, suggesting that only the 2⬘-hydroxyl group on the ribose of the far 3⬘ nucleotide is methylated by Pimet. (E) ssRNAs (26nt) were methylated by GST-Pimet as in B and subjected to oxidation and ␤-elimination treatment. Only methylated ssRNAs (notvisible in the bottom “TBO” panel) showed resistance (14C) to thechemical treatment.

as in the case of HEN1 (Yu et al. 2005). ssRNAs methylated by GST-Pimet show resistance to oxidation/␤elimination treatment (Fig. 4E). These results indicatethat Pimet in Drosophila is involved in piRNA methylation.
Recombinant Pimet methylates piRNAs with Aub
but not miRNAs with AGO1 from Pimet mutant ovary
In Pimet mutant ovary, piRNAs associated with Aub andPiwi were not methylated at the 3⬘ ends (Fig. 3B), most
likely due to loss of Pimet expression. We then examined
if GST-Pimet is able to methylate these piRNAs associated with the PIWI proteins from Pimet mutant ovary.
Aub–piRNA complexes were immunopurified with a
specific antibody against Aub (Gunawardane et al. 2007)
and subjected to in vitro methylation assays. As a control, miRNAs associated with AGO1 were also obtained
through immunoprecipitation using anti-AGO1 (Miyoshi et al. 2005) from ovary lysate. We found that piRNAs
were methylated even in a complex form with Aub (Fig.

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Figure 5. (A) Pimet methylates piRNAs associated with Aub obtained from Pimet mutant ovary, but does not methylate miRNAs
associated with AGO1. Aub–piRNA complexes were immunopurified from Pimet mutant ovary lysate with anti-Aub antibody (Gunawardane et al. 2007) and subjected to methylation by GST-Pimet.
miRNAs associated with AGO1 immunopurified from Pimet mutant ovary lysate were used as a control. Nonimmune IgG (n.i.) was
also used as a negative control. (Top panel) piRNAs were even methylated in a complex with PIWI protein (14C). By contrast, miRNAs
associated with AGO1 were not methylated, although thesemiRNAs are single-stranded in a complex form with AGO1 (Miyoshi et al. 2005). (Bottom panel) miRNA levels appearing in A wereseveral-fold higher than those of piRNAs (32P; 5⬘ end phosphorylation after CIP). (B) Aub–piRNA complexes immunopurified from
Pimet ovary lysate were subjected to methylation by GST-Pimet as
in A. piRNAs were then isolated and subjected to oxidation and␤-elimination treatment. piRNAs methylated in vitro showed resistance to the chemical treatment. (C) Association of Pimet with PIWI
proteins. GST-Pimet (Supplementary Fig. S4C) was incubated with
the Pimet mutant ovary lysate, and after extensive washing theeluates were probed with antibodies against Aub, Piwi, AGO3, and
AGO1. Aub, Piwi, and AGO3 are clearly detected in the boundfraction with GST-Pimet but not with GST. AGO1 was not observed in either lane.

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Pimet methylates fly piRNAs

posed to the surface of the protein. Alternatively, but notmutually exclusively, it is conceivable that Pimet may
interact only with PIWI proteins and not with AGO proteins, thereby methylating only small RNAs associatedwith PIWI proteins. To test this, we next examined if
Pimet associates with PIWI proteins or not. A GST pulldown assay was performed; GST-Pimet was first incubated with Pimet mutant ovary lysate, and after extensive washing the eluates were probed with PIWI protein
antibodies. Aub, Piwi, and AGO3 were clearly detectedin the bound fraction with GST-Pimet but not with GST
itself (Fig. 5C). By contrast, AGO1 was not observed ineither lane (Fig. 5C). These results indicated that Pimetis capable of physically interacting with PIWI proteins
containing piRNAs that can serve as substrates for Pimet
methylation. Addition of RNaseA did not affect the interaction of Pimet with Aub (Supplementary Fig. S4D),
suggesting that Pimet is able to associate directly with
the PIWI proteins. In Drosophila, piRNA methylation
may occur after matured piRNAs are loaded onto PIWI
proteins. If so, it clearly differs from the case of miRNA
methylation in plants, which likely occurs prior tomiRNA loading onto the AGO proteins when miRNAs
are still in a duplex form with the complementary
miRNA* molecules (Yu et al. 2005).
Mutations in Arabidopsis hen1 cause reduced fertility(Chen et al. 2002). Thus, is the piRNA methylation by
Pimet crucial in Drosophila? piRNAs function in genome surveillance in germlines in concert with PIWI
proteins (Saito et al. 2006; Vagin et al. 2006; Gunawardane et al. 2007). Mutations in aub, piwi, and others like
spindle-E (homeless) cause piRNAs not to be accumulated in gonads (Aravin et al. 2004; Savitsky et al. 2006;
Vagin et al. 2006), and lead to germ cell malformationand sterility (Gillespie and Berg 1995; Lin and Spradling
1997; Harris and Macdonald 2001). This clearly indicatesthat piRNAs are necessary for perpetuation of organisms. However, the Pimet mutant fly seems to be viable
and fertile. Steady-state levels of piRNAs in the methylation-defective mutant are also similar to those in wild
type (data not shown). Expression levels of retrotransposons do not seem to be changed by loss of Pimet expression (data not shown). Thus, the function of 3⬘ end
methylation is currently unknown. Further investigation such as by immunohistochemistry may be required
to obtain a more detailed morphology of the mutant.
Extensive analyses of the mechanisms underlying
piRNA methylation may also provide important clues tomore fully elucidating piRNA biogenesis. Aub andAGO3, which determine and form the 5⬘ end of piRNAs(Brennecke et al. 2007; Gunawardane et al. 2007) inpiRNA biogenesis, were shown to be in the protein fraction associated with Pimet (Fig. 4B). Identifying more
Pimet-associated proteins may reveal the factors required for formation of the 3⬘ end of piRNAs.
Materials and methods
Periodate oxidation and ␤-elimination
AGO1, Aub, and Piwi were immunopurified as described (Saito et al.
2006) from an ovary using anti-AGO1 (Miyoshi et al. 2005), anti-Aub(Gunawardane et al. 2007), and anti-Piwi (Saito et al. 2006) antibodies,
respectively. Small RNAs coimmunopurified with AGO1, Aub, and Piwi
were isolated from the immunoprecipitates using phenol and phenol:
chloroform and were ethanol-precipitated. RNAs isolated were labeledwith 32P-␥-ATP after CIP treatment. Ten-thousand counts per minute(cpm) of 32P-labeled RNAs were used per reaction. Periodate oxidationand ␤-elimination of RNAs were performed as described (Gunawardane
et al. 2007). Chemically synthesized luc guide siRNA (Okamura et al.
2004) was used as a control RNA.
Nucleoside analysis of piRNAs by capillary LC nano electrosprayionization (ESI)/MS
piRNAs associated with Aub in wild-type fly ovary were obtained as
described in the Periodate Oxidation/␤-Elimination section. piRNAs (∼5pmol) were digested by RNase T2 into 3⬘-, 5⬘-diphospho nucleotides,
3⬘-monophospho nucleotides, and 3⬘-terminal nucleosides for 3 h at 37°C
in 5 µL of reaction mixture containing 10 mM ammonium acetate (pH5.3) and 1.25 U/mL RNase T2 (GE Healthcare) as described (Ohara et al.
2007; Suzuki et al. 2007). The reaction solution was evaporated and dissolved in 1.5 µL of 50 mM ammonium acetate (pH 5.3). Synthetic 2⬘ or
3⬘-O-methyl nucleosides were obtained from ChemGenes Corporationand Sigma-Aldrich. The procedure for LC/MS analysis has been described(Ikeuchi et al. 2006). To analyze a limited quantity of nucleosides, we
devised our own system of capillary LC coupled with ESI/MS. A QStarXL Hybrid LC/MS/MS System (Applied Biosystems) equipped with a
NanoSpray II nanoelectrospray (Applied Biosystems) source, a Nanovolume Valve (Valco Instruments), and a splitless nano HPLC system DiNa
(KYA Technologies) were used to analyze RNase T2-digested piRNAs.
The digest (1.5 µL) was loaded onto an ODS capillary column (HiQsil
C18H-3, 0.15 × 150 mm; KYA Technologies) directly via a 1-µL loop
injection and was chromatographed at a flow rate of 500 nL/min using alinear gradient of 0%–60% of Solvent B (50% acetonitrile containing 5mM ammonium acetate at pH 5.3)over Solvent A (5 mM ammonium
acetate at pH 5.3) in 40 min. The chromatographic eluent was sprayedand ionized from an energized (+2.2-kV) sprayer tip attached to the capillary column. Throughout the separation, ionized molecules were
scanned in a positive polarity mode over an m/z range of 108–400. CID ofeach 2⬘-O-methylnucleoside was carried out with a collision energy of+25, and scanned in an m/z range of 50–300.
RT–PCR, in vitro small RNA methylation assay, and GST pull-down
assay were performed as described in the Supplemental Material.

Acknowledgments
We thank Xuemei Chen for the cDNA of Arabidopsis HEN1 and themiRNA methylation protocol. We also thank Tomoko Mori, Tomoko
Nagami, Kazumichi M. Nishida, and Yoshinori Kawamura for technicalassistance, and other members of the Siomi laboratory for discussions
and comments on the manuscript. K.S. is a post-doctoral fellow of the
21st century COE Program from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (MEXT). This work was supported in part by grants to T.S., M.C.S., and H.S. from MEXT and the
New Energy and Industrial Technology Development Organization(NEDO).

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