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Small RNA profiling and characterization of piRNA clusters
in the adult testes of the common marmoset, a model primate
TAKAMASA HIRANO,1,6 YUKA W. IWASAKI,1,6 ZACHARY YU-CHING LIN,2 MASANORI IMAMURA,2,3 NAOMI
M. SEKI,1,4 ERIKA SASAKI,2,5 KUNIAKI SAITO,1 HIDEYUKI OKANO,2 MIKIKO C. SIOMI,4 and HARUHIKO SIOMI1
1

Department of Molecular Biology, 2Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan
Molecular Biology Section, Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama,
Aichi 484-8506, Japan
4
Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
5
Central Institute for Experimental Animals, Kawasaki, Kanagawa 210-0821, Japan
3

ABSTRACT
Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here, we describe small RNA
profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis
was piRNAs, although 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse
MIWI and a very abundant PIWI in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As
in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated asintergenic regions. However, unlike in mice, marmoset piRNA clusters are also found on the X chromosome, suggesting escapefrom meiotic sex chromosome inactivation by the X-linked clusters. Some of the piRNA clusters identified contain antisenseorientated pseudogenes, suggesting the possibility that pseudogene-derived piRNAs may regulate parental functional proteincoding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition,
the strand bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. Thesefindings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, may regulate
protein-coding genes via pseudogene-derived piRNAs, and may even play roles in meiosis in the adult marmoset testis.
Keywords: miRNAs; piRNAs; PIWI; transposable elements; pseudogenes

INTRODUCTION
Eukaryotic cells express a large number of small noncoding
RNAs, 20–35 nucleotides (nt) in length, that are derived
from various parts of the genome, including intergenic regions, transposable elements (TEs) and their remnants, andalso from viruses (Girard and Hannon 2008; Ghildiyal andZamore 2009; Siomi and Siomi 2009; Ketting 2011). These
small RNAs associate with Argonaute proteins to form regulatory complexes known as RNA-induced silencing complexes (RISCs), which then pair with complementary RNA targets
to promote the inactivation of homologous sequences. Thesepathways are referred to as RNA interference (RNAi), or RNA
silencing, and help regulate protein levels and restrain the expression of parasitic and pathogenic invaders such as TEs andviruses (Bartel 2009; Saito and Siomi 2010). The RNAi pathway is present in a diverse range of eukaryotes, including someunicellular organisms, and is thought to have evolved as a

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These authors contributed equally to this work.
Corresponding author: awa403@z2.keio.jp
Article published online ahead of print. Article and publication date are at
http://www.rnajournal.org/cgi/doi/10.1261/rna.045310.114.

form of nucleic acid-based immunity to inactivate TEs and viruses (Buchon and Vaury 2006).
According to their biogenesis and associated Argonaute
proteins, small silencing RNAs are classified into three types:
microRNAs (miRNAs), endogenous small interfering RNAs
(endo-siRNAs), and PIWI-interacting RNAs (piRNAs) (Ghildiyal and Zamore 2009; Kim et al. 2009; Siomi and Siomi2009). miRNAs are 21–24 nt RNAs that derive from distinctive hairpin precursors and repress target messenger RNAs(mRNAs) either by transcript destabilization, translational inhibition, or both, by base-pairing to the “seed” sequence,
which is defined relative to the position of the miRNA 5′
end (Bartel 2009). Endo-siRNAs are 21–22 nt RNAs thatmainly derive from TEs and pair to their transcripts to induce
cleavage via small RNA-guided endonuclease activity or Slicer
activity of associated Argonautes, thereby silencing TEs(Ghildiyal et al. 2008; Kawamura et al. 2008; Okamura et al.
© 2014 Hirano et al. This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://
rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available
under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

RNA 20:1–15; Published by Cold Spring Harbor Laboratory Press for the RNA Society

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Hirano et al.

2008; Tam et al. 2008; Watanabe et al. 2008). In addition,some endo-siRNAs are derived from complementary annealed transcripts or long fold-back transcripts (Babiarz
et al. 2008; Kawamura et al. 2008; Okamura and Lai 2008;
Miyoshi et al. 2010). Most miRNAs and endo-siRNAs areproduced by Dicer and loaded onto the AGO subfamily of
Argonautes.
In contrast, piRNAs are longer, at 24–35 nt, and their sequences are more diverse than any other known class of cellular RNAs (Siomi et al. 2011; Pillai and Chuma 2012). piRNAs
are produced by Dicer-independent mechanisms from longsingle-stranded precursors mainly derived from active TEs
and intergenic unannotated large blocks known as piRNA
clusters, after which they are loaded onto the PIWI subfamilyof Argonautes (Malone and Hannon 2009; Siomi et al. 2011).
Some populations of these primary piRNAs are further amplified by a feed-forward amplification loop, the so-called pingpong cycle, in which sense and antisense transcripts of TEs arereciprocally cleaved by the Slicer activity of PIWI proteins(Brennecke et al. 2007; Gunawardane et al. 2007; Aravin
et al. 2008). The amplified secondary piRNAs have characteristic features with 1U/10A partners and a 10-nt 5′ overlap,
which is referred to as the ping-pong signature. Although
miRNAs and endo-siRNAs are expressed in both animals
and plants, piRNA expression is largely restricted to animal
gonads, where they primarily mediate TE silencing for genome integrity and germ cell development (Thomson and
Lin 2009; Siomi et al. 2011). piRNAs act as guides for thecleavage of TE transcripts by PIWI protein Slicer activity inthe cytoplasm, which often couples with the ping-pong cycle,
or for Slicer-independent heterochromatin formation at TEloci in the nucleus (Sienski et al. 2012; Dönertas et al. 2013;
Huang et al. 2013; Le Thomas et al. 2013; Muerdter et al.
2013; Ohtani et al. 2013).
In mice, piRNAs are most abundantly expressed in malegerm cells, especially during spermatogenesis, and are loaded
onto the three PIWI proteins: MIWI (PIWIL1), MILI(PIWIL2), and MIWI2 (PIWIL4) (Thomson and Lin 2009;
Siomi et al. 2011; Pillai and Chuma 2012). Depletion of individual Piwi genes causes male-specific sterility because of
severe defects in sperm formation, indicating their nonredundant functions of Piwi genes in the testes (Deng and Lin 2002;
Kuramochi-Miyagawa et al. 2004; Carmell et al. 2007). Two
distinct piRNA populations have been identified: the fetal prepachytene and adult pachytene piRNA pools. Prepachytene
piRNAs are enriched in TE- and other repeat-derived sequences and associate with MIWI2 and MILI proteins(Aravin et al. 2008; De Fazio et al. 2011), suggesting thatthey might play a role in silencing TEs. Moreover, as pingpong signatures are detected between MIWI2 and MILI,
this indicates that they are involved in amplification of prepachytene piRNAs. In contrast, pachytene piRNAs have a higher
proportion of intergenic, unannotated sequences, with a diminished contribution from TE-derived sequences (Aravin
et al. 2006, 2007; Girard et al. 2006; Beyret et al. 2012).
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They are loaded onto MILI and MIWI from pachytene spermatocytes to elongating spermatids that are not further amplified. Although the loss of genes required to generatepachytene piRNAs blocks the production of mature sperm
and results in TE deregulation (Deng and Lin 2002; Aravinand Hannon 2008; Reuter et al. 2011; Pillai and Chuma
2012; Vourekas et al. 2012), a biological role for pachytene
piRNA clusters has yet to be identified. It also remains unknown if the absence of these RNAs causes the severe defects
in spermatogenesis observed in mutant mice defective in thepachytene piRNA pathway.
Unlike rodents, primates possess four PIWI genes(PIWIL1–PIWIL4); thus, piRNA-mediated silencing may differ between rodents and primates (Sasaki et al. 2003; Ishizu
et al. 2012). However, little is known about primate PIWIpiRNA pathways. Herein, in an effort to understand them,
we analyze small RNAs in the adult testes of Callithrix jacchus,
the common marmoset. Marmosets are small New World
monkeys that have been extensively used in biomedical research and provide an attractive alternative to traditional nonhuman primate species. This is mainly because (1) they reachsexual maturity at 18 months of age, (2) they often bear twins
after a relatively short gestation period, and (3) transgenic animals can now be produced (Sasaki et al. 2009; Orsi et al. 2011;
Okano et al. 2012). To gain insights into the function of small
RNAs in the marmoset testis, we used small RNA sequencing(small RNA-seq) to generate extensive small RNA data, in
conjunction with RNA-seq data. We show that approximately
200 of nearly 700 miRNA genes identified are specific to marmosets. The most abundant class of small RNAs in the adulttestis is, however, piRNAs. Using a specific monoclonal antibody that recognizes the PIWI protein MARWI (marmoset
PIWIL1), a marmoset homolog of mouse MIWI, we generate
MARWI-associated piRNA data and find that most piRNAs
in adult testes are associated with MARWI, and they show
characteristics of mouse pachytene piRNAs. Most MARWIassociated piRNAs derive from conserved intergenic clusters,some of which are located on the X chromosome. This suggests that they escape transcriptional silencing by meiotic
sex chromosome inactivation (MSCI), which silences the X
and Y chromosomes shortly after the zygotene-to-pacytene
transition (Turner 2007; Heard and Turner 2011). Someclusters also contain pseudogenes of an antisense orientation,suggesting regulation of their cognate functional genes.
Interestingly, piRNAs tend to map to TEs with copies inpiRNA clusters much more than TEs with no copy in
piRNA clusters, indicating that pachytene piRNA clusters
act as traps that catch actively transposed TEs. Moreover,piRNAs corresponding to one subfamily are predominantly
antisense to the TE, whereas piRNAs corresponding to another subfamily are predominantly sense. This is consistent withthe sense/antisense bias of corresponding TE insertion topiRNA clusters, suggesting that the direction of TE insertion
into the piRNA clusters is one of the major factors for setting
the strong bias to the production of these piRNAs. Although aDownloaded from rnajournal.cshlp.org on February 2, 2015 - Published by Cold Spring Harbor Laboratory Press

Small RNA profiling in the marmoset adult testes

biological role for mouse pachytene piRNA clusters remains
unknown, our findings suggest that pachytene piRNA clusters
function in both TE silencing and regulation of protein-coding gene expression and may even have functions in meiosis.
RESULTS
Small RNA profiling in the marmoset adult testes
We sequenced total small RNAs of ∼18–35 nt from the testisof an adult marmoset (41 months old; note that marmosets
are normally reproductively active from 2 to ∼8 years ofage) (Abbott et al. 2003) using Illumina MiSeq, resulting ina total of 18,832,688 reads. We mapped the reads to the marmoset genome, which yielded 13,818,718 perfectly matchingreads, mostly in the size range of 21–33 nt (Supplemental Fig.
S1A; Supplemental Table S1). Among these, 28.1% mapped
to multiple regions within the genome (Supplemental Fig.
S1B), suggesting that they originate from repetitive elements
like TEs. Small RNA profiling of mapped reads revealed two
distinct peaks, one at the 21–23 nt position (6.7% of the total
mapped reads), corresponding to miRNA and endo-siRNA in
size, and another at the 24–33 nt position, which is the mostabundant class of small RNAs (83.5% of the total mapped
reads), corresponding to piRNAs in size (Supplemental Fig.
S1A). To annotate the genome-mapped small RNA reads,
we referred to the UCSC Genome Browser (Meyer et al.
2013) and Ensembl database (Flicek et al. 2013) for the annotation of marmoset genomic regions. We found that 19.28%
of small RNAs map to repetitive elements, 9.11% to cellularnoncoding (nc) RNAs, including miRNAs (3.60%) and transfer RNAs (tRNAs) (4.65%), 1.68% to coding genes, and69.96% to unannotated regions (Supplemental Fig. S1C).
miRNAs in the marmoset testis
To precisely analyze the first peak (21–23 nt) observed in marmoset total small RNAs (Supplemental Fig. S1A), the nucleotide bias was checked, and the first nucleotide at the 5′ end wasshown to be biased to “U” and “A” (U, 63.2%; A, 24.4%)(Supplemental Fig. S2A), which is a known characteristicof animal miRNAs (Seitz et al. 2011). The 21–23 nt length
reads were mapped to the genome and checked for annotation(Supplemental Fig. S2B), revealing that 43.7% match miRNA
coding regions registered in the Ensembl database (Flicek et al.
2013). However, more than half were annotated to other regions, such as TEs, repeats, and unannotated intergenic
regions, indicating that they may also be capable of producing miRNAs. To clarify this, we identified miRNAs with
miRDeep2 pipeline (Friedländer et al. 2012), using total small
RNA-seq reads ranging in length from 21–23 nt as input. Thissearch assigned 88.8% of 21–23 nt small RNA reads to 685
miRNAs, of which 332 were identical to miRNAs registeredin the Ensembl database and 353 miRNAs were novel (Supplemental Table S2). Of the novel miRNAs, 174 had seedsequences in common with other primate miRNA species.
The genomic regions from where these miRNAs originate
varied, and some were expressed from protein coding regions(Supplemental Fig. S2C).
A primate-specific X-linked miRNA cluster was of particular interest (Supplemental Fig. S2D; Zhang et al. 2007; Li et al.
2010; Meunier et al. 2013). This cluster is comprised of tandem repeats of the Medium Reiteration frequency (MER)
TE, which folds into a palindromic structure similar to thehairpin of the miRNA precursor and appears expressed almost
exclusively in the testis (Yuan et al. 2010; Ahn et al. 2013;
Meunier et al. 2013). During mammalian male germline development, the X and Y chromosomes are compartmentalized
into a perinuclear subdomain called the sex body and become
transcriptionally silenced from early pachytene onward,
through to the end of spermatogenesis. This process is known
as MSCI. X-linked miRNA cluster appears to be expressed
during pachytene, suggesting escape from MSCI (Song et al.
2009; Meunier et al. 2013). The expression of two miRNAs
from this cluster (miR-MER91C-5-3p and -21-3p) was confirmed by northern blotting (Supplemental Fig. S2E).
Consistent with the recent studies that have revealed rapid
evolution of this cluster in primates, including frequent tandem duplications and nucleotide substitutions (Zhang et al.
2007; Meunier et al. 2013; Sun et al. 2013), only two of the
23 seed sequences of the X-linked cluster-encoded miRNAs
were conserved between marmoset and human, indicating
the rapid divergence of this miRNA cluster (Supplemental
Tables S3, S4). These findings also suggest that this clusterof miRNAs has a testis-specific function and that a form of genetic conflict may be driving its rapid evolution in primates.
Lack of endo-siRNA in the marmoset testis
To identify endo-siRNAs, we used 21–23-nt long total small
RNA-seq reads that mapped to genomic regions other thanthe location of miRNA genes (476,689 reads). We searched
for complementary small RNAs with 2-nt 3′ overhangs corresponding to products excised from long double-stranded
precursors by Dicer in endo-siRNA biogenesis, and we also
manually searched for genomic regions that can potentiallygive rise to phased endo-siRNAs by folding into long stem–
loop secondary structures (Okamura and Lai 2008). However, we were unable to detect endo-siRNA-like small RNAs
with significant reads (data not shown; also see Supplemental
Material). This suggests that adult marmoset testes express
very few endo-siRNAs, although recent studies have shownthat murine adult spermatogenic cells express abundantendo-siRNAs (Song et al. 2011).
Abundant expression of MARWI, a homolog of mouse
MIWI, in the marmoset adult testis
As many marmoset small RNAs are derived from unannotated intergenic regions (Supplemental Fig. S1C), which iswww.rnajournal.org

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reminiscent of mouse MIWI- or MILIassociated piRNAs that start to be expressed at the pachytene stage of meiosis(pachytene piRNAs) (Li et al. 2013),
we analyzed the expression levels of
AGO/PIWI genes using RNA-seq data to
determine if marmoset homologs ofmouse Miwi and/or Mili are expressedin adult testes. Computational searchesof the marmoset genome (UCSC Genome Browser and Ensembl database) revealed eight Argonaute genes: four AGO
subfamily genes (AGO1–AGO4) and
four PIWI subfamily genes (PIWIL1–4).
We named the marmoset PIWI subfamily
genes MARWI, MARLI, MARWI2, and
MARWI3, which correspond to human
HIWI (PIWIL1), HILI (PIWIL2), HIWI2(PIWIL4), and HIWI3 (PIWIL3) (Sasaki
et al. 2003), respectively. We performed
directional RNA-seq using total RNA obtained from two adult marmoset testes(41 and 51 months old). Replicates werehighly correlated with each other (R 2 =0.50) (Supplemental Fig. S3A); therefore, we merged the reads, resulting in
168,398,076 RNA-seq reads (Supplemental Table S1). Reads were used to calculate
the RPKM value for each gene of the FIGURE 1. MARWI associates with piRNAs. (A) Western blotting was performed from marmoArgonaute family. RPKM values indicat- set testes and fibroblasts with anti-MARWI and anti-β-TUBULIN antibodies. MARWI protein is
only detected in the testis. The anti-MARWI antibody (1A5) clearly detects a single band in theed that expression levels of MARWI, a testis lysate. (B) Silver staining of protein components in the testis immunoprecipitant withmarmoset homolog of mouse Miwi, anti-MARWI antibody from testis identifies a 90-kDa band for MARWI protein by LC-MS/MS
without protein contamiwere vastly overrepresented among the (data not shown). MARWI protein is exclusively immunoprecipitated32
gene family in this tissue (Supplemental nants. (C) Isolated RNAs from MARWI immunoprecipitates were P-labeled and separated bya denaturing polyacrylamide gel. MARWI protein associates with ∼29–30-nt-long piRNAs. (D)Fig. S3B), suggesting that most observed The size distribution of MARWI-associated piRNAs, which peak at 29–30 nt. (E) Nucleotide
piRNA-like reads bind to MARWI. bias of 24–33 nt total small RNAs (piRNA-like) and MARWI-associated piRNAs. MARWI′
Expression levels of the AGO subfamily piRNAs have a uridine bias at their 5 end, consistent with the observation in 24–33 nt total small
RNA reads. (F ) Annotation of 24–33 nt total small RNAs and MARWI-associated piRNAs. Bothin the testis are relatively low, which cor- library compositions resemble each other. This, together with the length distribution and annotarelates with the fact that endo-siRNAs tion shown in E and F, indicates that MARWI-piRNA is the major component of total small RNA
were barely detectable in the small RNA in the marmoset testis.
library. MARWI3, which does not have amouse homolog, does not appear to be
expressed in the adult testis (Supplemental Fig. S3B).
at the 3′ terminal nucleotide with a 2′ -O-methyl marker(Kirino and Mourelatos 2007; Ohara et al. 2007; Saito et al.
2007; Simon et al. 2011; Siomi et al. 2011). The resistanceComparison of piRNAs in total testis small RNAsof MARWI piRNAs to periodate oxidation (NaIO4) and βwith MARWI-associated piRNAs
elimination reactions (Supplemental Fig. S4B) show thatthey also have the same 3′ modification.
To test whether MARWI binds to the majority of piRNAs
To further characterize MARWI piRNAs, we performedin adult testes, we immunopurified MARWI from adult testes
Illumina HiSeq sequencing using piRNA samples immunousing monoclonal antibodies we generated (Fig. 1A,B; Suppurified with an anti-MARWI antibody from two individual
plemental Fig. S4), in which it is expressed from spermatoadult marmoset testes (41 and 51 months old). Replicates
cytes to elongating spermatids (see below) and examined its
were highly correlated with each other (R 2 = 0.89) (Suppleassociated RNAs (Fig. 1C). piRNAs associated with MARWI
mental Fig. S5); therefore, we merged the reads, resulting(MARWI piRNAs) in testes were ∼30 nt in length (Fig. 1C,
in 140,662,017 marmoset MARWI-IP piRNA reads in total
D). piRNAs in fly and mouse are known to be modified
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5′ end of only five tRNA species (tRNA-GluUUC, tRNAGluCUC, tRNA-AspGUC, tRNA-GlyGCC, and tRNAValAAC) account for 99.6% of piRNA reads mapped totRNAs (Fig. 2A; Supplemental Table S5). The vast majorityof these tRNA-derived piRNAs mapped to tRNAs for Glu.
Moreover, the tRNAs with the most abundant piRNA species
mapped, tRNA-GluCUC, had ∼1.9 million reads mapped toits 5′ end, corresponding to 1.4% of the total number of
MARWI-associated piRNAs reads (Fig. 2B). Northern blotsof MARWI piRNAs revealed that the 5′ , but not the 3′ , fragments of the tRNA are loaded onto MARWI (Fig. 2C). We
further confirmed that the 5′ fragments of tRNA-AspGUC
are also detected by northern blotting analysis but not
the 5′ fragments of tRNAs, which hardly match MARWI
piRNA reads (Supplemental Fig. S6). These findings together
suggest that these tRNAs are specifically processed into
MARWI-bound piRNAs. Recent studies have revealed thatsome tRNA-derived small RNAs associate with AGO/PIWI
proteins and function as classical siRNAs or miRNAs, whereas others also inhibit translation in a complementary base-(Supplemental Table S1). Expression of highly detected
piRNA reads was confirmed by northern blotting (Supplemental Fig. S4C). As in the case of total small RNA reads (using the testis from the 41-month-old marmoset), the lengthof sequenced reads ranged from 24–33 nt with peaks at 29
and 30 nt (Fig. 1D) and possessed a uracil nucleotide bias
at the 5′ end (1U-bias) (Fig. 1E). Annotation of these
MARWI piRNA reads was similar to that of the 24–33 nt total
small RNA reads (Fig. 1F): Most (∼74.0%) mapped to unannotated intergenic regions and only ∼18.1% of the reads
originated from TEs. This profile is similar to that of pachytene piRNAs associated with mouse MIWI, in which ∼70%
derive from intergenic regions and ∼24% from TEs (Reuter
et al. 2011).
tRNA-derived piRNAs and genic piRNAs
Some (4.5%) piRNAs corresponds to tRNAs (Fig. 1F), which
has not been described in previous analyses of mammalian piRNAs. Surprisingly, MARWI piRNAs mapped to the
A

B

A

G
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C

Cys
Cys
TER
Trp
Arg
Arg
Arg
Arg
Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly

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tRNA mapped reads
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41%
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UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGCUCUCACCGCCGCGGCCCGGGUUCGAUUCCCGGUCAGGGAAUCCCUGGUGGUCUAGUGGUUAGGAUUCGGC..........................................
UCCCUGGUGGUCUAGUGGUUAGGAUUCGG...........................................
UCCCUGGUGGUCUAGUGGUUAGGAUUCG............................................
UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGCU.......................................
UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCG.........................................

IP

50 -

30

3’
5’ A
UA
tRNA-GluCUC CC GG
CG
UA
GC
UUA
G
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Non-immune

C

Tyr
Tyr
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TER
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His
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Gln
Asn
Asn
Lys
Lys
Asp
Asp
Glu
Glu

Ser
Ser
Ser
Ser
Pro
Pro
Pro
Pro
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala

Phe
Phe
Leu
Leu
Leu
Leu
Leu
Leu
Ile
Ile
Ile
Met
Val
Val
Val
Val

2nd

anti-MARWI

T

G

A

C

Fraction of tRNA mapped reads

T

75 tRNAGluCUC 5’50 -tRNAGluCUC 3’

40 30 -

20 20 -FIGURE 2. MARWI associates with tRNA-derived piRNAs. (A) Heat map showing abundance of MARWI-piRNA reads originating from tRNAs foreach codon: (red) higher fraction of reads; (white) lower fraction of reads. Codons for tRNAs with first U at 5′ end are shown in yellow. (B) Pie chart
showing the ratio of tRNA-derived piRNA reads mapped from the first nucleotide of mature tRNAs. The secondary structure of tRNA-GluCUC thatrecognizes the GAG codon is shown together with the region from where piRNAs are derived (in red). The number of reads and piRNA-read sequences are also indicated. (C) Northern blotting with mature tRNA-GluCUC probes targeting either the 5′ or 3′ region of tRNA. Signals were detected in
MARWI immunoprecipitant for 5′ but not 3′ probes.

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pairing-independent manner or modifyfunctions of associated AGO/PIWI proteins (Sobala and Hutvagner 2011).
Some mRNAs in flies and vertebrates
are known to be processed into piRNAs(Robine et al. 2009; Saito et al. 2009;
Siomi et al. 2011). We identified 73
mRNAs that map more than 1000MARWI piRNAs to their 3′ UTRs (Supplemental Fig. S7; Supplemental Table
S6). Marmoset genic piRNAs were also
found to originate from irregular positions in host 3′ UTRs forming pronounced peaks (or hot spots) and gapsof piRNA density as seen in flies andmice (Supplemental Fig. S7A). Similarly,
some transcripts (seven of 73) exhibited
piRNA enrichment in both the 3′ UTR
and protein coding sequence (Supplemental Fig. S7B; Supplemental Table S6;
Robine et al. 2009). We analyzed theexpression levels of these piRNA-generating mRNAs using RNA-seq data andfound that their median RPKM value(5.0) was only slightly higher than thatof total mRNAs (1.7), which is consistent
with previous findings showing thatmouse genic piRNA-generating transcripts are only slightly skewed toward
highly expressed genes, and house-keeping genes are underrepresented (Robine
et al. 2009). These observations suggest
a selective mechanism of genic piRNA
production, although the mechanismthat selects genic piRNA precursors
from all cellular RNAs and directs themfor piRNA processing remains to beelucidated.
piRNA clusters in the marmoset
genomeA
Reads/cluster
1-10,000
10,000 - 100,000
100,000 - 1,000,000> 1,000,000

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X

+ Y >3 1

1 >3Read count (log(10))+ Y -+ X >6 2

2 >6Read count (log(10))

>3 1

1 >3

Read count (log(10))

FIGURE 3. MARWI-associated piRNA cluster. (A) Location of MARWI-piRNA clusters on
marmoset chromosomes. Circles indicate the position of piRNA clusters within each chromosome, and the color of the circles indicates the number of piRNA reads per cluster. Left and rightbars on each chromosome show mapped positions of clusters in positive and negative strands,
respectively. Of genome-mapped reads, 83.4% were included within piRNA clusters and were distributed randomly within most chromosomes. (B) Genome-wide representation of the synteny ofclusters between humans, marmosets, and mice: (green) three species; (red) human and marmoset; (blue) marmoset and mouse. Synteny of piRNA clusters was conserved among species, andthe large ratio is conserved only between the human and marmoset. (C) Example of MARWIpiRNA cluster on chromosome X. Distributions of uniquely mapped MARWI-piRNAs andRNA-seq reads on the X chromosome located piRNA cluster are shown. Blue bar indicates sequence mapped to the sense orientation, and yellow bar indicates sequence in antisense orientation. (D) MARWI expresses from pachytene spermatocytes. Coimmunostaining with MARWI
and γH2AX are shown: (red) MARWI; (green) γH2AX; (blue) DAPI. γH2AX express mitotic
spermatocytes with different forms; some signals are spread around the nucleus, and the others
are restricted to the sex bodies (yellow arrowhead). MARWI protein signals are not detected in
γH2AX diffused cells but are found in sex body–formed cells, where transcription is silenced
on the sex chromosomes. Dashed line indicates MARWI expression boundary.

Genomic mapping of piRNAs in flies,
zebra fish, mice, and humans has shownthat multiple piRNAs map within discrete
genomic intervals, forming piRNA clusters that can exceed 100 kb (Aravin et al.
2006; Girard et al. 2006; Brennecke et al.
2007; Houwing et al. 2007). This pronounced clustering also suggests thatmultiple piRNAs are processed fromlong primary transcripts. To determine if this occurs in marmosets, we mapped MARWI piRNA reads and found a total of
199 piRNA clusters with an average size of ∼17 kb and thelargest at 132 kb (Fig. 3A; Supplemental Table S7; for details
6

7

to define piRNA clusters, see Supplemental Material). This
analysis also indicated that most MARWI piRNAs (83.4%
of piRNAs mapped to the genome) are produced in a clustered fashion, although piRNA clusters constitute just 0.1%

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Small RNA profiling in the marmoset adult testesof the marmoset genome. Both unidirectional clusters, inwhich piRNAs map to only one strand, and bidirectional clusters, in which the polarity of piRNA production switches
between plus and minus strands, were found in the marmoset
genome as seen in other species. Analysis of directional RNAseq data showed that the distribution of RNA-seq reads occurs
in regions where piRNA clusters are located, indicating thatclustered MARWI piRNAs are likely to be derived from thesame single-stranded precursor transcripts.
Although the primary sequences of piRNA clusters are notconserved, their genomic location is highly conserved fromrodents to humans (Aravin et al. 2006, 2008; Girard et al.
2006; Beyret et al. 2012). Indeed, only a small fraction of
MARWI piRNAs could be mapped to the genomes of humanand mouse (7.3% were mapped to the human genome withperfect matches and 4.5% to the mouse genome). To examine if the genomic locations of marmoset piRNA clusters areconserved, we searched for MARWI piRNA syntenic loci inhumans and mice, using a previously published data set(Girard et al. 2006). Chromosomal positions of most previously detected piRNA clusters from humans and mice wereconserved in the marmoset (Fig. 3B). We also detected a large
number of clusters that were likely to be conserved only between marmosets and humans, indicating the existence ofprimate-specific piRNA clusters. However, we also observedthat several clusters are conserved between humans and micebut apparently lost in marmosets, and several others wereconserved between marmosets and mice but apparently lostin humans.
Interestingly, we identified three piRNA clusters on X
chromosome (Fig. 3A,C). From pachytene spermatocyte onward, X and Y chromosome-linked genes are transcriptionally silenced owing to the MSCI (Turner 2007; Heard andTurner 2011). MIWI, the MARWI ortholog in mice, expresses from pachytene spermatocyte to elongating spermatids
during spermatogenensis (Deng and Lin 2002; Di Giacomo
et al. 2013). To determine MARWI expression in detail during spermatogenesis, coimmunostaining with meiotic marker γH2AX was performed. During leptotene to zygotene
spermatocyte, punctate staining of γH2AX is seen throughout the nucleus. In contrast, at pachytene spermatocyte,
γH2AX becomes restricted to the sex body (Mahadevaiah
et al. 2001; Fernandez-Capetillo et al. 2003; Di Giacomo
et al. 2013). MARWI protein signal is not detected in the earlyspermatocyte but is observed in the cytoplasm from thepachytene onward, which is similar to ortholog MIWI (Fig.
3D; Supplemental Fig. S8; Di Giacomo et al. 2013). Thus,
MARWI and MARWI-associated piRNAs express frompachytene onward, suggesting that the X-linked clusters aretranscribed during meiosis in spite of MSCI.
New classes of piRNA clusters
Neither the function of piRNA clusters nor the functional
implication of such extensive synteny in mammals are cur-rently understood, so we further characterized the marmoset piRNA clusters identified in the present study. We
found two new classes of piRNA clusters: clusters consisting of two segments in which the polarity of piRNA andmRNA production switches between the plus and minus
strands (Fig. 4A) and clusters with pseudogenes (Fig. 4B).
piRNA mapping, together with directional RNA-seq data,
revealed that piRNAs mapped to only one strand but not
to the mRNAs (Fig. 4A). In the former class, one of thesestrands encodes the gene named WD-repeat protein 1gene (WDR1), also called actin-interacting protein1 (AIP1),
which encodes a protein promoting cofilin-mediated actin
filament disassembly (Fujibuchi et al. 2005), thereby playing a likely role in round spermatid formation. Budding
uninhibited by benzimidazole 1 (BUB1), which encodes aserine/threonine protein kinase that activates spindle checkpoint assembly and is highly expressed in testes, also forms
a bidirectional cluster-like transcript (Fig. 4A). Importantly,
this gene is essential for spermatogenesis, because the testes
of deficient mice showed impaired normal chromosome
segregation (Perera et al. 2007). A large number of piRNA
clusters in mice appear to be transcribed bidirectionallyby the MYB family of transcriptional factors (Li et al.
2013). This suggests that a single transcriptional factor may
transcribe both piRNA clusters and protein-coding genes
bidirectionally from a single promoter in a coordinatedmanner.
piRNA clusters with pseudogenes
The second class of clusters contains processed pseudogenes.
We found five such piRNA clusters, of which four contain
pseudogenes with an antisense orientation relative to thepolarity of host clusters (Fig. 4B). piRNAs derived fromthese pseudogenes are therefore antisense-oriented relative
to their parental genes, suggesting that they regulate expression of their parental functional genes. The parental genesof these four pseudogenes in this class of cluster include
TROPOMYOSIN-1 (TMP1, also called α-TROPOMYOSIN)
encoding a coiled-coil protein that regulates actin filament
function (Lange et al. 2007), CACYBP (also called SIP) encoding a S100A6- and Siah-1 binding protein with unknown
function (Wasik and Filipek 2013), ANO6 (also called
Tmem16F) encoding a protein that forms a Ca2+-activatedcation channel required for phospholipid scrambling andlinked to Scott syndrome, a rare bleeding disorder caused bya defect in phospholipid scrambling activity (Kunzelmann
et al. 2014), and SSBP3 (also called SSDP1) encoding a nuclear protein that binds single-stranded pyrimidine-richsequences and is involved in transcription (Bayarasaihan
et al. 1998). An in vitro cleavage assay confirmed thatMARWI-piRNA complexes were capable of specifically cleaving target RNAs containing sequences that are perfectly complementary to piRNAs (Fig. 4C). We also confirmed thatMARWI was a cytoplasmic protein (Fig. 3D; Supplemental
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Hirano et al.

A

BpiRNA cluster

GenepiRNA cluster

Pseudogene

WDR1 mRNA

piRNA cluster

5000

5000

MARWI 0-piRNAMARWI 0-piRNA

5000
600

5000
750

RNA-seq 0

RNA-seq 0

600

WDR1

750

isoforms
chr3
181440000

CACYBP-P1
181500000

181560000

chr9111910000

111950000

piRNA cluster
5000

Gene

piRNA cluster

BUB1 mRNA

5000
500

2000

MARWI 0-piRNA

RNA-seq 0

2000500RNA-seq 0500

500

SSBP3-P1
chr1
2000
Chr1_GL284810_random-1456-17180

BUB1
15204000

15192000

nt
150 -

-

+

MARWI
-piRNA 0

-

5000
400

RNA-seq 0
+

EDTA

400

ANO6-P1
chr9

100 80 70 60 50 40 -

16000

5000

Non-immune

MW

C

10000

15216000

anti-MARWI

15180000

Target RNA only

chr14

MARWI 0-piRNA

15040000

15100000

15180000

5000
piR-2
32

P

MARWI 0-piRNA
5000
500

RNA-seq 0

30 32

P

20 -

500

TPM1-P1
chr2

24870000

24876000

FIGURE 4. piRNA clusters contain pseudogenes. (A) Distribution of MARWI-piRNAs and RNA-seq reads where unidirectional piRNA cluster andprotein-coding mRNAs are bidirectionally transcribed. WDR1 and BUB1 gene loci are shown. (B) Examples of piRNA clusters including pseudogenes
in the opposite orientation. CACYBP-P1 (ENSCJAG00000001398) pseudogene, SSBP3-P1 (ENSCJAG00000010873) pseudogene, ANO6-P1 pseudogene,and TMP1-P1 (ENSCJAG00000015442) pseudogene loci inserted in unidirectional and bidirectional piRNA clusters are shown. (C) MARWI-piR-2 complementary RNA incubation with MARWI immunoprecipitates. Cleavage product was found in the MARWI immunoprecipitates, where the cleavage
was inhibited by EDTA. piR-2 is a MARWI piRNA, which occupies the second largest number of reads according to MARWI-piRNA sequencing data.

Fig. S8). These findings together indicate that pseudogenederived piRNAs play important roles in the regulation ofprotein-coding gene expression in the cytoplasm, and alsosuggest a model in which pachytene piRNA clusters regulate
protein-coding genes through the processed pseudogenes
trapped within.
8

RNA, Vol. 20, No. 8

Strong strand bias of piRNAs derived from each
TE subfamily
To further understand TE-derived piRNAs, we first examined the read frequency and strand bias of piRNAs thatmapped to each of the TE subfamilies. We aligned MARWI
piRNA sequences to a comprehensive set of marmoset TE

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Small RNA profiling in the marmoset adult testes

sequences obtained from RepeatMasker (UCSC Genome
Browser track) (Smit et al. 2010), which includes each of
the TE sequences originating from different genomic positions. Reads aligned to multiple sequences were normalized
by the number of aligned sequences. Vertebrate TEs can be
assigned to one of four broad classes according to their mechanism of transposition: long interspersed elements (LINEs),
short interspersed elements (SINEs), long terminal repeat(LTR) retrotransposons, and DNA transposons (Mandal
and Kazazian 2008); they are further classified into distinct
subfamilies according to the degree of deviation from family
consensus sequences (Smit et al. 2010; Sookdeo et al. 2013).
The MARWI piRNA read counts for each TE sequence weregathered by subfamily classes to integrate TEs originatingfrom multiple regions within the genome as one.
We made a list of the top 100 TE subfamilies with highly
mapped piRNA reads and analyzed what fraction of the total
TE-mapped piRNA reads mapped to each TE subfamily (Fig.
5A). We found that most piRNA reads were accumulated at
specific TE subfamilies. Of 918 analyzed TE subfamilies,
88.2% of TE-derived piRNA reads were mapped to the top
100 TE subfamilies, and the top 20 TE subfamilies occupied
more than half (55.8%) of TE-derived piRNA reads. TE-derived piRNAs (∼24%) associated with MIWI in mice have astrand bias skewed to the sense orientation: Two-thirds aresense-oriented (Reuter et al. 2011). To determine whether
this is also true for MARWI piRNAs, we examined the strand
bias of the mapped piRNA reads (Fig. 5A), but found that
48.0% of TE-mapped piRNAs were sense-oriented, indicating no clear strand bias. However, 80.0% of TE subfamily
members were biased to either sense or antisense at a ratio
higher than 75%, suggesting that members within the same
TE subfamily tend to be transcribed unidirectionally independent of their genomic location (see also below).
A fraction of piRNAs originating from TEs is generated by
the ping-pong cycle, which produces secondary piRNAs withping-pong signatures (Brennecke et al. 2007; Gunawardane
et al. 2007). We found that both MARWI piRNAs and 24–
33-nt-long small RNAs were biased for a 5′ -terminal uracil(1U) with no 10th adenine (10A) bias, indicating that theyharbor characteristic of primary piRNAs and that secondary
piRNAs are absent or produced at only low levels (Fig. 1E).
To corroborate this, we searched for piRNAs with initial
10-nt complementarity in both MARWI piRNAs and total
small RNAs (Supplemental Fig. S9A). However, only 2.5%of total small RNAs and 0.2% of MARWI piRNAs had 10nt complementary partners, suggesting that piRNA biogenesis in the adult marmoset testis is mostly, if not completely,
independent of the ping-pong cycle, as in the case of adultmouse piRNAs (Beyret et al. 2012).
Trans-targeting by TE-derived piRNAs
Because these piRNAs are not amplified by the ping-pong cycle and the production of piRNAs was biased to particular TE

subfamilies (Fig. 5A), we hypothesized that these piRNAs may
not only be capable of regulating TEs within the same subfamily but also other subfamily members by mediating thecleavage of transcripts from corresponding opposite strands.
To test this hypothesis, we searched for TEs potentially targeted by MARWI-piRNA complexes (Supplemental Fig. S9B).
Recent studies have revealed that although mismatches at several 3′ -terminal nucleotides of piRNAs are tolerated, basepairing of nucleotides 2–21 is required for efficient target
cleavage by MIWI proteins (Reuter et al. 2011). We therefore
searched for TE partners with perfect matches at the first 2–
21 nt (see Supplemental Material) and found that 9.4% of
TE-derived MARWI piRNAs have potential target TEs otherthan those from the same subfamily. This also implies thatMARWI-piRNA complexes, with Slicer activity (Fig. 4C),
are capable of regulating TEs in trans by cleaving them.
These findings suggest a model in which the mutual cleavageof TE transcripts originating from each subfamily member
sets a threshold of expression for the entire TE family.
piRNA clusters as the major source of TE-derived piRNAs
We found that the amount of piRNA reads produced per TE
was higher for some members of LINEs and SINEs comparedwith LTR retrotransposons and DNA transposons (Fig. 5A;
Supplemental Fig. S10A; Supplemental Table S8). As thismight be caused by the higher activity of recently moved
and therefore relatively younger LINEs and SINEs, wechecked this for each LINE and SINE with higher piRNA production (Fig. 5A). However, both the evolutionarily younger
members (that is, still actively transposing; e.g., L1PA, L1PB,
AluS) and older and ancient members (that is, transpositionincompetent; e.g., L2, L1ME, MIR) (Giordano et al. 2007)
were equally identified as the source of higher piRNA production. Therefore, we concluded that the amount ofpiRNAs produced per TE does not rely on recent TE activity.
We next checked for the correlation between genomic
copy numbers of TEs and mapped piRNA reads per TEs(Fig. 5B) and observed a positive correlation for LINEs and
SINEs. This suggests that the more copies in the genome,
the more piRNAs they generate. On the other hand, the correlation was relatively low for LTR retrotransposons andDNA transposons (see below). In addition, we examined
the annotation of MARWI piRNA cluster regions and foundthat up to 33.7% of the regions were occupied by TEs, suggesting that piRNA clusters serve as a major source for TEderived piRNAs. This is similar to the degree of TE occupancy in mouse pachytene piRNA clusters, although mouse fetal
prepachytene clusters are full of TEs (Lakshmi and Agrawal2008). Thus, we analyzed the correlation between genomic
copy numbers of TEs and copy numbers of the same classof TEs in piRNA clusters (Supplemental Fig. S10B) andfound that they were highly correlated, especially for LINEs
and SINEs. This result together with the observation thatpiRNA production from TEs is dependent on their genomic
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B
SINE

2
R = 0.486

642

n = 146

Mapped piRNA reads (log10)882
R = 0.476

6

4

2

2
4
6
8
Genomic copy number (log10)

LINEn = 52
2
4
6
8
Genomic copy number (log10)

LTR
8

DNA

2

R = 0.058
642

n = 5122
4
6
8
Genomic copy number (log10)

Mapped piRNA reads (log10)

SINE

8

2

R = 0.096
642

n = 2082
4
6
8
Genomic copy number (log10)

C
Average number of piRNA reads per TE subfamily

LINE

SINE

LTR

DNA

131,678

75,21630,463

8,172

905

66

483

240

TEs in piRNA clusters

LTR

piRNA cluster
TEs out of piRNA clusters
piRNA cluster

DpiRNA cluster

piRNA cluster
piRNAs
L1MB7 piRNA cluster

Low copy TEs

High copy TEsex.) L1MB7

DNA

L1:L1PA7
L1:L1MB7
L1:L1MB8
L2:L2
L2:L2b
L1:L1PB1
L2:L2c
L2:L2aL1:L1P2
L1:L1M5
CR1:L3
L1:L1M4
L1:L1ME1
L1:L1ME4a
L1:L1M2
L1:L1P3
L1:L1MA7
RTE:L4L1:L1PB4
L1:HAL1
CR1:L3b
L1:L1MD2
L1:L1ME3A
L1:L1MCL1:L1PA13
L1:L1ME3E
L1:L1PA16
L1:L1MA6
L1:L1MC2
L1:L1MC5
L1:L1MA8
L1:L1MEd
L1:L1PREC2
L1:L1MEc
L1:L1MCa
MIR:MIRb
MIR:MIR
MIR:MIR3
MIR:MIRc
Alu:AluJr
Alu:AluSg
Alu:AluSx
Alu:AluJb
ERV1:HERVS71-int
ERV1:HUERS-P2-int
ERV1:LTR1
ERV1:HERVE-int
ERVK:LTR22B
ERV1:MER4E
ERVL:MER21A
ERVL-MaLR:MLT1C
ERV1:LTR10E
ERV1:LTR10C
ERVL-MaLR:THE1B
ERV1:MER49
ERVL-MaLR:MSTB
ERVL-MaLR:MSTA
ERV1:PABL_A
ERVL-MaLR:MLT1A0
ERV1:HERVH-int
ERV1:MER66D
ERV1:HERV9-int
ERV1:LTR19A
ERV1:MER52AERVL-MaLR:MLT1J
ERV1:MER51AERVK:LTR14
ERVL-MaLR:MLT1F-int
ERV1:MER50-int
ERVL:MLT2A2
ERV1:LTR31
ERV1:LTR26
ERV1:LTR2752
ERV1:MER90a
ERVL-MaLR:MLT1J-int
ERVL-MaLR:MLT1D
ERV1:LTR7C
ERVL-MaLR:THE1D
ERV1:MER51B
ERV1:MER50
ERV1:MER61A
ERV1:LTR9D
ERVL-MaLR:MSTA-int
ERV1:HERV3-int
ERVL-MaLR:THE1A-int
ERV1:MER4D0
ERVL:LTR62
ERVL-MaLR:THE1B-int
ERV1:LTR7B
ERV1:MER52-int
ERV1:MER66AERVL:MER21B
ERV1:HERVFH21-int
ERVK:HERVK14-int
hAT-Charlie:MER103C
hAT-Tip100:MER115
TcMar-Mariner:HSMAR1
hAT-Tip100:MER45A
hAT-Charlie:MER5A
hAT-Charlie:Charlie18a

Mapped piRNA reads (log10)

LINE

Mapped piRNA reads (log10)

Read freaquency (%)
6
4 2
0piRNA reads

A

Cluster insertion

Hirano et al.

Strand-bias
sense
100%

anti-sense
50%

100%

FIGURE 5. Transposable element insertion to piRNA clusters gives rise to the source of piRNA production. (A) Heat maps show strand bias of transposon-derived MARWI-piRNAs for each TE subfamily (left) and the direction of each TE subfamily insertion into piRNA clusters (right).
Transposons are grouped into LINEs, SINEs, LTRs, and DNA transposons. Color intensities indicate the degree of strand bias: (blue) sense; (yellow)
antisense; (gray) unbiased. The frequencies of piRNAs mapped to each TE subfamily over the total TE-mapped piRNAs are shown in the bar graph.
(B) Dot plot shows the relationship between genomic copy number and mapped piRNA reads for LINE, SINE, LTR, and DNA transposons. (C) Table
showing average number of piRNA reads obtained from each group of transposon subfamilies. The values are averaged over transposon subfamilies
with or without insertions into piRNA clusters. (D) Schematic of marmoset piRNA clusters working as “TE-traps.”

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Small RNA profiling in the marmoset adult testes

copy number, suggest that the number of elements transposed to piRNA clusters might determine the piRNA production from each TE.
In the case of LTR retrotransposons, some of the memberswith lower copy numbers are capable of producing higher
amounts of piRNA (Fig. 5B). We surmised that this might
be caused by the genomic position at which LTR retrotransposons have integrated. To test this, we normalized theamounts of piRNAs by the total length of each TE and compared them to the ratio of the TE insertion to piRNA clusters(Supplemental Fig. S10C). Interestingly, we found that LTR
retrotransposons can efficiently produce piRNAs comparedto the other TE family members. Additionally, although theratio of LINEs and SINEs integrated into piRNA clusters
was similar to the ratio of piRNA clusters within the genome(∼0.1%), LTR retrotransposons were enriched in piRNA
clusters. This suggests that LTR retrotransposons might
have preferentially integrated into piRNA clusters.
To ask if the TE copy numbers transposed into piRNA
clusters are correlated with levels of piRNA production, we
analyzed the amounts of piRNAs derived from groups ofTE subfamilies with or without copies in piRNA clusters(Fig. 5C; Supplemental Fig. S10D). We divided TEs into
two groups: one with at least one copy of elements integrated
in a piRNA cluster and the other with no such copies. Whenthe production of piRNAs was compared, the former groupwas shown to produce many more piRNAs. We therefore hypothesize that actively transposed TEs are more likely to jump
into piRNA clusters, and therefore start to produce more
piRNAs. Moreover, when we checked the direction of TE
insertion into each of the piRNA clusters, we found thatthe strand bias was similar to that of highly biased piRNAs(R 2 = 0.48) (Fig. 5A). Therefore, the direction of TE insertion
may be one of the main factors that set the strong sense/antisense bias in piRNA production (Supplemental Fig. S10E).
These findings also suggest a model in which TEs that happen
to jump into piRNA clusters in the opposite direction regulate other subfamily members expressed from different genomic positions (Fig. 5D; Supplemental Fig. S10F). Although
the function of mouse pachytene piRNA clusters is unknown,
our findings also suggest that they act as TE-traps, as seen in
fly piRNA clusters (Malone and Hannon 2009; Karginov and
Hannon 2010).
DISCUSSION
In the present study, we describe the small RNA profile fromthe adult testes of the common marmoset, a small New World
primate, which has been attracting much attention in the research field of biomedical sciences as a model primate species(Sasaki et al. 2009; Orsi et al. 2011; Okano et al. 2012). Our
small RNA profiling confirmed the importance of using thecommon marmoset to analyze primates. Our main findings
were (1) marmoset-specific miRNAs, including a primatespecific X-linked cluster; (2) piRNAs may mediate in transregulation among TE subfamilies; (3) TE-derived piRNAs
biased to either sense or antisense strands of each element;(4) abundantly expressed tRNA-derived piRNA species; and(5) piRNA clusters on the X chromosome, suggesting thatthese clusters are able to evade MSCI, which can be informative for studies of the primate germ line and spermatogenesis. We also revealed some interesting findings that may becommon to species other than primates. These include (1)MARWI piRNAs as the most abundant class of small RNAsin the marmoset adult testis, which show characteristics ofmouse pachytene piRNAs with unknown functions; (2) bidirectionally transcribed coding genes and piRNA clusters; (3)some pseudogenes integrated into piRNA clusters that may
regulate cognate functional genes; and (4) piRNA clusters,
mostly localized to intergenic regions, serving as “traps” forTEs (see below).
piRNAs in the adult testis of the common
marmosetMarmosets encode four AGO and four PIWI subfamily members of Argonautes, of which the expression of MARWI is
overrepresented in adult testes (Supplemental Fig. S3B).
Consistent with this, most piRNAs associate with MARWI(Fig. 1D–F; Supplemental Fig. S1A). Moreover, the predominant class of small RNAs expressed in the marmoset adulttestis is likely to be MARWI piRNAs. These have characteristics of mouse pachytene piRNAs that associate with MIWI
(Aravin et al. 2006; Girard et al. 2006; Reuter et al. 2011;
Beyret et al. 2012), including a higher proportion of intergenic, unannotated sequences (74.0%) with a diminishedcontribution of TE sequences (18.1%) and very few piRNAs
with ping-pong signatures. In addition, mouse piRNA clusters constitute just 0.3% of the genome yet account for 95%
of piRNAs in the adult mouse testis (Li et al. 2013). Similarly,
marmoset piRNA clusters constitute only 0.1% of the genome
yet account for 83.4% of MARWI piRNAs.
We found that within the same TE family (for example,
L1), some subfamily members generate larger amounts ofpiRNAs than others (Fig. 5A). These subfamily-derived
piRNAs, regardless of their strand biases, establish an expression threshold for TE families by cleaving homologous TEtranscripts. Recently, MIWI was shown to be necessary tomaintain the Slicer-dependent silencing of the L1 retrotransposon in the mouse testis after birth (Reuter et al.
2011). This indicates that MIWI Slicer activity directly cleaves
TE mRNAs, thereby silencing TE without piRNA amplification. Two-thirds of TE-derived piRNAs in pachytene piRNAs
that associate with MIWI derive from the sense strand of TEs,
suggesting that the antisense piRNA dosage required for silencing of the L1 is relatively low, probably because of the catalytic nature of Slicer. These findings also suggest that the
predominant generation of antisense piRNAs from some
TEs observed in the adult marmoset testis contribute to silencing of the other family members in that organ.
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Hirano et al.

piRNA clusters in adult marmoset testes stipulatecharacteristics of pachytene piRNAs
We found that the TE group with copies in piRNA clusters
produces larger amounts of piRNAs (Fig. 5C; Supplemental
Fig. S10D). This suggests a model in which TEs that happen
to jump into piRNA clusters regulate other elements in thesame TEs expressed from different genomic positions (Fig.
5D). As originally proposed by Bergman and colleagues
and later elaborated by Hannon and colleagues following Drosophila analysis, piRNA clusters act as “TE traps”
that rely on the innate mobility of TEs to passively acquire
new content by chance transposition (Bergman et al. 2006;
Malone and Hannon 2009; Karginov and Hannon 2010;
Sabin et al. 2013). TEs that have jumped into the clusters
can then become fixed by evolutionary selection and become
part of a silencing program that instructs a small RNA-based
immunity to selectively silence homologous elements ingerm cells, thereby providing an adaptive immunity to thehost genome (Khurana et al. 2011). Although the functionof mouse pachytene piRNA clusters is unknown, our findings
suggest that pachytene piRNA clusters can also capture andefficiently regulate TEs that express in meiotic or post-meiotic cells during spermatogenesis. Indeed, 33.7% of MARWI
piRNA clusters are composed of TEs. Moreover, from theobservation that pseudogenes are found in piRNA clusters(Fig. 4B), we hypothesize that piRNA clusters trap not only
TEs but also other movable genetic elements. Thus, piRNA
clusters may enable the genome to recognize self and nonself and regulate expression of deleterious transposable genetic elements by determining the quality and quantity ofpiRNAs.
On the other hand, we found that LTR retrotransposons
were enriched in piRNA clusters regardless of their abundance in the genome (Fig. 5B; Supplemental Fig. S10C).
The reason for this remains unclear, but one hypothesis isthat there is a specific preference of the region into which
LTR retrotransposons tend to integrate. A previous study indicated that LTR retrotransposons are enriched in open chromatin sites (Jacques et al. 2013), which may include highly
transcribed piRNA cluster transcripts. An alternative explanation would be that LTR retrotransposons might haveplayed the role in generation of piRNA clusters. In fact, it
has been reported that LTR retrotransposons contribute asa source of long ncRNAs (Kapusta et al. 2013). Further analysis would be necessary to reveal this point.

piRNA clusters as possible regulators
for protein-coding genes
Marmoset piRNA clusters harbor not only TEs but also pseudogenes, which tend to be antisense-oriented relative to thepolarity of host clusters (Fig. 4B). Thus, these clusters generate
antisense piRNAs relative to the pseudogenes and their parental genes. Historically, pseudogenes have been regarded
12

RNA, Vol. 20, No. 8

as nonfunctional evolutionary relics (Balakirev and Ayala
2003). Recent evidence indicates that endo-siRNAs arisingfrom pseudogenes in mouse oocytes can adjust the level ofthe founding source mRNA by mediating mRNA cleavage(Tam et al. 2008; Watanabe et al. 2008). By analogy,
piRNAs originating from processed pseudogenes may also
regulate the expression of their parental genes post-transcriptionally by cleaving mRNAs in the marmoset adult testis.
Processed pseudogenes are generated by the reverse transcription of mRNA transcripts followed by integration of thecDNAs into the genome. It is tempting to speculate that justlike TEs, random trapping of the cDNAs into piRNA clusters
can then become fixed by evolutionary selection to selectively
regulate homologous elements in germ cells. In addition,none of the pseudogenes we identified in marmoset piRNA
clusters are found in known mouse piRNA clusters, suggesting that pseudogene-derived piRNAs may have species-specific functions in the testis.
Taken together, the piRNAs and the set of piRNA clusters in marmoset testes defined by our work provide an important insight into the evolutionary origins and functionsof pachytene piRNAs. Future work should analyze small
RNAs associated with each of the eight marmoset Argonautes
and corresponding human proteins in gonads to determine
whether primate-specific mechanisms are deployed to regulate gene expression during germ cell development.
MATERIALS AND METHODS
Animals and tissue collection
This study was approved by the animal ethics committee of theCentral Institute for Experimental Animals (CIEA) and Keio University, and it was performed in accordance with CIEA and Keio
University guidelines. Mice were obtained from CREA Japan, Inc.,and Japan SLC, Inc. Information on individual marmosets is described in Supplemental Table S9.

Generation of anti-MARWI/PIWIL1 monoclonal
antibodies
Anti-MARWI monoclonal antibodies were produced essentially as
described previously (Ishizuka et al. 2002; Nishida et al. 2007).
Briefly, monoclonal mouse antibodies against MARWI were generated by injecting mice with GST-MARWI-N (1–204 amino acids)
and then fusing lymph node and spleen cells with the myeloma
cell line P3U1 to produce hybridomas. Note that the anti-MARWI
antibodies produced also recognize mouse MIWI/PIWIL1 protein(Supplemental Fig. S11).

Small RNA-seq and data processing
To prepare total small RNA libraries, total RNAs from the marmoset testis were extracted using the miRNeasy mini kit (QIAGEN,
217004), and gel-purified small RNAs were cloned using the
NEBNext Small RNA Library Sample Prep Set (NEB, E7330) with

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Small RNA profiling in the marmoset adult testes

slight modifications. Detailed protocols are described in Supplemental Material. Total small RNA reads were sequenced using
MiSeq (illumina), obtaining 18,832,666 reads in total.
To identify MARWI associated piRNAs, coimmunoprecipitated
RNA was extracted from MARWI immunoprecipitates with phenol
and chloroform, and gel-purified MARWI-associated piRNAs werecloned. MARWI-associated piRNA libraries were analyzed by
HiSeq2000 (Illumina) using two replicates from different samples.
Replicates were highly correlated (R 2 = 0.89), so the reads weremerged giving a total of 140,662,017.
Adapter sequences were removed from obtained reads andmapped to the marmoset reference genome (calJac3, WUSTL 3.2)
using Bowtie (Langmead et al. 2009), allowing zero mismatches.
Annotation of the reads was determined according to UCSC
(Meyer et al. 2013) and Ensembl databases (Flicek et al. 2013).
Details of further sequence analysis can be found in Supplemental
Material.

DATA DEPOSITION
Total small RNA sequences, MARWI-associated small RNA sequences, and total RNA sequences data have been deposited in the
NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.
nih.gov/geo/) under accession no. GSE52927.

SUPPLEMENTAL MATERIAL
Supplemental material is available for this article.

ACKNOWLEDGMENTS
We thank members of the Siomi laboratory for helpful discussions
and support, especially Kazumichi M. Nishida for technical assistance with monoclonal antibody generation and Soichiro Yamanaka
for critical reading of the manuscript. We thank Yukiteru Ono forbioinformatic support and Ikuko Sagawa for mass spectrometry analysis. We are very grateful to Cedric Feschotte for critical reading of themanuscript, to Yasunori Aizawa and Hervé Seitz for useful discussions on TE-derived piRNA sequences and MSCI, and to Satomi
Kuramochi-Miyagawa for fruitful discussions on piRNA clusters
and reagents. This work was supported by Grants-in-Aid for Scientific Research to H.S. and M.C.S.; a Grant-in-Aid for the Global
COE program to Keio University to H.S., T.H., Z.Y.-C.L., E.S., andH.O. from the Ministry of Education, Culture, Sports, Science, andTechnology (MEXT) of Japan; the Research Fellowships for Young
Scientists to T.H.; Grants-in-Aid for Young Scientists to Y.W.I.; theFunding Program for Next-Generation World-Leading Researchers
to K.S.; and the “Funding Program for World-leading Innovative
R&D on Science and Technology” to H.O. from the Japan Society
for the Promotion of Science (JSPS).
Received March 14, 2014; accepted May 5, 2014.

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Small RNA profiling and characterization of piRNA clusters in theadult testes of the common marmoset, a model primate
Takamasa Hirano, Yuka W. Iwasaki, Zachary Yu-Ching Lin, et al.
RNA published online June 9, 2014

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