Supplementary information
Materials and Methods
Reference
Supplementary Figures S1~S5Materials and Methods
Pull-down assay
AGO3-2-sDMA (without R72-sDMA) and Aub-sDMA (without R72-sDMA) peptides
were synthesized by Operon.
Western blot analysis
Western blot analysis was performed as described previously (Miyoshi et al, 2005).
Anti-Spn-E monoclonal antibody was used at 1:2,000 dilution. Culture supernatant ofhybridomas producing anti-Tud monoclonal antibody was used without dilution as a
primary antibody.
RNAi
RNAi in S2 cells was performed as described (Ishizuka et al, 2002). The primers
utilized to produce dsRNAs were;
T7-spnE3501: 5'-TAATACGACTCACTATAGGGAGTGGTCTAGTCAAAAGCTG-3'andT7-spnE2848:
5'-TAATACGACTCACTATAGGGAAACGCGATAGTTATTTCCA-3'.
TnT
The cDNA encoding the ORF of dTdrd1 (nucleotide number; 4-4809) was amplified by
PCR using primers;
BamHI-dTdrd1:
5’-GGGGGGATCCGCTATCTTCGTCCAGCTAACCACAGATAAT-3' and
SacI-dTdrd1:

5'-GGGGGAGCTCTTATGATTTTTTCAGGAACATCCAATTTTC-3'. The resultant
DNA fragment was inserted into pET-28a (Novagen) to produce pET-dTdrd1.
35

S-methonine labeled dTdrd1 protein was produced using the TNT Coupled

Reticulocyte Lysate Systems (Promega), according to the manufacturer’s instructions.
Sucrose gradient
Ovary lysates prepared as described in the Materials and Methods section were resolved
on a linear sucrose gradient (5 to 30%). Besides sucrose, Gradient buffer contained 30
mM HEPES-KOH (pH 7.3), 100 mM KOAc, 4 mM MgOAc, 1 mM DTT, 2 mg/mL
Pepstatin, 2 mg/mL Leupeptin, and 0.5% Aprotinin. The gradient was centrifuged at
4°C in a Beckman MLS-50 rotor at 23,000 rpm for 15 hr and 10 min. Following
centrifugation, fractions were collected (200 µl each) and proteins in the fractions were
TCA-precipitated and subjected to western blot analyses using anti-Tudor, anti-AGO3
and anti-Aub antibodies.
RT-PCR
Total RNAs were isolated from the immunopurified complexes by phenol: chloroform
extraction and precipitated with ethanol. Reverse transcription (RT) was performed
using Accuscript RT (Stratagene) according to the manufacturer’s protocol. Primers
used were;roo-primer-1761: 5’-AATAAAATTGAATTTTTATGGCATAAAATA-3’
roo1960-rev: 5’-GGTAACTCCTCCGCCTTAAC-3’.
PCR was performed using KOD plus (TOYOBO) with the primers for RT and primers
indicated below;
roo2060-rev: 5’-ATCCTCTCTCACATGTATGA-3’
roo2160-rev: 5’-CATAATTTTCTCCAGAGCCG-3’
roo2260-rev: 5’-TCTAAATTTAAATTTGGCAT-3’
roo-primer-1791: 5’-GATAACCTGATTGAACAGGTGAATAGTCGT-3’.
Deep-sequencing
Small RNA libraries were prepared as described previously (Morin et al, 2008).
Deep-sequencing was performed on an Illumina Genome Analyzer at Hokkaido systemscience. For bioinformatics analyses (Figure S5), we randomly picked up 30,000 readsequences from each of tud heterozygous and homozygous mutant libraries.
Cloning frequency and strand bias
Short reads ranging from 20 to 30 nt were extracted from original sequencing results of
tud heterozygous and homozygous mutants, respectively. The extracted short reads weremapped to the fruit fly genome sequence (dm3) without allowing any base mismatches.
We obtained 27,197 and 28,721 perfectly matched reads from tud heterozygous andhomozygous mutants, respectively. By comparing the mapped reads to RepeatMasker
tracks of the UCSC Genome Browser (http://genome.ucsc.edu/), 19,738 and 21,716
reads were found to be transcripts of transposons. The transposon-derived short reads
were annotated and assigned to transposon families. Per family population wascomputed for each transposon family. The population was normalized to a percentile
over the total transposon-derived reads. We visualized the percentile population oftransposon families as a heatmap image. For visual simplicity, minor families whichappear less than 1% of the total population are not shown. For each transposon family, aratio between sense and antisense reads was computed using the following equation:(sense reads – antisense reads)/total reads, which ranges from -1.0 to 1.0. The ratio
represents strand bias of transposon-derived short reads for each transposon family. We
visualized the ratio per transposon family as a heatmap image by using a two-color
microarray-like color scheme. An antisense-biased family is rendered as a red-colored
box whereas a sense-biased family is rendered green.
Population pie-chart
The mapped short reads were annotated by comparison to the following data tracks of
the UCSC Genome Browser, where various gene classes are represented:
flyBaseNoncoding (for tRNA, rRNA, snoRNA, snRNA, miRNA, and other ncRNA),rmsk, and simpleRepeat. After the annotation, the population of each gene class wascomputed and visualized as a pie-chart.

Reference
Morin RD, O'Connor MD, Griffith M, Kuchenbauer F, Delaney A, Prabhu AL, Zhao Y,McDonald H, Zeng T, Hirst M, Eaves CJ, Marra MA. 2008. Application of massively
parallel sequencing to microRNA profiling and discovery in human embryonic stem
cells. Genome Res. 18: 610-621

Fig. S1
Nishida et al.

Cbeads only

120k-

Aub-sDMA peptide- Spn-EInput

220k160k-

Aub peptide

Pull-downof TnT productovaryS2A

100k- dTdrd1

80k70k60k50k(Da)- Tubulin

BSpn-E RNAiin S2 cells-+- Spn-E

Figure S1. (A) Western blot analysis of S2 cells and ovary lysates using anti-Spn-E antibodies raised in our laboratory.
Tubulin was detected as an internal control. (B) Western blot analysis of S2 cells, with and without RNAi treatment against
the spn-E gene, shows the specificity of the anti-Spn-E antibodies utilized in (A). (C) dTdrd1 does not show a specificinteraction with the peptides. 35S-labeled dTdrd1 was produced in rabbit reticulocyte lysates and utilized in pull-down
assays using Aub and Aub-sDMA peptides (Figure 2A). The signals were slightly higher in both Aub and Aub-sDMA
peptide lanes compared with the negative control lane (beads only), indicating that dTdrd1 might be able to bind weakly
with the peptides; however, the sDMA-dependency was not observed. Input shows 10% of dTdrd1 used for each pull-down
reaction.

Fig. S2
Nishida et al.

Aｚ+2(13)ｚ+2(14)LL

N

RA2meR G 2meR G VS I N V HQ L T T ILD

ｚ+1(25) 2+ｚ+1(26) 2+T EI

ｚ+2(17)ｚ+2(12)

NF

ｚ+2(15)ｚ+2(11)Sｚ+1(22) 2+ｚ+1(23) 2+ｚ+1(8)2+ｚ+1(9)2+ｚ+1(10)2+ｚ+1(11)2+

L

ｚ+2(10)K

ｚ+1(19)2+ｚ+1(20) 2+ｚ+2(7)G A N

ｚ+2(8)ｚ+2(4)ｚ+2(5)ｚ+2(6)KGoxiM N

ｚ+1(13)2+ｚ+1(14)2+ｚ+1(15)2+ｚ+1(16)2+ｚ+2(3)C

ｚ+1(3)2+ｚ+1(4)2+ｚ+1(5)2+ｚ+1(6)2+BG2meR

G AcS

N HS S LL

Figure S2. Analysis of AGO3 modifications by LC-MS/MS. (A) AGO3 peptide was identified by MASCOT database searching.
Letters with yellow background are search hits. Those with green background indicate amino acids with modifications; of those,
M, R, and C indicate methionine with oxidation, arginine with dimethylation, and cysteine with carbamidomethylation, respectively.
The N-terminal serine (S2) was found to be N-terminally acetylated. (B) MS spectrum of the AGO3 peptide; amino acid sequencesfrom S2 to K20 (S2-K20; m/z = 688.6937, theoretical value = 688.6938) and from L45 to R72 (L45-R72; m/z = 789.9459,
theoretical value = 789.9460). (C) ETD MS/MS spectrum of the AGO3 peptides S2-K20 and L45-R72 with Mascot Ion scores of
82 and 90, respectively. 2meR indicates an arginine with dimethylation. Ac indicates an N-terminal acetylation. z-series ionsassigned to the amino acid sequences are indicated.

AGO3-2-sDMA peptide (without R72-sDMA)C

IP from ovary
antiInput n.i. Aubyw
tud-/yw
tud-/yw
tud-/-

AGO3-2-sDMA peptide

AGO3-2 peptide

Aub peptide

Input

A

Aub-sDMA peptide (without R17-sDMA)

Fig. S3
Nishida et al.

-Tud-Tud-Aub
Aub-sDMA peptide (without R17-sDMA)-AGO3

MNLPPNPVIARGRGRGRKPNNVEAN
AGO3-2-sDMA peptide (without R72-sDMA)TLQHVNISVGRGRARLIDTLKTDDH

B
InputSucrose gradient
5%130%
23456789 10 11 12 13 14 15 16-20-Tud-Aub-AGO3

Figure S3. (A) Pull-down assays were performed as in Figure 2A, from ovary lysates using Aub-sDMA (without R17-sDMA) andAGO3-2-sDMA (without R72-sDMA) peptides. The sequences of the peptides are shown below. Rs in red indicate Rs that weresDMA-modified. The protein pools obtained were probed with anti-Tud antibodies. (B)Linear gradient sedimentation experiments
were performed using wt ovary lysates. Western blot analyses show that Tud, Aub and AGO3 co-sediment even in relatively heavier
fractions. (C) Immunoprecipitation was performed from ovary lysates of wt and tud mutants using anti-Aub antibodies. Non-immune
IgG (n.i.) was used as a negative control. The immunopurified complexes were probed with anti-Tud, anti-Aub and anti-AGO3
antibodies. Tud was observed only in the Aub complex obtained from wt ovaries. It is noted that Aub is able to interact with AGO3
even in tud mutants.

Fig. S4
Nishida et al.

A
[1]

[I]

[II]

[III]

roo antisense transcript
[2]

[3]

[1] roo-primer-1761 (RT primer)[2] roo2060-rev[3] roo2160-rev[4] roo2260-rev[I] roo-primer-1791[II] roo#3 piRNA-as[III] roo#4 piRNA-as[1]+[4][1]+[3][1]+[2][1]+[4]anti-Tud[1]+[3][1]+[2]n.i.

[4]primers

700 -B

500 -

500bp

400 -

400bp

300 (bp)

300bp

[2]

roo sense transcript[1][1] roo1960-rev (RT primer)[2] roo-primer-1791

200 175 -anti-Tudn.i.

[1]+[2]

170bp

150 125 (bp)

Figure S4. (A) RT-PCR showed that the anti-Tud immunoprecipitates (Figure 4B) contain part of the roo antisense transcript.
Non-immune IgG (n.i.) was used as a negative control. The primer used for the RT (reverse transcription) reaction was roo-primer-1761(RT primer). roo-primer-1761 was also utilized for PCR in combination with either roo2060-rev, roo2160-rev, or roo2260-rev primer.
The positions of oligos used for northern blot analysis in Figure 4B (roo-primer-1791, roo#3 piRNA-as and roo#4 piRNA-as) are also
indicated in this figure. (B) RT-PCR shows that the anti-Tud immunoprecipitates contain sense roo piRNA precursor-like molecules.

Fig. S5Nishida et al.

A

B

C

0.6%
0.3%
8.7%

24.2%

46.4%

Homo

19.8%

0.3%

0.1%
8.2%

22.4%
46.2%

Hetero

22.9%

Figure S5. (A) Strand bias shows a comparison of strand preferences between transposon-derived short reads from tud 
heterozygous and homozygous mutants. The ﬁgure uses a color scheme analogous to that of two-color microarray data presentation. A red-colored box represents the corresponding transposon family that expresses more antisense transcripts than sense transcripts. A green-colored box represents the reverse situation. (B) Cloning frequency shows population differences between transposon-derived short reads from tud heterozygous and homozygous mutants. The population of each transposon family is normalized as a percentile over total reads. A darker box represents a greater percentile. (C) The 
pie-charts show the contents of short reads from tud heterozygous and homozygous mutants. It should be noted that transposon-derived piRNAs are omitted from the charts.