BI84CH25-Siomi ARI 24 February 2015 12:7

Review in Advance first posted online

V I E W
E on March 5, 2015. (Changes may
R

still occur before final publication

S
online and in print.)

C E
I N

A
D V A

PIWI-Interacting RNA: Its

Biogenesis and Functions
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

Yuka W. Iwasaki,1 Mikiko C. Siomi,2,∗

and Haruhiko Siomi1,∗
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

1
Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582,
Japan; email: awa403@[Link]
2
Department of Biological Sciences, Graduate School of Science, University of Tokyo,
Tokyo 113-0032, Japan; email: siomim@[Link]

Annu. Rev. Biochem. 2015. 84:25.1–25.29 Keywords

The Annual Review of Biochemistry is online at piRNA, RNA silencing, RNAi, PIWI proteins, transposon, gene regulation
[Link]

This article’s doi: Abstract

10.1146/annurev-biochem-060614-034258
PIWI-interacting RNAs (piRNAs) are a class of small RNAs that are 24–31
Copyright  c 2015 by Annual Reviews. nucleotides in length. They associate with PIWI proteins, which constitute
All rights reserved
a germline-specific subclade of the Argonaute family, to form effector com-
∗
Corresponding authors plexes known as piRNA-induced silencing complexes, which repress trans-
posons via transcriptional or posttranscriptional mechanisms and maintain
germline genome integrity. In addition to having a role in transposon silenc-
ing, piRNAs in diverse organisms function in the regulation of cellular genes.
In some cases, piRNAs have shown transgenerational inheritance to pass on
the memory of “self ” and “nonself,” suggesting a contribution to various
cellular processes over generations. Many piRNA factors have been identi-
fied; however, both the molecular mechanisms leading to the production of
mature piRNAs and the effector phases of gene silencing are still enigmatic.
Here, we summarize the current state of our knowledge on the biogenesis
of piRNA, its biological functions, and the underlying mechanisms.

25.1

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

Contents
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.2
PIWI PROTEINS AND piRNAs FOR MAINTAINING
GENOME INTEGRITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.4
BIOGENESIS OF piRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.6
Primary piRNA Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.6
Epigenetic Regulation of Transposable Elements by piRNAs . . . . . . . . . . . . . . . . . . . . . 25.8
Secondary piRNA Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25.10
Other Factors Involved in piRNA Biogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25.10
SOURCE OF piRNA PRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25.11
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

piRNA Clusters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25.11

Molecular Process to Define piRNA Clusters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25.13
RECOGNITION OF SELF AND NONSELF BY piRNAs. . . . . . . . . . . . . . . . . . . . . . . . .25.14
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

Acquisition of Novel piRNAs for Regulation of Transposons . . . . . . . . . . . . . . . . . . . . .25.14

Hybrid Dysgenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25.15
Use of Small RNAs for Genome-Wide Recognition of Self and Nonself . . . . . . . . . . .25.17
THE IMPACT OF piRNAs ON PROTEIN-CODING GENES . . . . . . . . . . . . . . . . . . .25.19
piRNA-Mediated Control of Nontransposon Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25.19
Sex Determination and Mating-Type Determination by piRNAs . . . . . . . . . . . . . . . . . .25.20
Nongonadal Function of PIWI Proteins and piRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25.21
CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25.22

INTRODUCTION
Our genome encodes thousands of genes responsible for various cellular functions, and the regula-
tion of the expression levels and patterns of these genes is crucial for development and homeostasis.
This regulation is performed by a collection of intramolecular and intermolecular events. RNA
silencing, also referred to as RNA interference (RNAi), has emerged as one of the key gene regu-
latory pathways in most eukaryotes (1, 2). Central to RNA silencing pathways is the generation of
small RNAs of 20 to 31 nucleotides (nt). These form an RNA-induced silencing complex (RISC)
with Argonaute proteins and recognize their targets via Watson–Crick base pairing. The Slicer en-
donuclease activity of Argonaute proteins cleaves target transcripts to accomplish gene silencing;
posttranscriptional gene silencing, such as translational repression, or transcriptional gene silenc-
ing via specific chromatin modifications is performed through recruitment of other proteins (3).
Argonaute proteins are divided into two subfamilies, the Argonaute (AGO) and PIWI families
(4). AGO subfamily proteins are ubiquitously expressed and can bind to microRNAs (miRNAs) and
small interfering RNAs (siRNAs), both of which are processed from double-stranded precursors
into mature small RNAs of 20 to 22 nt in length in a Dicer-dependent manner (Figure 1) (3).
PIWI subfamily proteins (PIWI proteins) are expressed mainly in germline cells and form specific
RISCs with a small RNA population known as PIWI-interacting RNAs (piRNAs); these RISCs
are termed piRISCs.
piRNAs are slightly longer (24–31 nt) than miRNAs and siRNAs, possess 2 -O-methyl mod-
ification sites at the 3 terminus, and are processed from single-stranded precursor transcripts
expressed from intergenomic regions termed piRNA clusters via a Dicer-independent mecha-
nism (5, 6). piRNA clusters harbor a large number of and various types of transposons; therefore,

25.2 Iwasaki · ·Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

Small RNA loci

Transcription and processing

miRNA precursors esiRNA precursors piRNA precursors
Small RNA precursors

Hairpin-RNA dsRNA or Hairpin-RNA ssRNA

Dicer-dependent Dicer-independent
processing processing

miRNA esiRNA piRNA

~22 nt ~21 nt 24–31 nt Mature small RNAs
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

P OH P OH P OCH3

Formation of RISC
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

U Human RISC
AGO1–4 AGO1–4 HIWI HILI HIWI2 PIWIL3

U U U Mouse RISC
AGO1–4 AGO1–4 MIWI MILI MIWI2

U U U Drosophila RISC
AGO1 AGO2 Piwi Aub AGO3

Argonaute subfamily PIWI subfamily

(ubiquitously expressed) (mainly enriched in germline)

Argonaute family

Coding Transposons Transposons

genes and and Major target genes
exogenous other genes
genes

Figure 1
RNA silencing by small RNAs and their partner Argonaute family proteins in human, mouse, and Drosophila.
Expression of a fourth PIWI protein (PIWIL3) has been detected specifically in humans. Characteristics of
piRNA precursors, mature sequences, RISC formation, and target genes are summarized for miRNAs,
esiRNAs, and piRNAs. An association between piRNAs and human PIWI proteins has not yet been
identified. Abbreviations: dsRNA, double-stranded RNA; esiRNA, endogenous small interfering RNA
(siRNA); miRNA, microRNA; nt, nucleotide; piRNA, PIWI-interacting RNA; RISC, RNA-induced
silencing complex; ssRNA, single-stranded RNA.

piRNAs regulate mainly the activity of transposons, namely their expression and transposition
within the genome. Because transposition of transposons has a high risk of damaging the genome
intracellularly, the piRNA-mediated regulation of transposons is essential, especially for preserving
normal gametogenesis and reproduction. Owing to their processing mechanisms and the variety
of source transposons, piRNA sequences are much more diverse than those of any other known

[Link] • Biogenesis and Functions of piRNA 25.3

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

class of cellular RNAs and constitute the largest class of noncoding RNAs (6, 7). Transposon
regulation by piRNAs is conceptually similar to that in immune systems, which can achieve “self ”
and “nonself ” recognition. As with our immune systems, piRNAs use a complex mechanism to
effectively select and regulate the nonself genes for regulation (8).
Recent studies have revealed a large number of cytoplasmic factors that support and maintain
piRNA biogenesis, as well as some nuclear factors that either recognize and transcribe piRNA
clusters to produce piRNA precursors or function in piRNA-mediated transcriptional silencing.
Additionally, analyses of various eukaryotes have identified piRNAs that target protein-coding
genes and piRNAs that are passed through generations to transmit a memory of past transposon
activity (9–11). Here, we discuss how piRNAs are generated and how they function to recognize
and regulate their target genes. We focus mostly on recent research in the model animals Drosophila
melanogaster and mouse, but we also discuss important findings in other model animals.
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

PIWI PROTEINS AND piRNAs FOR MAINTAINING

Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

GENOME INTEGRITY
PIWI proteins and piRNAs are conserved in a wide range of eukaryotes, from sponges to humans,
and they are expressed mainly in the gonads (6, 12). The prototype of PIWI proteins is encoded
by the Drosophila piwi (P-element-induced wimpy testes) gene, which was originally identified as
an essential gene for germline development (13, 14). In Drosophila, which contains three distinct
PIWI genes [ago3, aubergine (aub), and piwi], piwi and aub are required for both male and female
fertility, whereas ago3 is essential for female fertility (14–17). Derepression of transposons is
observed in each of these PIWI mutant ovaries, indicating that all three PIWI proteins have
nonredundant roles in gonad development and transposon silencing. Aub and AGO3 cleave their
target transposon transcripts in the cytoplasm, whereas Piwi can regulate its target transposons at
the transcriptional level in the nucleus (15, 18–22). Interestingly, the Drosophila piRNA pathway
also regulates transposon activity to maintain the telomeres. Unlike most other eukaryotes, the
transposition of a distinct set of transposons to the chromosomal ends maintains the telomeres of
Drosophila chromosomes, whereas defects of the piRNA pathway in gonads reduce expression of
telomere-specific piRNAs and disrupt assembly of the telomere protection complex. Meanwhile,
piRNA pathway defects do not affect transposon expression or telomere structure in somatic
tissues (23–25).
Mice also express three PIWIs (MIWI, MIWI2, and MILI). All three PIWI proteins are
expressed at different stages during spermatogenesis (Figure 2), but only MILI is expressed,
albeit weakly, in female germ cells. Mutations in mouse Piwi genes affect the male germline but
not the female germline (26–29). Deficiency in MILI or MIWI2 leads to the activation of long
interspersed nuclear element and long terminal repeat (LTR) retrotransposons including L1 and
IAP elements, and spermatogenic stem cell arrest is observed. During MIWI depletion, the L1
transposon is also dysregulated, and spermatogenesis is arrested at the early spermatid stage (26,
28, 30–32).
Mouse PIWI proteins are bound to piRNAs expressed in two phases: prepachytene piRNAs
and pachytene piRNAs. Prepachytene piRNAs are derived mostly from transposable elements and
are associated with MILI and MIWI2 in the gonocyte stage, whereas pachytene piRNAs originate
from piRNA clusters located in various regions of the genome and bind to both MILI and MIWI in
pachytene spermatocytes to the round spermatid stage. Although a fraction of pachytene piRNAs
originates from transposons, the largest fraction comprises those originating from an unannotated
region (27–29, 31, 33). MIWI and MILI are necessary to maintain the Slicer-dependent silencing
of the L1 transposon in the mouse testis after birth, indicating that Slicer activity directly cleaves

25.4 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

Germ cell

Primordial germ cell

migration E7.5
Prepachytene piRNA cluster
Transposons
Genome
demethylation E12.5
PTGS

Ping-
pong
Cell cycle arrest E15.5
MILI MILI
Gonocyte

De novo DNA Prepachytene

methylation Birth

MIWI2
piRNA
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

P3
MIWI2
Nuclear
import TGS
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

5meC
Spermatogonia P6.5 De novo DNA
methylation

Leptotene Pachytene piRNA cluster

spermatocyte P10
A-MYB
Meiosis

Pachytene Intergenic
P14 regions
spermatocyte
Pachytene
piRNA
Round
MILI

spermatid P20
MILI MIWI
MIWI

Elongated
P27 PTGS
spermatid
PIWI protein
expression

piRNA
expression

Sperm P30

Figure 2
Expression patterns of MILI, MIWI, MIWI2, and mouse piRNAs during spermatogenesis. piRNAs are
classified into pachytene piRNAs and prepachytene piRNAs on the basis of the stage in spermatogenesis
when they are expressed. In gonocytes, MILI and MIWI2 bind to piRNAs from a prepachytene piRNA
cluster, which consists mainly of transposons. MILI performs the homotypic ping-pong cycle to silence
targets by PTGS and produce piRNAs that associate with MIWI2. MIWI2 localizes to the nucleus upon
piRNA loading to accomplish nuclear silencing by de novo DNA methylation. Beyond the pachytene stage,
MIWI and MILI are bound to piRNAs from pachytene piRNA clusters, a large fraction of which consists of
intergenic regions. Pachytene piRNAs regulate their target genes by PTGS in the cytoplasm. Abbreviations:
piRNA, PIWI-interacting RNA; PTGS, posttranscriptional gene silencing; TGS, transcriptional gene
silencing.

[Link] • Biogenesis and Functions of piRNA 25.5

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

transposon messenger RNAs (mRNAs) (Figure 2) (34, 35). Moreover, mouse PIWI proteins
function not only in posttranscriptional gene silencing by cleaving transposon transcripts, but
also in transcriptional silencing by directing CpG DNA methylation on transposon loci. Indeed,
silencing and de novo DNA methylation of L1 and IAP elements are decreased in male germlines
that are defective for the activity of the MILI or MIWI2 gene (27–29). This finding indicates
that mouse piRNAs guide specific de novo DNA methylation to silence their target transposons.
Because mouse PIWI proteins are expressed in a developmental stage–specific manner (29), the
proper removal of PIWI proteins is essential for male germ-cell development. It was recently
reported that ubiquitination of piRNA-associated MIWI triggers degradation of the MIWI–
piRNA complex via the APC/C–26S proteasome pathway (36).
Although transposons are the major targets of PIWI–piRNA complexes, how regulation of
transposons is connected to defects in gametogenesis remains unknown. Activation of transposons
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

in PIWI protein mutants may lead to the generation of double-stranded DNA breaks during
abortive or successful transposition that activate a DNA damage checkpoint, resulting in a sterile
phenotype (37). Additionally, loss of the piRNA biogenesis factor Maelstrom (Mael) in mouse
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

results in acrosome and flagellum defects that lead to spermiogenic arrest (38). Thus, tissue-
specific and developmental timing–specific expression of PIWI proteins and piRNAs may play
major roles in maintaining the integrity of the genome and fertility of the organism. However, a
mutation in Drosophila piwi that leads to transposon activation does not directly affect germline
development (39). Also, derepression of L1 transposons by deletion of a part of a pachytene piRNA
cluster does not affect spermatogenesis in mouse (40), suggesting that transposon silencing and
germline development can be separated.

BIOGENESIS OF piRNAs
Two major pathways generate piRNAs: the primary processing pathway and the ping-pong cycle
that amplifies secondary piRNAs (Figure 3). Primary piRNAs have a bias toward having uridine
(U) at their 5 nucleic acid (1U bias), whereas secondary piRNAs show 10-nt complementarity with
primary piRNAs at their 5 ends and possess a sense bias with adenosine at the tenth nucleotide
(10A bias) (5, 21, 22, 29–31).

Primary piRNA Biogenesis

In Drosophila ovaries, the primary pathway operates in both germline and surrounding somatic
cells, whereas the ping-pong cycle operates only in germline cells. Within the primary pathway
in Drosophila ovarian somatic cells, only Piwi is expressed among PIWI subfamily members, and
piRNA precursors are transcribed from piRNA clusters such as the flamenco ( flam) locus as long
unidirectional single-stranded transcripts and processed in multiple steps (Figure 4a) (21). The
flam locus harbors a large number of truncated transposons, most of which are antisense oriented
relative to transposon coding strands (21, 41, 42). Long primary transcripts from the piRNA
clusters are exported to the cytoplasm and possibly processed into intermediates, but the detailed
processes are still largely unknown. Although an endonuclease, Zucchini (Zuc), which is located
on the surface of mitochondria, is required for processing precursor RNAs (43–46), whether Zuc
preferentially produces precursor RNAs with U at the 5 end remains to be addressed. The loading
and subsequent maturation steps probably occur within perinuclear granules termed Yb bodies,
which are also formed on the surfaces of mitochondria (Figure 4b); moreover, components of
Yb bodies, including fs(1)Yb (Yb), Armitage (Armi), Vreteno (Vret), Shutdown, and Sister of Yb
(SoYb), are essential for producing mature piRNAs (Table 1) (47–52). Additionally, Minotaur

25.6 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

Primary pathway
Nucleus Cytoplasm
Single-stranded Dual-stranded
piRNA clusters Genic piRNAs piRNA clusters Transposons
3' UTR
Argonaute3 (AGO3)

piRNA
precursor Cytoplasmic silencing
U
U
piRNA A ?
intermediates U U

Piwi Aubergine U A
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

U (Aub) Maternal
U inheritance Ping-pong pathway
?
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

A
Nuclear silencing U piRNA precursor

Red: sense to transposons U

Blue: antisense to transposons
A

Figure 3
Biogenesis pathway of Drosophila piRNAs, consisting of the primary and ping-pong pathways. In the primary pathway, piRNAs are
transcribed from genomic regions called piRNA clusters, processed, and loaded onto Piwi or Aub. 3 -UTR sequences of some
protein-coding genes can also serve as a source of primary piRNAs. Silencing takes place both in the cytoplasm and nucleus (also see
Figure 4). Piwi performs transcriptional gene silencing in the nucleus. Together with AGO3, the Aub–piRNA complex serves as a
trigger to start the ping-pong amplification pathway. The ping-pong pathway silences the target transposon sequence and amplifies the
piRNA sequence at the same time. Note that some Aub–piRNA complexes are also maternally inherited. Abbreviations: piRNA,
PIWI-interacting RNA; UTR, untranslated region.

(Mino) and GasZ are localized to mitochondria and function in primary piRNA processing (53,
54). Although these connections between piRNA biogenesis and mitochondria have emerged,
whether some mitochondrial activity is required for piRNA biogenesis is unclear. Within the
process, however, the precursors are further trimmed to the mature piRNA size by the activity of
an unknown 3 –5 exonuclease (55). The DmHen1/Pimet methyltransferase then 2 -O-methylates
the 3 ends of piRNAs to produce mature Piwi–piRNA complexes or Piwi–piRISCs (56, 57). Piwi–
piRNA complexes are then imported into the nucleus to transcriptionally regulate target genes
(58). piRNA-free Piwi stays in the cytoplasm, suggesting that the Yb body is where functional
piRISC formation is assessed: Only functional complexes can be imported into the nucleus (47,
48). Interestingly, transcripts (or processed intermediates of them) derived from the flam locus
accumulate at perinuclear foci adjacent to the Yb body, termed Flam bodies (59). The formation
of a Flam body depends on the RNA-binding activity of Yb, indicating that Yb integrates primary
piRNA transcripts or their intermediates into the Flam body.
In Drosophila germline cells, primary piRNAs are also derived from dual-stranded piRNA
clusters such as the 42AB locus and loaded onto both Aub and Piwi to form piRISCs (Figure 1). The
amount of germline piRNAs is reduced in Drosophila mutants upon Armi depletion (60), indicating
that some of the somatic factors described above are also required for germline piRNA biogenesis.
However, the germline counterparts for components of Yb bodies remain to be identified. Thus,
the primary piRNA pathway partly differs between somatic and germline cells, and the details of
how the primary pathway operates in Drosophila germline cells are still unknown.

[Link] • Biogenesis and Functions of piRNA 25.7

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

a
piRNA cluster (e.g., flam) Neighboring coding gene Histone marks (H3K9me3)
Pol II
Pol II
Transposons Epigenetic regulation
Mostly antisense-oriented by Piwi–piRNA
transposable elements

piRNA Mael HP1a

precursor
DmGTSF1
U
Nucleus
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

Cytoplasm
piRNA
intermediate
U Piwi b Mito
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

Yb N

Flam body ? 3' trimming

and
2'-O-methylation Mito
HSP83 Hen1
Mito
Zuc Vret
Yb Shu
U
Yb body
Armi Piwi
Mitochondrion
GasZ

Figure 4
Epigenetic silencing by Piwi–piRNA in Drosophila. (a) Piwi, a Drosophila PIWI protein, is localized to the nucleus and can epigenetically
silence target genes. Transcripts of piRNA clusters, which contain numerous sequences complementary to transposons, serve as
precursors of piRNAs. piRNA precursors are processed into piRNA intermediates and exported to the cytoplasm. Intermediates are
processed by the endonuclease Zuc near the mitochondria, localized to granules termed Flam bodies, and then to Yb bodies, where
factors such as Yb, Armi, Vret, and Shut are localized. Armi is recruited to mitochondria by Gasz. Here, piRNAs are processed and
loaded onto Piwi. Then, piRNAs are 3 trimmed and 2 -O-methylated by Hen1 and transferred into the nucleus. Within the nucleus,
Piwi–piRNA complexes regulate their target genes by modifying histone marks and association of Pol II with target genes. Several
factors, such as DmGTSF1, Mael, and HP1a, are involved in this process, but the regulatory mechanism remains to be revealed.
(b) Immunoelectron microscopy using an anti-Yb antibody shows a perinuclear Yb body in an ovarian somatic cell (red arrowhead ).
Abbreviations: Armi, Armitage; Mael, Maelstrom; Mito, mitochondria; N, nucleus; piRNA, PIWI-interacting RNA; Pol II, RNA
polymerase II; Shut, Shutdown; TE, transposable element; Vret, Vreteno; Yb, fs(1)Yb; Zuc, Zucchini. Scale bar, 0.2 μm.

Mammalian orthologs of Drosophila somatic primary piRNA biogenesis factors have been iden-
tified (Table 1) (61–65). For example, a mouse ortholog of Zuc is MitoPLD, which is also a
mitochondrial protein involved in piRNA production (63, 65). Additionally, MOV10L1, a mouse
ortholog of Armi, is an RNA helicase that is essential for piRNA biogenesis (62, 66). Thus, pri-
mary piRNAs in mammals may be produced via pathways that are similar to the Drosophila somatic
primary pathway, although the details have not been explored.

Epigenetic Regulation of Transposable Elements by piRNAs

After Piwi–piRISCs are imported to the nucleus, they direct methylation of histone 3 lysine
9 (H3K9me3) on chromatin at target transposon loci to induce heterochromatin formation,

25.8 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

Table 1 Factors involved in the piRNA pathway

Interacting Homolog in
Protein name Domain PIWI protein Expression mouse Reference(s)
Piwi Paz, Mid, Piwi ND Gonad MIWI 83, 91, 128
Aub Paz, Mid, Piwi Ago3 Gonad MILI 15, 128
Ago3 Paz, Mid, Piwi Aub Gonad MIWI2 15, 47, 48
Tud Tudor Aub, Ago3 Ubiquitous TDRD6 85
PAPI Tudor, KH Ago3, Piwi Ubiquitous TDRD2 88
Spn-E Tudor, DEAD, Hel-C, HA2 Aub Gonad TDRD9 76, 91
Qin/Kumo Tudor, RING, B-box Piwi, Aub Gonad TDRD4 89, 90
Tejas Tudor, Lotus Aub Gonad TDRD5 91
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

Yb Tudor, Hel-C, DEAD Piwi Gonad ND 47–50

BoYb Tudor, DEAD, Hel-C NA Gonad ND 50
SoYb Tudor, DEAD, Hel-C NA Gonad TDRD12 50
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

Vret Tudor, RRM, MYND Piwi, Aub, Ago3 Gonad ND 49, 50

Krimp Tudor NA Gonad ND 76, 92
Egg/SetDB1 Tudor, MBD, preSET, SET, NA Ubiquitous SETDB1/ 74
postSET SETDB2
Vasa DEAD, Hel-C Aub Gonad MVH 91
Armi P-loop NTPase, Uvr_D_C_2 Piwi Gonad MOV10 47–50
Shu PPlase, TPR Piwi Gonad FKBP6 87, 51
Zuc PLD-like Piwi, Aub Gonad MitoPLD 43–47
Squ RNase HII-like Piwi, Aub Gonad ND 45, 46
Mino GPAT NA Ubiquitous GPAT1, GPAT2 54
GasZ Ankyrin-rpt, SAM, NA Ubiquitous GASZ 53
Mael Mael NA Gonad Mael 58
DmGTSF1/Arx UPF0224_CHHC_Znf Piwi Gonad GTSF1 70–72
Rhi Chromo NA Gonad Cbx5 95–97
Cuff RAI1 NA Ubiquitous Dom3z 95, 96
Del NA NA Gonad ND 95, 96

Abbreviations: NA, not available; ND, not determined; piRNA, PIWI-interacting RNA.

thereby transcriptionally silencing transposons (Figure 4) (39, 58, 67–69). A nuclear protein
termed Asterix/DmGTSF1 associates with Piwi–piRISCs to mediate the addition of this silencing
histone mark (70–72). Mael is also involved in Piwi-piRISC-mediated transposon silencing in the
nucleus, but it is not required for the establishment of H3K9me3 on silenced transposon loci
(58).
How H3K9me3 marks are deposited at piRNA target regions is still being discussed, but
the involvement of the methyltransferases Eggless (Egg)/SetDB1 and Su(var)3–9, as well as the
heterochromatin protein HP1a, has been suggested (71, 73–75). However, it is not clear whether
Piwi directly associates with HP1a, which triggers recruitment of methyltransferases, or if Piwi
first recruits methyltransferase or some other factor to deposit the H3K9me3 mark, resulting in
an association between Piwi and HP1a (10). Therefore, the precise mechanisms have yet to be
revealed.

[Link] • Biogenesis and Functions of piRNA 25.9

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

Secondary piRNA Biogenesis

Aub–piRISCs initiate the ping-pong cycle in the cytoplasm together with AGO3 to produce sec-
ondary piRNAs (Figure 3). The ping-pong pathway is believed to take place at an electron-dense,
nonmembranous perinuclear structure known as the nuage because both PIWI proteins and many
other factors involved in piRNA biogenesis accumulate in the structure (76). In the ping-pong
pathway, AGO3 and Aub act in a complementary fashion to cleave sense and antisense transposon
transcripts via their Slicer activities. piRNA generation in a feed-forward mechanism results in the
consumption of transposon transcripts, thereby silencing transposons (21, 22). piRNAs produced
in the ovary are also deposited in the embryo as Aub–piRISCs, which additionally boost piRNA
production by initiating the ping-pong cycle in the ovaries of the offspring (77).
Slicer activity directs cleavage of its cognate target RNAs across the position between 10 and
11 nt, measured from the 5 end of the associated small RNA (78). Thus, reciprocal cleavage of
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

transposon transcripts by AGO3–piRISCs and Aub–piRISCs determines the 5 end of secondary

piRNAs, although how their 3 ends are formed is still unknown. As Aub-bound primary piRNAs
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

show an antisense and 1U bias, secondary piRNAs loaded onto AGO3 show 10-nt complemen-
tarity at their 5 ends with Aub-bound piRNAs and possess a 10A bias. The characteristic features
of piRNAs with 1U/10A partners and a 10-nt 5 overlap in the pathway are often referred to
as the ping-pong signature (21, 22). Although the precise mechanism remains unknown, recent
studies (79, 80) have begun to address how Slicer-mediated cleaved products are handed to a ping-
pong partner PIWI: Vasa, a conserved RNA helicase and a component of the nuage, facilitates
release of 5 sliced piRNA precursors from piRISCs via an ATP-dependent mechanism, sug-
gesting that Vasa promotes transfer of cleaved piRNA intermediates between ping-pong partner
PIWIs.
In mouse testis, MILI associates with primary piRNAs and hands secondary piRNAs to MIWI2.
However, heterotypic MILI–MIWI2 ping-pong may not operate, because, once loaded with sec-
ondary piRNAs, MIWI2 is imported into the nucleus to direct specific DNA methylation on
transposon loci (28, 29, 34, 35). Therefore, it would be difficult to provoke another round of
ping-pong with MILI, which is localized to the cytoplasm. This finding suggests that mouse ping-
pong may be a one-way process (Figure 2). However, some evidence supports the possibility
that MILI–piRNA complexes perform homotypic ping-pong to amplify piRNAs (35). Deviations
from the primary and ping-pong paradigms have also been observed in several organisms including
nematodes and cilia (see below) (9, 10).

Other Factors Involved in piRNA Biogenesis

In addition to the factors described above, the functions of various proteins, such as Tudor domain–
containing (TDRD) proteins, are indispensable for the piRNA biogenesis pathway (Table 1).
TDRD proteins interact with proteins with symmetrical dimethyl arginine (sDMA) or asymmet-
rical dimethyl arginine (aDMA) modifications. Moreover, some TDRD proteins also function
in RNA metabolism (81). PIWI proteins harbor sDMA modifications in their N-terminal re-
gions, and the modifications are likely to be substrates for TDRD proteins (82–86). TDRD
proteins that are involved in the Drosophila piRNA pathway include Tudor, Partner of PIWIs
(PAPI), Qin/Kumo, Tejas, Spindle-E (Spn-E), Yb, SoYb, Brother of Yb (BoYb), Krimper (Krimp),
Egg/SetDB1, and Vret (47–51, 64, 76, 85, 87–92). As described above, some of these proteins
function in the primary pathway, and the others function to perpetuate the ping-pong pathway.
For example, Tudor, a large TDRD protein containing 11 Tudor domains, interacts with Aub
and AGO3 in an sDMA-dependent manner to provide a platform for ping-pong amplification.

25.10 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

Although piRNAs are still loaded onto Aub and AGO3 in tudor mutants, the identity of piRNA
sequences is significantly changed from that in the wild type, suggesting that Tudor is essen-
tial for quality control of piRNAs produced from the ping-pong cycle (85). Qin/Kumo, another
TDRD protein containing five Tudor domains, also functions to maintain the ping-pong cycle.
In qin/kumo mutants, Aub and AGO3 cannot localize to the nuage (90), and Aub starts to perform
the homotypic ping-pong cycle, which is weaker than the ping-pong cycle of Aub and AGO3 (89).
This finding suggests that Qin/Kumo plays an essential role in maintaining the heterotypic ping-
pong of Aub and AGO3 (89, 90). Interestingly, in silkworms, Qin/Kumo, together with a silkworm
homolog of Spn-E, functions in primary piRNA processing (80). Mutations in genes encoding
TDRD proteins have relatively mild effects on piRNA production compared with mutations in
other piRNA biogenesis factors, probably because some TDRD proteins cooperatively function to
maintain piRNA biogenesis. Therefore, it would be important to understand the precise function
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

of each TDRD protein and reveal links between them to fully understand piRNA biogenesis.
To gain comprehensive knowledge of the factors involved in piRNA biogenesis, several large-
scale screenings of factors involved in the Drosophila piRNA pathway have been performed
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

(Figure 5). Hannon’s group (71, 75) has screened for germline piRNA pathway factors using
transgenic RNAi flies and for somatic piRNA pathway factors using double-stranded RNA library
transfection into an ovarian somatic sheet cell line. These screenings were performed indepen-
dently, but many common factors were identified. The genes identified as being involved in the
germline piRNA pathways included those related to transcriptional elongation, RNA export, pro-
tein nuclear import, and chromatin modification. The somatic piRNA factors identified included
those related to functions such as exon junction complex formation, general RNA metabolism
factors, and those involved in the SUMOylation machinery.
Brennecke’s group (53) has identified ∼50 factors involved in piRNA biogenesis. For each line
of transgenic RNAi flies, these authors performed primary screening by analyzing a β-galactosidase
reporter system constructed using the LTR from the gypsy transposon, which serves as a source of
piRNAs in both the soma and germline piRNA pathways. The results suggest connections between
the piRNA pathway and mitochondrial metabolism, nuclear pore complex factors, transcriptional
elongation factors, and chromatin biology factors, among others. Using the burdock transposon, an
element that is involved only in the germline piRNA pathway, as a reporter, these researchers also
identified factors specifically involved in the germline piRNA pathway. They revealed factors that
are involved in ping-pong amplification by Aub/AGO3 and processing of dual-stranded piRNA
clusters, such as Rhino (Rhi) (see below). As a result of these reports, we now have a good blueprint
for what types of genes and machinery are involved in piRNA pathways. These factors must now
be analyzed in greater depth if we are to understand how they are involved in piRNA production
and to elucidate the mechanisms by which piRNAs silence transposons.

SOURCE OF piRNA PRODUCTION

piRNA Clusters
piRNA clusters have been identified as genomic regions where a large number of piRNA reads are
uniquely mapped. They often reside within or close to heterochromatin. The length of piRNA
clusters ranges from a few kilobases to hundreds of kilobases (21, 60). For example, the flam locus,
a major somatic primary piRNA cluster in the Drosophila X chromosome, is transcribed into an
∼180-kb-long single-stranded transcript. As described above, most of the flam transcript sequences
correspond to transposons, including gypsy, idefix, and ZAM, in an antisense orientation (Figure 6a)
(42). Thus, piRNAs derived from the cluster can target sense transcripts produced from cognate

[Link] • Biogenesis and Functions of piRNA 25.11

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

Transcriptional elongation Transcriptional regulation Transcriptional silencing

Atu CG9915 lin-52 NSL2 wde Cul-4
CG3909 CG40228 MBD-R2 NSL3 His2Av Mi-2
TfII-S SPT6 mof NSL1 asf1
Rcd5
Pol II H3K9me3
Nuclear cap binding
Transposons
Ars2 cbp20
cbp80
Piwi
Pol II piRNA cluster H3K4 methylation
SUMOylation Set1 wds
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

Hcf Rbbp5
smt3 Uba2
Wdr82 Cfp1
lwr Ulp1
dpy30 ash2
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

piRNA Aos1
precursor
RNA export Exon junction complex
NXT1 thoc5 mago Acn
NXF1 thoc7 tsu(Y14) RNPS1 Nuclear pore complex
NXF2 thoc6 eIF4AIII Bin1
NUP154 tho2 btz Upf3 Nup58 CG11092
NUP43 Hpr1 Pnn Upf2 Nup54 CG14749
aly SRm160 Nup214

Nucleus
Nuclear import
Cytoplasm
karybeta3
piRNA 3'-end formation
intermediate
Rrp6 Csl4

Mitochondrial function Yb body

Mitochondrial function genes

Piwi–piRNA
Mitochondrion

Figure 5
Examples of biological functions and factors newly identified by genome-wide screening to be involved in the piRNA pathway. Factors
identified by screenings performed by Hannon’s group ( green) (71, 75), Brennecke’s group (blue) (53), and both groups (red ) are listed
along with their annotated functions. Abbreviations: piRNA, PIWI-interacting RNA, Pol II, RNA polymerase II; Yb, fs(1)Yb.

transposons dispersed throughout the genome. Another type of piRNA cluster, namely the 42AB
cluster in germline cells, is transcribed from both strands as a dual-stranded transcript. piRNAs
generated from this type of cluster are integrated into both the primary pathway and the ping-
pong cycle (21). Unlike the flam locus, dual-stranded piRNA clusters contain transposons that
are sense and antisense oriented, mostly in a random manner, relative to the polarity of the
locus transcription; therefore, it is unclear how the antisense bias observed for the germline
primary piRNAs is generated initially. Perhaps Aub may associate with both sense and antisense
primary piRNAs. Because the ping-pong cycle requires ongoing expression of the cluster and target

25.12 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

transposons, the amplification loop steers piRNA production toward transcriptionally active and
highly expressed transposons (Figure 3). Therefore, as long as there is an input of active transposon
transcripts, antisense piRNAs are preferentially produced for Aub and the bias can be maintained.
However, how the antisense bias for Piwi-bound piRNAs is enforced is enigmatic because, as in
MIWI2, once loaded with piRNAs, Piwi is imported into the nucleus (47, 48); thus, it is unlikely
to participate in the ping-pong cycle with AGO3, which is localized to the cytoplasm.
In mammals, the genomic location or synteny of piRNA clusters is highly conserved, although
their primary sequences are not conserved (27, 30, 31, 93). Two types of piRNA clusters exist in
mouse: unidirectional piRNA clusters and bidirectional piRNA clusters (Figure 2) (27, 29, 31, 33).
As in Drosophila, some mouse piRNA clusters are transcribed as a single strand, frequently spanning
a long region of the genome; these are termed unidirectional piRNA clusters. Bidirectional piRNA
clusters are transcribed both from sense and antisense strands, but unlike dual-stranded piRNA
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

clusters in Drosophila, they are transcribed from a single central promoter. Transcription then
switches to the opposite direction. A MYB-related protein underlies transcription of these piRNA
clusters in mouse (94).
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

Molecular Process to Define piRNA Clusters

It was unknown for a long time how transcripts of the piRNA clusters can produce piRNAs
and how they are protected against degradation to be relatively long transcripts. Dual-stranded
piRNA cluster transcripts have unique characteristics: They lack a clear promoter, 5 methyl-
guanosine caps, and clear transcription termination (95). Also, they seem not to be alternatively
spliced, although they harbor intron-like sequences (96). These transcripts somehow escape from
transcription termination and RNA decay and are processed into mature piRNAs.
Recent studies have shed some light on how piRNA generation from dual-stranded piRNA
clusters in Drosophila is initiated (Figure 6b). Rhi, an HP1a family gene, plays a major role in
the identification of dual-stranded piRNA clusters in germline cells (97). Furthermore, Rhi forms
a complex together with Deadlock (Del) and Cutoff (Cuff ), and this complex is anchored to
H3K9me3-marked chromatin, where it would be defined as a piRNA cluster (95). Cuff protects the
5 ends of piRNA precursor transcripts from the cap-binding complex. This results in inhibition of
alternative splicing as well as polyadenylation and transcription termination, leading to continuous
transcription of the piRNA cluster transcript. Importantly, depletion of Piwi results in the loss
of the Rhi–Del–Cuff complex at a subset of piRNA clusters. Although it seems counterintuitive,
the competence of genomic regions to generate piRNAs appears to depend on the presence of
a high level of H3K9me3 marks on the cluster. Association of the Rhi–Del–Cuff complex would
allow piRNA clusters to be transcribed and would also protect the transcripts from transcription
termination. Therefore, Piwi can specify piRNA clusters by guiding H3K9me3 marks to recruit the
Rhi–Del–Cuff complex. Rhi also functions together with Cuff and UAP56 to suppress alternative
splicing of piRNA clusters (96). The suppression of alternative splicing would distinguish piRNA
clusters from mRNAs, although the underlying mechanism is not yet clear.
Single-stranded primary piRNA clusters, such as flam, seem to be independent of this transcrip-
tion mechanism, because the loss of Rhi leads to loss of piRNAs only from dual-stranded piRNA
clusters. Indeed, Rhi is not expressed within ovarian somatic cells, where only single-stranded
piRNA clusters are active. The molecular mechanism underlying transcription of single-stranded
piRNA clusters remains unknown. Additionally, how piRNA clusters in different species are tran-
scribed and whether they also have specific factors for the identification of piRNA clusters are
issues that have only just begun to be addressed (98).

[Link] • Biogenesis and Functions of piRNA 25.13

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

RECOGNITION OF SELF AND NONSELF BY piRNAs

Acquisition of Novel piRNAs for Regulation of Transposons

Regulation of transposons by piRNAs can be described as a recognition system of “self ” genes
(coding and essential genes) and “nonself ” sequences (transposons and repeat sequences). Because

a flam cluster:
single-stranded cluster stalker4 10 kb
>
stalker2 stalker2 fw stalker4 mdg1 mdg1 blood gypsy6 hmsbeagle gypsy10
<< < >> << < > << << << <<
412 gypsy2 doc mdg1 mdg1 G6 gypsy6 fw gypsy3 gypsy3 zam tirant
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

<< < >> << >> << << < < < < <
flam
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

DIP1

Chr X: 21.505 21.550 21.595 21.640 21.685

(Mbp)

42AB cluster: 10 kb
dual-stranded cluster
cr1a batumi gypsy12 copia2 stalker4 fw doc6 invader6 invader4 RT1b invader3 G6 fw
> >> << < << < < > < < > > <
RT1a transib2
bs2 max roo bs fw gypsy12 1731 invader1 frogger circle batsumi G4 mdg3
< >> > < > < << > > > >> > > > >
42AB

42AB
Chr 2R: 2.144 2.205 2.265 2.326 2.386
(Mbp)

b Piwi Cuff

Rhi
Pol II Del
H3K9me3 Polyadenylation
Transcription termination
Splicing
Dual-stranded
piRNA cluster locus
UAP56
CBC

c Transposition of TE Transposition of TE Regulation of TEs

to piRNA cluster by novel piRNAs

Novel piRNAs
from inserted TEs
<<< Insertion of TE

TE
> < > <<< <
piRNA cluster region piRNA cluster piRNA cluster
Active TEs Silenced TEs

25.14 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

transposons do not have structural or sequence characteristics in common, the PIWI–piRNA

pathway must somehow recognize self and nonself sequences to selectively silence the latter.
Transposons fall into several types, such as DNA transposons, LTR transposons, and non-LTR
transposons, and they require different strategies to spread their copies around the genome (99).
Even within the same types, they can be subdivided into many different families/subfamilies.
To discriminate among these sequences, the Drosophila PIWI–piRNA pathway makes use of an
essential characteristic of transposons: the mobility of the elements. As described above, piRNAs
arise from piRNA clusters encoding long piRNA precursors. Because transposons move into
different regions in the genome, they will eventually jump into a piRNA cluster, which serves
as a transposon trap (8, 100). By falling into a piRNA cluster region, novel piRNAs targeting
original copies of transposons will be produced and possibly amplified by the ping-pong pathway
(77, 101). In this way, piRISCs can selectively and effectively target harmful transposons with
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

higher mobility (Figure 6c). Numerous transposons are found within piRNA cluster regions in
nested form (42), indicating that piRNA clusters have successfully trapped a number of mobile
elements at different time points. A DNA structural characteristic or epigenetic status of piRNA
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

cluster regions may be necessary to effectively attract transposons (98), but this remains to be
determined. The fact that mammalian species also have piRNA clusters with large numbers of
embedded transposons leads us to speculate that this system is conserved in a wide range of species.
In fact, a recent finding has suggested that it may also be conserved in adult primates, which express
mainly pachytene-piRNA-like clusters (93).

Hybrid Dysgenesis
Hybrid dysgenesis is a phenomenon within correlated genetic traits that is spontaneously induced
in hybrids between certain mutually interacting strains (102). It includes the sterility syndrome
observed between two fly strains of the same species, where a particular transposon is expressed
within one but not the other. In this case, silencing of the transposon depends on the direction
in which the two fly strains are crossed (Figure 7). The transposon is activated if it was inher-
ited from the father, leading to a sterile phenotype (dysgenic cross). Conversely, transposons
inherited from the mother are silenced, leading to a fertile phenotype (reciprocal cross), even
though the genotypes are common between dysgenic cross and reciprocal cross progeny. This
phenomenon was first observed in the early 1970s, but it has taken a while to determine that the
activation of transposons in a dysgenic cross correlates with the absence of piRNAs targeting those
elements.

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 6
Transposons in Drosophila piRNA clusters and generation of novel piRNAs. (a) The structure of the flam piRNA cluster
(single-stranded piRNA cluster) and 42AB piRNA cluster (dual-stranded piRNA cluster) is illustrated with embedded transposons. In
the case of flam, the transposons are frequently inserted in the antisense direction, resulting in production of piRNAs complementary to
the original transposon. In contrast, no significant bias could be observed for 42AB. Transposons longer than 2 kb are illustrated with
approximate length and genomic position. The information on transposons was obtained from the UCSC Genome Browser
([Link] (b) Noncanonical transcription of dual-stranded piRNA cluster transcripts. The dual-stranded piRNA
cluster locus is H3K9me3 marked by Piwi, and Rhi is recruited together with Del and Cuff to the region. Cuff then binds to the newly
formed 5 end of a nascent piRNA cluster transcript, preventing polyadenylation and termination of Pol II by competing with the CBC.
Cuff also inhibits splicing together with UAP56, leading to export and processing of piRNA cluster transcripts. (c) Model showing
integration of transposons into piRNA clusters. Transposon integration results in acquisition of novel piRNA sequences, which can
regulate the original transposon. Abbreviations: CBC, cap-binding complex; Cuff, Cutoff; Del, Deadlock; piRNA, PIWI-interacting
RNA; Pol II, RNA polymerase II; Rhi, Rhino; TE, transposable element.

[Link] • Biogenesis and Functions of piRNA 25.15

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

Dysgenic cross Reciprocal cross

× ×

piRNA piRNA
Maternal Maternal
deposition deposition
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

Regulation of
transposons
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

Aub–piRNA Aub–piRNA
complex complex piRNA
Transposition of
transposons

Sterile progeny Fertile progeny

Figure 7
Hybrid dysgenesis. Sterility and abnormality occur from crosses between different strains of the same Drosophila species. When female
flies lacking a transposon and male flies carrying a transposon are crossed, the progeny becomes sterile owing to activation of the
transposon (dysgenic cross). Conversely, crosses between male flies lacking the transposon and female flies carrying the transposon
result in fertile progeny (reciprocal cross) because of the maternal transmission of piRNAs, which can target the transposon. The PIWI
protein Aub is localized to the posterior pole of the oocyte and embryo, indicating that the Aub–piRNA complex mediates maternal
inheritance. Abbreviations: Aub, Aubergine; piRNA, PIWI-interacting RNA.

In a dysgenic cross or horizontal transmission, transposons cannot be regulated by the PIWI–

piRNA pathway, resulting in a sterile phenotype (77). Surprisingly, the PIWI–piRNA pathway
can establish silencing of newly invading transposons and restore fertility in a single generation
(101). This has been made feasible by using piRNA clusters, which can trap active transposons. As
described above, newly invading transposons jump into piRNA clusters as they increase their copy
numbers in the genome, triggering production of novel piRNAs (Figure 6c). This heritable change
in the genome structure results in silencing of transposons over generations. Similarly, maternal in-
heritance of lacZ piRNAs could convert the lacZ transgene into a piRNA-generating locus, which
can transmit the acquired silencing capacity through generations (103). Additionally, inherited
piRNAs provide a trigger for piRNA production by enhancing production of homologous tran-
scripts by the ping-pong cycle in the cytoplasm and by inducing installment of H3K9me3 marks
on piRNA cluster sequences so that Rhi can bind to those genomic regions in the nucleus (104).
In Drosophila, germ-cell fate determinants are inherited in pole cells from the pole plasm at the
posterior of the oocyte, and the primordial germ cells of the offspring are produced from these
pole cells (105). Aub and Piwi, Drosophila PIWI proteins, are detected at the posterior pole of the
oocyte and embryo, suggesting that Aub–piRNA and Piwi–piRNA complexes are inherited from
the mother (21, 85, 106). Indeed, Aub-bound piRNAs produced in the ovary of the mother are
also deposited into offspring embryos (77). Thus, piRNAs are inherited together with transposons
from the mother, resulting in inheritance of the silencing machinery for transposons and the fertile

25.16 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

phenotype. This indicates that piRNAs can serve as a molecular memory, which can be transmitted
from one generation to the next. Because piRNAs are most likely inherited as a complex associated
with Aub, it may be possible to amplify the silencing signal via the ping-pong pathway.

Use of Small RNAs for Genome-Wide Recognition of Self and Nonself

As discussed above, Drosophila and mammals use some piRNAs, if not all, to recognize transposons
as nonself sequences. In contrast, ciliates and nematodes use piRNAs to recognize both nonself
and self sequences in order to identify which sequences to silence. Unlike most other eukaryotes,
ciliates have two distinct sorts of nuclei: the germline micronucleus for reproduction and a large
somatic macronucleus for general cell regulation (107). These nuclei have discrete functions: Only
the micronucleus DNA is passed on during conjugation (i.e., the sexual reproduction process), and
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

only the macronucleus provides nuclear RNA for vegetative growth and determines the phenotypes
of individuals. Extensive DNA rearrangement and an amplification process would be necessary
to maintain this nuclear dimorphism. Formation of the macronucleus requires the elimination
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

of much of the repetitive content and intergenic sequences found in the micronucleus, and the
coding genes need to be rearranged for their expression to occur.
A PIWI ortholog in the ciliate Tetrahymena (Twi1p) is essential for proper genome rearrange-
ment (108). Twi1p-bound small RNAs named scan RNAs (scnRNAs) are similar to piRNAs be-
cause they are associated with a PIWI-related protein. However, unlike piRNAs, they are produced
via a Dicer-dependent mechanism from dual-stranded precursors encoded in the micronucleus
genome. These scnRNAs are able to scan through developing macronucleus RNAs to mark homol-
ogous sequences for elimination. scnRNAs use genomic information obtained from the micronu-
cleus to recognize unwanted repeated and intergenic sequences within the macronucleus. Interest-
ingly, the silencing of marked genes is likely mediated by histone modifications, such as H3K9me
and H3K27me, as in Drosophila piRNAs (109). These modifications are required for proper genome
rearrangements and marking sequences for elimination in Tetrahymena (Figure 8a).
DNA elimination in Oxytricha trifallax, another ciliate protozoan species that uses piRNAs for
the elimination and reconstruction of DNA in the macronucleus, revealed the possibility of piRNA
recognition (110). Oxytricha PIWI ortholog (Otiwi-1)-bound piRNAs are ∼27 nt long and possess
a 1U bias, as in the primary piRNAs in other species, but they lack 3 -terminal modifications. Like
Tetrahymena, piRNAs are expressed during conjugation to play a role in the selection of self
and nonself genes in the macronucleus. However, deep sequencing of Otiwi-1-bound piRNAs
revealed a striking characteristic of these piRNA populations: They map to the macronucleus
somatic genome, not to the micronucleus somatic genome (110). In other words, these piRNAs
recognize self sequences to specify genome retention, but not elimination (Figure 8a).
As in ciliates, Caenorhabditis elegans recognizes self and nonself sequences not only via piRNAs
that can recognize nonself genes for elimination, but also through cooperation with small RNAs
in other categories, which can protect and promote self gene expression (Figure 8b). C. elegans
expresses 21U-RNAs and 22G-RNAs. 21U-RNAs are so named because of their 21-nt length
and their 1U bias. RNA polymerase II transcribes these RNAs from a piRNA cluster region as an
∼26-nt transcript. These RNAs are then loaded onto the PIWI protein PRG-1 (111–114). The
population of 21U-RNAs transcribed from a piRNA cluster region on chromosome IV contains
the conserved sequence motif CTGTTTCA ∼42 nt upstream of the coding region. Forkhead
family transcription factors recognize this sequence motif. As a result, ∼26-nt-long precursors are
decapped, and 2 nt at the 5 end are removed (111, 115). Afterwards, the sequences are loaded
onto PRG-1, along with 3-nt trimming at the 3 end, forming the PRG-1–21U-RNA complex.
Recently, several factors, such as PRDE-1, PID-1, and TOFUs, were identified as regulators of

[Link] • Biogenesis and Functions of piRNA 25.17

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

a DNA region in developing macronucleus b piRNA cluster in C. elegans

Self genes
Transcription and
21U-coding loci processing of 21U-RNA

U PRG-1–21U-RNA
U complex
U
Nonself genes

Regulation of nonself Protection of self Regulation of Protection and activation of

genes in Tetrahymena genes in Oxytricha trifallax nonself transcripts self transcripts

scnRNAs (from soma) piRNAs (from germline) Cytoplasm 22G-RNA

21U-RNA
G
PRG-1 U G
U
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

G G CSR-1
U U
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

Mark with repreressive RdRP G

Elimination of DNA G 22G-RNA Nucleus
histone modifications G
unrecognized by piRNAs
and unscrambling WAGO G G
Pol II
G
PTGS
Elimination of DNA G
recognized by scnRNAs G
Transcriptional silencing of
nonself genes
Reorganized DNA region in mature macronucleus

Figure 8
Recognition of self and nonself by piRNAs. Some organisms use small RNAs for recognition of both nonself and self sequences in the
genome. (a) In the case of Tetrahymena, scnRNAs recognize nonself sequences in the macronucleus, which leads to epigenetic
regulation of the targeted region. However, in the case of Oxytricha, piRNAs recognize the self gene coding sequence in the
macronucleus genome. (b) Caenorhabditis elegans also recognize self and nonself sequences to effectively downregulate nonself genes.
21U-RNA is expressed from a piRNA cluster region where 21U-RNA is tandemly coded. The PRG-1–21U-RNA complex leads to
transcription of 22G-RNAs, which bind to WAGO for posttranscriptional and transcriptional silencing of nonself genes. Conversely,
22G-RNA bound to another protein, CSR-1, recognizes self genes and acts to protect the recognized region from regulation.
Abbreviations: piRNA, PIWI-interacting RNA; PTGS, posttranscriptional gene silencing; scnRNA, scan RNA.

these steps (116–118). Whether there is a direct target of PID-1-associated 21U-RNAs is still
unknown. However, an important indirect silencing mechanism of 21U-RNAs has been revealed.
PRG-1–21U-RNA complexes can scan through the genome, recognize foreign RNA sequences,
and initiate production of RdRP-dependent siRNAs known as 22G-RNAs (113, 119, 120). 22G-
RNAs are loaded onto worm-specific Argonaute members, the WAGO proteins, which mediate
transcriptional silencing via histone modifications such as H3K9me3. In this way, PRG-1–21U-
RNAs can silence their target sequences via WAGO–22G-RNA complex–mediated gene silencing.
Moreover, 22G-RNAs bound to another protein, CSR-1, can recognize and protect mRNAs
that should not be silenced. piRNAs sharing a common target with 22G-RNA bound to CSR-1
cannot efficiently downregulate their targets (120–124), perhaps because CSR-1–22G-RNA acts
as a dominant negative form of WAGO–22G-RNA. Interestingly, the next generation inherits
these phenomena (121–126). Therefore, in C. elegans, several types of small RNA–mediated gene
regulatory systems have been combined to identify nonself as well as self sequences and to efficiently
control the gene expression network over generations.

25.18 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

THE IMPACT OF piRNAs ON PROTEIN-CODING GENES

piRNA-Mediated Control of Nontransposon Genes

In retrospect, the first piRNAs found in Drosophila were those derived from the Suppressor of Stellate
locus on the Y chromosome (127). These piRNAs were suggested to target Stellate, a repetitive
but protein-coding gene on the X chromosome. Similarly, Aub-associated piRNAs from a repet-
itive region on the X chromosome target the essential maternal effector gene Vasa in Drosophila
testis (128, 129). Additionally, some piRNAs produced from transposons, roo and 412, induce
deadenylation and degradation of maternally deposited mRNAs, such as nanos, in Drosophila
embryos (130). Defects in piRNA-mediated nanos regulation result in head-development
defects.
Genome-wide mapping of piRNAs has revealed that transposons as well as the 3 UTRs of cod-
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

ing genes serve as sources of piRNAs, specifically termed genic piRNAs. For example, the traffic
jam (tj) gene in Drosophila is one source of genic piRNAs (131, 132). A search for the sequence com-
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

plements to tj-derived piRNAs identified Fasciclin 3 (Fas3), which encodes an immunoglobulin-like

cell adhesion molecule, as a potential target gene. Indeed, expression of Fas3 is increased in piwi
mutant gonads (132). In mammals, including primates, expression of piRNAs from a certain pop-
ulation of coding genes is also conserved, suggesting that genic piRNAs may have a conserved
function, such as regulation of coding genes (93, 131). In piRNAs produced from protein-coding
genes, the molecular mechanism underlying their recognition to initiate piRNA production re-
mains to be revealed.
Pachytene piRNAs bound to MIWI are responsible for eliminating the expression of bulk
mRNAs in spermatids (34). These piRNAs form a complex with CAF1, a catalytic subunit of
the CCR4–CAF1–NOT deadenylase complex; select their target mRNA by partial sequence
complementarity to the 3 UTR; and promote deadenylation and decay of targets, similar to
the workings of the miRNA machinery (133). Importantly, ∼5,000 protein-coding genes were
significantly upregulated upon MIWI or CAF1 knockdown, indicating that this mechanism may
play a critical role in the maintenance of reproductive tissue.
Involvement of the mouse PIWI–piRNA pathway in genomic imprinting has also been sug-
gested (134). Genomic imprinting causes silencing of one of the two alleles by epigenetically
marking the region through de novo methylation. The components of the piRNA pathway are
required for de novo methylation of the differentially methylated region (DMR) located near
the imprinted Rasgrf1 gene locus in chromosome 9. piRNAs derived from transposons coded
in chromosome 7 target noncoding RNA (piRNA-targeted noncoding RNA, also known as
pit-RNA) derived from the DMR of Rasgrf1. The promoter of pit-RNA is a direct repeat in
the DMR, which is required for the de novo methylation and imprinting of Rasgrf1, and the
Rasgrf1 gene fails to undergo de novo methylation when piRNAs target pit-RNA. Although
the mechanism remains unknown, piRISCs may recruit DNA methyltransferase to the DMR of
Rasgrf1.
Meanwhile, in Drosophila, H3K9me3 modifications to regulate transposons via the Piwi–piRNA
pathway can spread around neighboring chromosome regions and epigenetically regulate coding
genes in the region (58, 70). Whether regulation of transposon neighboring genes also possesses
an important function is still unknown. However, the number of regulated genes can be large
given that regulated transposons are spread around the genome. McClintock (135) observed the
developmental consequences of transposon activity and referred to transposons as “controlling
elements.” Similarly, the cases described in this section suggest that transposons can regulate
genes from a distance through the PIWI–piRNA pathway.

[Link] • Biogenesis and Functions of piRNA 25.19

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

Sex Determination and Mating-Type Determination by piRNAs

As described thus far, piRNAs can regulate both transposons and protein-coding genes. However,
in many cases, the biological significance of this regulation remains elusive. A recent study indi-
cated that sex determination in the silkworm Bombyx mori is accomplished by piRNA-mediated
protein-coding gene regulation (Figure 9a) (137). B. mori makes use of a WZ sex determination
mechanism: Males have two Z chromosomes, whereas females have both W and Z chromosomes.

a Sex determination by piRNAs

B. mori
Male Female Tandemly coded Fem
chr W
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

Fem
piRNA precursor
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

Siwi/Fem–piRNA
complex
Ping-
Masc Masc pong?
(from chr. Z) (from chr. Z)
Masc
protein BmAGO3/Masc–
piRNA complex
Male-specific splicing Female-specific splicing

bmdsx bmdsx

b Mating-type determination by scnRNAs

P. tetraurelia mtA P. septaurelia mtA
type E type E

mtB mtC mtB mtC

scnRNA
mtA mtA
type O type O

?
mtB mtC mtB mtC

Figure 9
Sex determination and mating-type determination by piRNAs. (a) A piRNA-mediated sex determination
model in Bombyx mori. In the case of B. mori, a piRNA transcribed from the Fem gene regulates a gene
termed Masc. This gene is essential for regulation of male-specific splicing of the Bmdsx gene, which acts as a
determinant gene of sex. (b) In the case of the ciliate Paramecium tetraurelia, the mtA gene serves as a
determinant of mating type. This gene is regulated by scnRNAs of P. tetraurelia in the mating type O gene.
In the case of another ciliate, Paramecium septaurelia, the mtA gene is regulated not by scnRNAs, but by
expression of another coding gene called mtB. Abbreviations: Bmdsx, doublesex; Fem, Feminizer; Masc,
Masculinizer; piRNA, PIWI-interacting RNA; scnRNA, scan RNA.

25.20 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

Therefore, W chromosomes are expected to play an important role in sex determination. Interest-
ingly, the W chromosome is largely occupied by transposons, and protein-coding genes have not
been identified in the chromosome. Feminizer (Fem), which is tandemly found on the W chromo-
some, encodes a noncoding RNA, which was identified as a piRNA precursor. After processing,
piRNAs derived from Fem are loaded onto the B. mori PIWI protein Siwi. Analyses suggest that a
target of Fem–piRNA is a newly identified gene on the Z chromosome termed Masculinizer (Masc),
a factor necessary for the production of a male-specific splice variant of B. mori doublesex (Bmdsx).
The specific splice variant of Bmdsx serves as a determinant of global gene expression in a sex-
specific manner. In females, Masc is downregulated by Fem–piRNAs, resulting in a female-specific
splicing pattern of Bmdsx. Interestingly, piRNAs are also produced from Masc, and Masc–piRNAs
are loaded onto another PIWI protein, BmAGO3. Masc–piRNAs possess a ping-pong signature
(10-nt overlap between sense and antisense piRNAs) with Fem–piRNAs, suggesting that they may
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

be generated through ping-pong amplification. In this way, female-specific piRNAs can target
the coding gene Masc and serve as a feminizing factor. This system is somewhat similar to the
Suppressor of Stellate and Stellate system that Drosophila uses in testis (127); that is, piRNAs derived
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

from a noncoding RNA–encoding gene on a male-specific or female-specific sex chromosome are

loaded onto a particular PIWI protein, and the resultant piRISCs downregulate a homologous
protein-coding gene on the other sex chromosome via piRISC-mediated cleavage of the target
mRNA.
Another example of clear phenotypic regulation by piRNAs was reported from a study using
ciliate Paramecium tetraurelia, which showed that piRNAs can serve as an essential factor for
epigenetic mating-type inheritance via regulation of a coding gene (Figure 9b). As described
above, scnRNAs excise transposons from germline micronuclei to form new somatic macronuclei.
During this process, the mating type (type E or type O in Paramecium) is determined by expression
of the mtA gene (136). mtA is expressed in type E but repressed in type O. The repression of mtA
in type O P. tetraurelia was due to scnRNAs, which can be maternally inherited by the next
generation. Interestingly, in the case of a sibling species, Paramecium septaurelia, expression of
mtA is regulated not by scnRNAs but by another gene, mtB. Despite mtA serving as a common
factor determining E/O expression, the mechanisms used to produce O clones differ even among
closely related species. This may indicate the frequent exaptation of the scnRNA pathway for
gene silencing. The sequences excised from mtA genes are functional parts of coding genes and
are not derived from the insertion of transposons, leaving a big mystery as to how this gene got
“selected” as a piRNA target. As with Masc regulation by B. mori piRNAs, this is another good
example showing the possible capacity of piRNAs to inactivate single-copy coding genes, leading
to phenotypic variation.

Nongonadal Function of PIWI Proteins and piRNAs

Some studies have suggested that the PIWI–piRNA complex possesses regulatory functions in
germ cells and somatic cells (9, 10). In Aplysia, memory storage in the brain requires PIWI–
piRNA function (138). Aplysia piRNAs are expressed from piRNA clusters in the genome. These
piRNAs are localized to the nucleus and can induce DNA methylation to regulate their target
genes. The targets include CREB2, a transcriptional repressor of memory. Analyses suggest that
the promoter region of CREB2 is methylated by piRNAs, leading to its regulation. Interestingly,
piRNAs were also observed in mouse hippocampus (139). These piRNAs associate with MIWI
and may be required for the development of dendritic spines.
In ascidians, PIWI orthologs (Bl-Piwi and Piwi) are involved in whole-body regeneration,
which is a regeneration phenomenon of functional adults from minute vasculature fragments. In

[Link] • Biogenesis and Functions of piRNA 25.21

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

Botrylloides leachi, whole-body regeneration occurs in cells expressing Bl-Piwi, and knockdown
of Bl-Piwi results in cell regeneration arrest (140). In another ascidian, Botryllus schlosseri, Piwi
depletion causes the gradual loss of cycling stem cells, resulting in regeneration arrest (141).
Thus, Piwi plays an essential role in maintaining the functionality of stem cells during whole-body
regeneration of ascidians. It still remains unclear whether piRNAs are involved in this process.
PIWI function may also be involved in human cancer development. In Drosophila, a loss-of-
function screening for the factors responsible for malignant brain tumors has demonstrated that
Piwi and Aub contribute to tumor growth (142). Human PIWI proteins are expressed in a variety
of human cancer cells. The first example of this was the finding of overexpression of HIWI in
seminomas, a male germ-cell cancer (143). Since then, ectopic expression of PIWI proteins has
been observed within cell lines and tissue samples of a variety of cancer cells, including those
associated with breast, cervical, gastric, and liver cancers, among others (reviewed in Reference
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

144). Additionally, HILI overexpression in NIH3T3 cells, a mouse fibroblast cell line, results in
activation of the transcription activator STAT3 and the antiapoptotic factor BCLX, suggesting that
HILI may be involved in cell growth, adhesion, and apoptosis (145). However, piRNA expression
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

and piRISC formation, for example, have not been convincingly demonstrated in cancer cells.
Therefore, further investigations are awaited to conclude whether the expression of PIWI proteins
in cancer cells is biologically significant.

CONCLUSION
We now know that a large number of factors cooperate to generate piRNAs and assist them
in selectively regulating nonself genes. Nevertheless, their functional roles are largely unknown,
especially with respect to the epigenetic modifications induced by piRNAs within the nucleus.
Moreover, high-throughput screening has identified numerous factors that may be involved in
piRNA production and/or transposon silencing, and their precise contribution within the piRNA
biogenesis pathway remains to be revealed. Fundamentally, we still do not know how regulation
of transposons by piRNAs is linked to fertility. It may be necessary to consider the regulation of
not only transposons but also coding genes by the PIWI–piRNA pathway to elucidate how defects
in piRNA pathways lead to germline development.
A variety of genes are involved in the PIWI–piRNA pathway, and examples show that inheri-
table PIWI–piRNAs have functions inside and outside the germline. Thus, cross talk may occur
between PIWI–piRNA pathways and other biological pathways, a suggestion that awaits further
investigation. Exploring novel model organisms may be a useful approach, as in the case of the
somatic genome rearrangement discovered by analyses of Tetrahymena piRNAs. For example, a
naked mole rat can live for more than 30 years without any sign of cancer, and its genome possesses
mostly old, inactivated transposons, although it encodes PIWI proteins (146, 147). Therefore, an-
alyzing this species may reveal a novel role other than regulation of transposons for mammalian
PIWI proteins and piRNAs.
Additionally, because piRNAs are involved in epigenetic modifications of gene expression,
PIWI proteins and piRNAs may have further roles in maintaining genomic structure. It will be in-
teresting to analyze the effect of PIWI protein expression on genomic structure by approaches such
as 3C (chromosome conformation capture)-based technologies, which enable us to determine the
three-dimensional architecture of a genome (148, 149). Because piRNAs also have a flexible mecha-
nism for identifying self and nonself genes that are transgenerationally inherited, molecular signals
that define the epigenetic status of cells can achieve long-lasting effects for transgenerational inher-
itance by getting integrated into the piRNA pathway. Therefore, we may be able to develop an ar-
tificial piRNA-producing system to transgenerationally regulate certain genes or groups of genes.

25.22 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS
We thank Kuniaki Saito and Soichiro Yamanaka for critical reading of the manuscript and Yukiko
Murota and Shinsuke Shibata for the immunoelectron microscopy image. Current research in
the authors’ labs is supported by a Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science, and Technology (MEXT) of Japan to Y.W.I., H.S., and
M.C.S.
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

LITERATURE CITED
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

1. Siomi H, Siomi MC. 2009. On the road to reading the RNA-interference code. Nature 457:396–404
2. Ghildiyal M, Zamore PD. 2009. Small silencing RNAs: an expanding universe. Nat. Rev. Genet. 10:94–
108
3. Kim VN, Han J, Siomi MC. 2009. Biogenesis of small RNAs in animals. Nat. Rev. Mol. Cell Biol. 10:126–
39
4. Peters L, Meister G. 2007. Argonaute proteins: mediators of RNA silencing. Mol. Cell 26:611–23
5. Vagin VV, Sigova A, Li C, Seitz H, Gvozdev V, Zamore PD. 2006. A distinct small RNA pathway
silences selfish genetic elements in the germline. Science 313:320–24
6. Siomi MC, Sato K, Pezic D, Aravin AA. 2011. PIWI-interacting small RNAs: the vanguard of genome
defence. Nat. Rev. Mol. Cell Biol. 12:246–58
7. Moazed D. 2009. Small RNAs in transcriptional gene silencing and genome defence. Nature 457:413–20
8. Malone CD, Hannon GJ. 2009. Small RNAs as guardians of the genome. Cell 136:656–68
9. Ishizu H, Siomi H, Siomi MC. 2012. Biology of PIWI-interacting RNAs: new insights into biogenesis
and function inside and outside of germlines. Genes Dev. 26:2361–73
10. Ross RJ, Weiner MM, Lin H. 2014. PIWI proteins and PIWI-interacting RNAs in the soma. Nature
505:353–59
11. Stuwe E, Toth KF, Aravin AA. 2014. Small but sturdy: small RNAs in cellular memory and epigenetics.
Genes Dev. 28:423–31
12. Grimson A, Srivastava M, Fahey B, Woodcroft BJ, Chiang HR, et al. 2008. Early origins and evolution
of microRNAs and Piwi-interacting RNAs in animals. Nature 455:1193–97
13. Thomson T, Lin H. 2009. The biogenesis and function of PIWI proteins and piRNAs: progress and
prospect. Annu. Rev. Cell Dev. Biol. 25:355–76
14. Cox DN, Chao A, Baker J, Chang L, Qiao D, Lin H. 1998. A novel class of evolutionarily conserved
genes defined by piwi are essential for stem cell self-renewal. Genes Dev. 12:3715–27
15. Li C, Vagin VV, Lee S, Xu J, Ma S, et al. 2009. Collapse of germline piRNAs in the absence of Argonaute3
reveals somatic piRNAs in flies. Cell 137:509–21
16. Schmidt A, Palumbo G, Bozzetti MP, Tritto P, Pimpinelli S, Schäfer U. 1999. Genetic and molecular
characterization of sting, a gene involved in crystal formation and meiotic drive in the male germ line of
Drosophila melanogaster. Genetics 151:749–60
17. Lin H, Spradling AC. 1997. A novel group of pumilio mutations affects the asymmetric division of
germline stem cells in the Drosophila ovary. Development 124:2463–76
18. Kalmykova AI, Klenov MS, Gvozdev VA. 2005. Argonaute protein PIWI controls mobilization of retro-
transposons in the Drosophila male germline. Nucleic Acids Res. 33:2052–59
19. Vagin VV, Klenov MS, Kalmykova AI, Stolyarenko AD, Kotelnikov RN, Gvozdev VA. 2004. The RNA
interference proteins and vasa locus are involved in the silencing of retrotransposons in the female
germline of Drosophila melanogaster. RNA Biol. 1:54–58

[Link] • Biogenesis and Functions of piRNA 25.23

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

20. Sabin LR, Delas MJ, Hannon GJ. 2013. Dogma derailed: the many influences of RNA on the genome.
Mol. Cell 49:783–94
21. Brennecke J, Aravin AA, Stark A, Dus M, Kellis M, et al. 2007. Discrete small RNA–generating loci as
master regulators of transposon activity in Drosophila. Cell 128:1089–103
22. Gunawardane LS, Saito K, Nishida KM, Miyoshi K, Kawamura Y, et al. 2007. A Slicer-mediated mech-
anism for repeat-associated siRNA 5 end formation in Drosophila. Science 315:1587–90
23. Savitsky M, Kwon D, Georgiev P, Kalmykova A, Gvozdev V. 2006. Telomere elongation is under the
control of the RNAi-based mechanism in the Drosophila germline. Genes Dev. 20:345–54
24. Khurana JS, Xu J, Weng Z, Theurkauf WE. 2010. Distinct functions for the Drosophila piRNA pathway
in genome maintenance and telomere protection. PLOS Genet. 6:e1001246
25. Pardue ML, DeBaryshe PG. 2003. Retrotransposons provide an evolutionarily robust non-telomerase
mechanism to maintain telomeres. Annu. Rev. Genet. 37:485–511
26. Carmell MA, Girard A, van de Kant HJ, Bourc’his D, Bestor TH, et al. 2007. MIWI2 is essential for
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

spermatogenesis and repression of transposons in the mouse male germline. Dev. Cell 12:503–14
27. Aravin AA, Sachidanandam R, Girard A, Fejes-Toth K, Hannon GJ. 2007. Developmentally regulated
piRNA clusters implicate MILI in transposon control. Science 316:744–47
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

28. Kuramochi-Miyagawa S, Watanabe T, Gotoh K, Totoki Y, Toyoda A, et al. 2008. DNA methylation
of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes.
Genes Dev. 22:908–17
29. Aravin AA, Sachidanandam R, Bourc’his D, Schaefer C, Pezic D, et al. 2008. A piRNA pathway primed
by individual transposons is linked to de novo DNA methylation in mice. Mol. Cell 31:785–99
30. Aravin A, Gaidatzis D, Pfeffer S, Lagos-Quintana M, Landgraf P, et al. 2006. A novel class of small
RNAs bind to MILI protein in mouse testes. Nature 442:203–7
31. Girard A, Sachidanandam R, Hannon GJ, Carmell MA. 2006. A germline-specific class of small RNAs
binds mammalian Piwi proteins. Nature 442:199–202
32. Grivna ST, Beyret E, Wang Z, Lin H. 2006. A novel class of small RNAs in mouse spermatogenic cells.
Genes Dev. 20:1709–14
33. Lau NC, Seto AG, Kim J, Kuramochi-Miyagawa S, Nakano T, et al. 2006. Characterization of the
piRNA complex from rat testes. Science 313:363–67
34. Reuter M, Berninger P, Chuma S, Shah H, Hosokawa M, et al. 2011. Miwi catalysis is required for
piRNA amplification-independent LINE1 transposon silencing. Nature 480:264–67
35. De Fazio S, Bartonicek N, Di Giacomo M, Abreu-Goodger C, Sankar A, et al. 2011. The endonuclease
activity of MILI fuels piRNA amplification that silences LINE1 elements. Nature 480:259–63
36. Zhao S, Gou LT, Zhang M, Zu LD, Hua MM, et al. 2013. piRNA-triggered MIWI ubiquitination and
removal by APC/C in late spermatogenesis. Dev. Cell 24:13–25
37. Klattenhoff C, Bratu DP, McGinnis-Schultz N, Koppetsch BS, Cook HA, Theurkauf WE. 2007.
Drosophila rasiRNA pathway mutations disrupt embryonic axis specification through activation of an
ATR/Chk2 DNA damage response. Dev. Cell 12:45–55
38. Castañeda J, Genzor P, van der Heijden GW, Sarkeshik A, Yates JR 3rd, et al. 2014. Reduced pachytene
piRNAs and translation underlie spermiogenic arrest in Maelstrom mutant mice. EMBO J. 33:1999–2019
39. Klenov MS, Sokolova OA, Yakushev EY, Stolyarenko AD, Mikhaleva EA, et al. 2011. Separation of stem
cell maintenance and transposon silencing functions of Piwi protein. PNAS 108:18760–65
40. Xu M, You Y, Hunsicker P, Hori T, Small C, et al. 2008. Mice deficient for a small cluster of Piwi-
interacting RNAs implicate Piwi-interacting RNAs in transposon control. Biol. Reproduct. 79:51–57
41. Prud’homme N, Gans M, Masson M, Terzian C, Bucheton A. 1995. Flamenco, a gene controlling the
gypsy retrovirus of Drosophila melanogaster. Genetics 139:697–711
42. Zanni V, Eymery A, Coiffet M, Zytnicki M, Luyten I, et al. 2013. Distribution, evolution, and diversity
of retrotransposons at the flamenco locus reflect the regulatory properties of piRNA clusters. PNAS
110:19842–47
43. Nishimasu H, Ishizu H, Saito K, Fukuhara S, Kamatani MK, et al. 2012. Structure and function of
Zucchini endoribonuclease in piRNA biogenesis. Nature 491:284–87
44. Ipsaro JJ, Haase AD, Knott SR, Joshua-Tor L, Hannon GJ. 2012. The structural biochemistry of Zucchini
implicates it as a nuclease in piRNA biogenesis. Nature 491:279–83

25.24 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

45. Pane A, Wehr K, Schupbach T. 2007. zucchini and squash encode two putative nucleases required for
rasiRNA production in the Drosophila germline. Dev. Cell 12:851–62
46. Haase AD, Fenoglio S, Muerdter F, Guzzardo PM, Czech B, et al. 2010. Probing the initiation and
effector phases of the somatic piRNA pathway in Drosophila. Genes Dev. 24:2499–504
47. Saito K, Ishizu H, Komai M, Kotani H, Kawamura Y, et al. 2010. Roles for the Yb body components
Armitage and Yb in primary piRNA biogenesis in Drosophila. Genes Dev. 24:2493–98
48. Olivieri D, Sykora MM, Sachidanandam R, Mechtler K, Brennecke J. 2010. An in vivo RNAi assay
identifies major genetic and cellular requirements for primary piRNA biogenesis in Drosophila. EMBO
J. 29:3301–17
49. Zamparini AL, Davis MY, Malone CD, Vieira E, Zavadil J, et al. 2011. Vreteno, a gonad-specific
protein, is essential for germline development and primary piRNA biogenesis in Drosophila. Development
138:4039–50
50. Handler D, Olivieri D, Novatchkova M, Gruber FS, Meixner K, et al. 2011. A systematic analysis of
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

Drosophila TUDOR domain–containing proteins identifies Vreteno and the Tdrd12 family as essential
primary piRNA pathway factors. EMBO J. 30:3977–93
51. Preall JB, Czech B, Guzzardo PM, Muerdter F, Hannon GJ. 2012. shutdown is a component of the
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

Drosophila piRNA biogenesis machinery. RNA 18:1446–57

52. Qi H, Watanabe T, Ku HY, Liu N, Zhong M, Lin H. 2011. The Yb body, a major site for Piwi-associated
RNA biogenesis and a gateway for Piwi expression and transport to the nucleus in somatic cells. J. Biol.
Chem. 286:3789–97
53. Handler D, Meixner K, Pizka M, Lauss K, Schmied C, et al. 2013. The genetic makeup of the Drosophila
piRNA pathway. Mol. Cell 50:762–77
54. Vagin VV, Yu Y, Jankowska A, Luo Y, Wasik KA, et al. 2013. Minotaur is critical for primary piRNA
biogenesis. RNA 19:1064–77
55. Kawaoka S, Izumi N, Katsuma S, Tomari Y. 2011. 3 end formation of PIWI-interacting RNAs in vitro.
Mol. Cell 43:1015–22
56. Saito K, Sakaguchi Y, Suzuki T, Siomi H, Siomi MC. 2007. Pimet, the Drosophila homolog of HEN1,
mediates 2 -O-methylation of Piwi-interacting RNAs at their 3 ends. Genes Dev. 21:1603–8
57. Horwich MD, Li C, Matranga C, Vagin V, Farley G, et al. 2007. The Drosophila RNA methyltransferase,
DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC. Curr. Biol. 17:1265–72
58. Sienski G, Donertas D, Brennecke J. 2012. Transcriptional silencing of transposons by Piwi and
Maelstrom and its impact on chromatin state and gene expression. Cell 151:964–80
59. Murota Y, Ishizu H, Nakagawa S, Iwasaki YW, Shibata S, et al. 2014. Yb integrates piRNA intermediates
and processing factors into perinuclear bodies to enhance piRISC assembly. Cell Rep. 8:103–13
60. Malone CD, Brennecke J, Dus M, Stark A, McCombie WR, et al. 2009. Specialized piRNA pathways
act in germline and somatic tissues of the Drosophila ovary. Cell 137:522–35
61. Soper SF, van der Heijden GW, Hardiman TC, Goodheart M, Martin SL, et al. 2008. Mouse maelstrom,
a component of nuage, is essential for spermatogenesis and transposon repression in meiosis. Dev. Cell
15:285–97
62. Frost RJ, Hamra FK, Richardson JA, Qi X, Bassel-Duby R, Olson EN. 2010. MOV10L1 is necessary for
protection of spermatocytes against retrotransposons by Piwi-interacting RNAs. PNAS 107:11847–52
63. Huang H, Gao Q, Peng X, Choi SY, Sarma K, et al. 2011. piRNA-associated germline nuage formation
and spermatogenesis require MitoPLD profusogenic mitochondrial-surface lipid signaling. Dev. Cell
20:376–87
64. Xiol J, Cora E, Koglgruber R, Chuma S, Subramanian S, et al. 2012. A role for Fkbp6 and the chaperone
machinery in piRNA amplification and transposon silencing. Mol. Cell 47:970–79
65. Watanabe T, Chuma S, Yamamoto Y, Kuramochi-Miyagawa S, Totoki Y, et al. 2011. MITOPLD is a
mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline. Dev.
Cell 20:364–75
66. Zheng K, Xiol J, Reuter M, Eckardt S, Leu NA, et al. 2010. Mouse MOV10L1 associates with Piwi
proteins and is an essential component of the Piwi-interacting RNA (piRNA) pathway. PNAS 107:11841–
46

[Link] • Biogenesis and Functions of piRNA 25.25

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

67. Le Thomas A, Rogers AK, Webster A, Marinov GK, Liao SE, et al. 2013. Piwi induces piRNA-guided
transcriptional silencing and establishment of a repressive chromatin state. Genes Dev. 27:390–99
68. Wang SH, Elgin SC. 2011. Drosophila Piwi functions downstream of piRNA production mediating a
chromatin-based transposon silencing mechanism in female germ line. PNAS 108:21164–69
69. Huang XA, Yin H, Sweeney S, Raha D, Snyder M, Lin H. 2013. A major epigenetic programming
mechanism guided by piRNAs. Dev. Cell 24:502–16
70. Ohtani H, Iwasaki YW, Shibuya A, Siomi H, Siomi MC, Saito K. 2013. DmGTSF1 is necessary for
Piwi-piRISC-mediated transcriptional transposon silencing in the Drosophila ovary. Genes Dev. 27:1656–
61
71. Muerdter F, Guzzardo PM, Gillis J, Luo Y, Yu Y, et al. 2013. A genome-wide RNAi screen draws a genetic
framework for transposon control and primary piRNA biogenesis in Drosophila. Mol. Cell 50:736–48
72. Donertas D, Sienski G, Brennecke J. 2013. Drosophila Gtsf1 is an essential component of the Piwi-
mediated transcriptional silencing complex. Genes Dev. 27:1693–705
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

73. Brower-Toland B, Findley SD, Jiang L, Liu L, Yin H, et al. 2007. Drosophila PIWI associates with
chromatin and interacts directly with HP1a. Genes Dev. 21:2300–11
74. Rangan P, Malone CD, Navarro C, Newbold SP, Hayes PS, et al. 2011. piRNA production requires
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

heterochromatin formation in Drosophila. Curr. Biol. 21:1373–79

75. Czech B, Preall JB, McGinn J, Hannon GJ. 2013. A transcriptome-wide RNAi screen in the Drosophila
ovary reveals factors of the germline piRNA pathway. Mol. Cell 50:749–61
76. Lim AK, Kai T. 2007. Unique germ-line organelle, nuage, functions to repress selfish genetic elements
in Drosophila melanogaster. PNAS 104:6714–19
77. Brennecke J, Malone CD, Aravin AA, Sachidanandam R, Stark A, Hannon GJ. 2008. An epigenetic role
for maternally inherited piRNAs in transposon silencing. Science 322:1387–92
78. Elbashir SM, Lendeckel W, Tuschl T. 2001. RNA interference is mediated by 21- and 22-nucleotide
RNAs. Genes Dev. 15:188–200
79. Xiol J, Spinelli P, Laussmann MA, Homolka D, Yang Z, et al. 2014. RNA clamping by vasa assembles a
piRNA amplifier complex on transposon transcripts. Cell 157:1698–711
80. Nishida KM, Iwasaki YW, Murota Y, Nagao A, Mannen T, et al. 2015. Respective functions of two
distinct Siwi complexes assembled during PIWI-interacting RNA biogenesis in Bombyx germ cells. Cell
Rep. 10:193–203
81. Chen C, Nott TJ, Jin J, Pawson T. 2011. Deciphering arginine methylation: Tudor tells the tale. Nat.
Rev. Mol. Cell Biol. 12:629–42
82. Chen C, Jin J, James DA, Adams-Cioaba MA, Park JG, et al. 2009. Mouse Piwi interactome identifies
binding mechanism of Tdrkh Tudor domain to arginine methylated Miwi. PNAS 106:20336–41
83. Kirino Y, Kim N, de Planell–Saguer M, Khandros E, Chiorean S, et al. 2009. Arginine methylation of
Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability. Nat. Cell Biol. 11:652–58
84. Reuter M, Chuma S, Tanaka T, Franz T, Stark A, Pillai RS. 2009. Loss of the Mili-interacting Tudor
domain–containing protein 1 activates transposons and alters the Mili-associated small RNA profile.
Nat. Struct. Mol. Biol. 16:639–46
85. Nishida KM, Okada TN, Kawamura T, Mituyama T, Kawamura Y, et al. 2009. Functional involvement
of Tudor and dPRMT5 in the piRNA processing pathway in Drosophila germlines. EMBO J. 28:3820–31
86. Vagin VV, Wohlschlegel J, Qu J, Jonsson Z, Huang X, et al. 2009. Proteomic analysis of murine Piwi
proteins reveals a role for arginine methylation in specifying interaction with Tudor family members.
Genes Dev. 23:1749–62
87. Munn K, Steward R. 2000. The shut-down gene of Drosophila melanogaster encodes a novel FK506-binding
protein essential for the formation of germline cysts during oogenesis. Genetics 156:245–56
88. Liu L, Qi H, Wang J, Lin H. 2011. PAPI, a novel TUDOR-domain protein, complexes with AGO3,
ME31B and TRAL in the nuage to silence transposition. Development 138:1863–73
89. Zhang Z, Xu J, Koppetsch BS, Wang J, Tipping C, et al. 2011. Heterotypic piRNA ping-pong requires
qin, a protein with both E3 ligase and Tudor domains. Mol. Cell 44:572–84
90. Anand A, Kai T. 2012. The Tudor domain protein Kumo is required to assemble the nuage and to
generate germline piRNAs in Drosophila. EMBO J. 31:870–82

25.26 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

91. Patil VS, Kai T. 2010. Repression of retroelements in Drosophila germline via piRNA pathway by the
Tudor domain protein Tejas. Curr. Biol. 20:724–30
92. Olivieri D, Senti KA, Subramanian S, Sachidanandam R, Brennecke J. 2012. The cochaperone shutdown
defines a group of biogenesis factors essential for all piRNA populations in Drosophila. Mol. Cell 47:954–69
93. Hirano T, Iwasaki YW, Lin ZY, Imamura M, Seki NM, et al. 2014. Small RNA profiling and char-
acterization of piRNA clusters in the adult testes of the common marmoset, a model primate. RNA
20:1223–37
94. Li XZ, Roy CK, Dong X, Bolcun-Filas E, Wang J, et al. 2013. An ancient transcription factor initiates
the burst of piRNA production during early meiosis in mouse testes. Mol. Cell 50:67–81
95. Mohn F, Sienski G, Handler D, Brennecke J. 2014. The Rhino–Deadlock–Cutoff complex licenses
noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157:1364–79
96. Zhang Z, Wang J, Schultz N, Zhang F, Parhad SS, et al. 2014. The HP1 homolog Rhino anchors a
nuclear complex that suppresses piRNA precursor splicing. Cell 157:1353–63
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

97. Klattenhoff C, Xi H, Li C, Lee S, Xu J, et al. 2009. The Drosophila HP1 homolog Rhino is required for
transposon silencing and piRNA production by dual-strand clusters. Cell 138:1137–49
98. Yamanaka S, Siomi MC, Siomi H. 2014. piRNA clusters and open chromatin structure. Mobile DNA
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

5:22
99. Kazazian HH Jr. 2004. Mobile elements: drivers of genome evolution. Science 303:1626–32
100. Bergman CM, Quesneville H, Anxolabehere D, Ashburner M. 2006. Recurrent insertion and duplication
generate networks of transposable element sequences in the Drosophila melanogaster genome. Genome Biol.
7:R112
101. Khurana JS, Wang J, Xu J, Koppetsch BS, Thomson TC, et al. 2011. Adaptation to P element transposon
invasion in Drosophila melanogaster. Cell 147:1551–63
102. Kidwell MG, Kidwell JF, Sved JA. 1977. Hybrid dysgenesis in Drosophila melanogaster: a syndrome of
aberrant traits including mutation, sterility and male recombination. Genetics 86:813–33
103. de Vanssay A, Bouge AL, Boivin A, Hermant C, Teysset L, et al. 2012. Paramutation in Drosophila linked
to emergence of a piRNA-producing locus. Nature 490:112–15
104. Le Thomas A, Stuwe E, Li S, Du J, Marinov G, et al. 2014. Transgenerationally inherited piRNAs trigger
piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing. Genes
Dev. 28:1667–80
105. Rongo C, Lehmann R. 1996. Regulated synthesis, transport and assembly of the Drosophila germ plasm.
Trends Genet. 12:102–9
106. Megosh HB, Cox DN, Campbell C, Lin H. 2006. The role of PIWI and the miRNA machinery in
Drosophila germline determination. Curr. Biol. 16:1884–94
107. Jahn CL, Klobutcher LA. 2002. Genome remodeling in ciliated protozoa. Annu. Rev. Microbiol. 56:489–
520
108. Mochizuki K, Fine NA, Fujisawa T, Gorovsky MA. 2002. Analysis of a piwi-related gene implicates small
RNAs in genome rearrangement in Tetrahymena. Cell 110:689–99
109. Mochizuki K. 2010. DNA rearrangements directed by non-coding RNAs in ciliates. Wiley Interdiscip.
Rev. RNA 1:376–87
110. Fang W, Wang X, Bracht JR, Nowacki M, Landweber LF. 2012. Piwi-interacting RNAs protect DNA
against loss during Oxytricha genome rearrangement. Cell 151:1243–55
111. Ruby JG, Jan C, Player C, Axtell MJ, Lee W, et al. 2006. Large-scale sequencing reveals 21U-RNAs
and additional microRNAs and endogenous siRNAs in C. elegans. Cell 127:1193–207
112. Batista PJ, Ruby JG, Claycomb JM, Chiang R, Fahlgren N, et al. 2008. PRG-1 and 21U-RNAs interact
to form the piRNA complex required for fertility in C. elegans. Mol. Cell 31:67–78
113. Das PP, Bagijn MP, Goldstein LD, Woolford JR, Lehrbach NJ, et al. 2008. Piwi and piRNAs act
upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis
elegans germline. Mol. Cell 31:79–90
114. Wang G, Reinke V. 2008. A C. elegans Piwi, PRG-1, regulates 21U-RNAs during spermatogenesis. Curr.
Biol. 18:861–67
115. Cecere G, Zheng GX, Mansisidor AR, Klymko KE, Grishok A. 2012. Promoters recognized by forkhead
proteins exist for individual 21U-RNAs. Mol. Cell 47:734–45

[Link] • Biogenesis and Functions of piRNA 25.27

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

116. Goh WS, Seah JW, Harrison EJ, Chen C, Hammell CM, Hannon GJ. 2014. A genome-wide RNAi
screen identifies factors required for distinct stages of C. elegans piRNA biogenesis. Genes Dev. 28:797–
807
117. Weick EM, Sarkies P, Silva N, Chen RA, Moss SM, et al. 2014. PRDE-1 is a nuclear factor essential for
the biogenesis of Ruby motif–dependent piRNAs in C. elegans. Genes Dev. 28:783–96
118. de Albuquerque BF, Luteijn MJ, Cordeiro Rodrigues RJ, van Bergeijk P, Waaijers S, et al. 2014. PID-1 is
a novel factor that operates during 21U-RNA biogenesis in Caenorhabditis elegans. Genes Dev. 28:683–88
119. Bagijn MP, Goldstein LD, Sapetschnig A, Weick EM, Bouasker S, et al. 2012. Function, targets, and
evolution of Caenorhabditis elegans piRNAs. Science 337:574–78
120. Lee HC, Gu W, Shirayama M, Youngman E, Conte D Jr, Mello CC. 2012. C. elegans piRNAs mediate
the genome-wide surveillance of germline transcripts. Cell 150:78–87
121. Shirayama M, Seth M, Lee HC, Gu W, Ishidate T, et al. 2012. piRNAs initiate an epigenetic memory
of nonself RNA in the C. elegans germline. Cell 150:65–77
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

122. Conine CC, Moresco JJ, Gu W, Shirayama M, Conte D Jr, et al. 2013. Argonautes promote male fertility
and provide a paternal memory of germline gene expression in C. elegans. Cell 155:1532–44
123. Seth M, Shirayama M, Gu W, Ishidate T, Conte D Jr, Mello CC. 2013. The C. elegans CSR-1 Argonaute
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

pathway counteracts epigenetic silencing to promote germline gene expression. Dev. Cell 27:656–63
124. Wedeles CJ, Wu MZ, Claycomb JM. 2013. Protection of germline gene expression by the C. elegans
Argonaute CSR-1. Dev. Cell 27:664–71
125. Ashe A, Sapetschnig A, Weick EM, Mitchell J, Bagijn MP, et al. 2012. piRNAs can trigger a multigen-
erational epigenetic memory in the germline of C. elegans. Cell 150:88–99
126. Luteijn MJ, van Bergeijk P, Kaaij LJ, Almeida MV, Roovers EF, et al. 2012. Extremely stable Piwi-
induced gene silencing in Caenorhabditis elegans. EMBO J. 31:3422–30
127. Aravin AA, Naumova NM, Tulin AV, Vagin VV, Rozovsky YM, Gvozdev VA. 2001. Double-stranded
RNA–mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster
germline. Curr. Biol. 11:1017–27
128. Nishida KM, Saito K, Mori T, Kawamura Y, Nagami-Okada T, et al. 2007. Gene silencing mechanisms
mediated by Aubergine piRNA complexes in Drosophila male gonad. RNA 13:1911–22
129. Nagao A, Mituyama T, Huang H, Chen D, Siomi MC, Siomi H. 2010. Biogenesis pathways of piRNAs
loaded onto AGO3 in the Drosophila testis. RNA 16:2503–15
130. Rouget C, Papin C, Boureux A, Meunier AC, Franco B, et al. 2010. Maternal mRNA deadenylation and
decay by the piRNA pathway in the early Drosophila embryo. Nature 467:1128–32
131. Robine N, Lau NC, Balla S, Jin Z, Okamura K, et al. 2009. A broadly conserved pathway generates
3 UTR-directed primary piRNAs. Curr. Biol. 19:2066–76
132. Saito K, Inagaki S, Mituyama T, Kawamura Y, Ono Y, et al. 2009. A regulatory circuit for piwi by the
large Maf gene traffic jam in Drosophila. Nature 461:1296–99
133. Gou LT, Dai P, Yang JH, Xue Y, Hu YP, et al. 2014. Pachytene piRNAs instruct massive mRNA
elimination during late spermiogenesis. Cell Res. 24:680–700
134. Watanabe T, Tomizawa S, Mitsuya K, Totoki Y, Yamamoto Y, et al. 2011. Role for piRNAs and
noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus. Science 332:848–
52
135. McClintock B. 1956. Controlling elements and the gene. Cold Spring Harb. Symp. Quant. Biol. 21:197–216
136. Singh DP, Saudemont B, Guglielmi G, Arnaiz O, Gout JF, et al. 2014. Genome-defence small RNAs
exapted for epigenetic mating-type inheritance. Nature 509:447–52
137. Kiuchi T, Koga H, Kawamoto M, Shoji K, Sakai H, et al. 2014. A single female-specific piRNA is the
primary determiner of sex in the silkworm. Nature 509:633–36
138. Rajasethupathy P, Antonov I, Sheridan R, Frey S, Sander C, et al. 2012. A role for neuronal piRNAs in
the epigenetic control of memory-related synaptic plasticity. Cell 149:693–707
139. Lee EJ, Banerjee S, Zhou H, Jammalamadaka A, Arcila M, et al. 2011. Identification of piRNAs in the
central nervous system. RNA 17:1090–99
140. Rinkevich Y, Rosner A, Rabinowitz C, Lapidot Z, Moiseeva E, Rinkevich B. 2010. Piwi positive cells
that line the vasculature epithelium, underlie whole body regeneration in a basal chordate. Dev. Biol.
345:94–104

25.28 Iwasaki · ·
Siomi Siomi

Changes may still occur before final publication online and in print
 BI84CH25-Siomi ARI 24 February 2015 12:7

141. Rinkevich Y, Voskoboynik A, Rosner A, Rabinowitz C, Paz G, et al. 2013. Repeated, long-term cycling
of putative stem cells between niches in a basal chordate. Dev. Cell 24:76–88
142. Janic A, Mendizabal L, Llamazares S, Rossell D, Gonzalez C. 2010. Ectopic expression of germline genes
drives malignant brain tumor growth in Drosophila. Science 330:1824–27
143. Qiao D, Zeeman AM, Deng W, Looijenga LH, Lin H. 2002. Molecular characterization of hiwi, a human
member of the piwi gene family whose overexpression is correlated to seminomas. Oncogene 21:3988–99
144. Suzuki R, Honda S, Kirino Y. 2012. PIWI expression and function in cancer. Front. Genet. 3:204
145. Lee JH, Schutte D, Wulf G, Fuzesi L, Radzun HJ, et al. 2006. Stem-cell protein Piwil2 is widely expressed
in tumors and inhibits apoptosis through activation of Stat3/Bcl-XL pathway. Hum. Mol. Genet. 15:201–
11
146. Kim EB, Fang X, Fushan AA, Huang Z, Lobanov AV, et al. 2011. Genome sequencing reveals insights
into physiology and longevity of the naked mole rat. Nature 479:223–27
147. Edrey YH, Park TJ, Kang H, Biney A, Buffenstein R. 2011. Endocrine function and neurobiology of the
Access provided by University of California - Santa Cruz on 03/07/15. For personal use only.

longest-living rodent, the naked mole-rat. Exp. Gerontol. 46:116–23

148. de Wit E, de Laat W. 2012. A decade of 3C technologies: insights into nuclear organization. Genes Dev.
26:11–24
Annu. Rev. Biochem. 2015.84. Downloaded from [Link]

149. Dekker J, Rippe K, Dekker M, Kleckner N. 2002. Capturing chromosome conformation. Science
295:1306–11

[Link] • Biogenesis and Functions of piRNA 25.29

Changes may still occur before final publication online and in print