INSIGHTS | P E R S P E C T I V E S

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10.1126/science.aab0234

756

RNA

Phased piRNAs tackle
transposons
Tertiary piRNAs help silence dangerous DNA elements
By Haruhiko Siomi1 and Mikiko C. Siomi2Transposable elements are dominant
inhabitants of the eukaryotic genome.
Their ability to mobilize and insert
anywhere in the genome can cause
mutations that are generally detrimental to the host. This makes them
key targets for silencing. Small RNAs called
PIWI-interacting RNAs (piRNAs) are onemeans to attack transposons. They constitute the largest class of small RNAs, but themechanisms by which they are generated
are not completely clear. On pages 812 and817 of this issue, Mohn et al. (1) and Han etal. (2), respectively, propose a revised modelfor piRNA biogenesis that increases the diversity of piRNA sequences and thereby expands the range of targets they can silence.
In animals, piRNAs are generated in germline cells, which means their effects can betransmitted to offspring. Most piRNAs arederived from genomic regions called piRNAclusters, which also harbor a large numberof truncated transposable elements (3). In
the germ cells of Drosophila melanogaster,
single-stranded precursor transcripts (antisense) are produced from these clusters (seethe figure). This precursor piRNA is thencleaved by an endonuclease Zucchini (Zuc),to generate short primary piRNAs (26 to 31
nucleotides long). The processing of clustertranscripts occurs in the cytoplasm, andmature piRNAs are loaded onto the PIWI
proteins Ago3, Aub, and Piwi (4). PrimarypiRNAs have a bias toward having uridine attheir 5´ ends (1U bias), are largely antisensewith regard to corresponding transposable
element sequences, and are loaded onto
Aub and Piwi. Aub-bound primary piRNAs,
together with Ago3, then initiate a “pingpong cycle,” which generates secondary
piRNAs by cleaving complementary targets(both sense and antisense single-stranded
RNA). Cleavage not only determines the 5ⴕ
end of secondary piRNAs but also consumes
transposable element transcripts, thereby
silencing transposons (3, 5). Secondary piRNAs loaded onto Ago3 show 10-nucleotide
complementarity at their 5ⴕ ends with Aubbound primary piRNAs and possess a sense
bias with adenosine at the 10th nucleotide(10A bias). Piwi does not participate in the
ping-pong cycle because once loaded withpiRNAs, it is imported into the nucleus torepress transposable elements by modifying
chromatin (6). This cycling model amplifies only ping-pong pairs of piRNAs and istherefore insufficient to explain the extraordinary sequence diversity of piRNAs.
Using state-of-the art bioinformatics,Mohn et al. and Han et al. independently
found that Piwi- and Aub-, but not Ago3bound piRNAs, displayed notable sequence“phasing.” By analyzing small RNA populations from Drosophila germ cells (fromovaries) that are depleted of Rhino, a factor
required for dual strand piRNA cluster transcription (7–9), Mohn et al. noticed a distinct
nucleotide start site in piRNA profiles. Thesesites may correspond to piRNA biogenesis
initiation sites, and the piRNAs that containthem are called “responder piRNAs.” Most
responder piRNAs occupy Aub and are com-“…PIWI-interacting RNAs
(piRNAs) are one means toattack transposons.”
plementary to Ago3-bound piRNAs, with a
10-nucleotide overlap at their 5ⴕ ends. These
Ago3-bound secondary piRNAs may trigger
production of responder piRNAs. Surprisingly, piRNAs originating downstream of theresponder piRNAs, called “trailer piRNAs,”
mostly associate with Piwi.
Mohn et al. observed that the 3ⴕ ends ofresponder piRNA are immediately followedby 5ⴕ ends of Piwi-bound trailer piRNAs.
Remarkably, trailer piRNAs show phasing
with a ~27-nucleotide interval, though boththe amounts of trailer piRNA and phasing
accuracy decrease as the distance from thetrigger site increases. Phased trailer piRNAs
also show a striking 1U bias. These findings support an intriguing model in which
piRNA 3ⴕ ends are defined by a cleavage
event that preferentially occurs immediately upstream of a U residue, which corresponds to the 5ⴕ end of the next piRNA.
1Department of Molecular Biology, Keio University Schoolof Medicine, Tokyo 160-8582, Japan. 2Graduate School ofScience, The University of Tokyo, Tokyo 113-0032, Japan.
E-mail: awa403@keio.jp, siomim@bs.s.u-tokyo.ac.jp

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toaurignacian, although at sites including Le
Piage and Isturitz in France, the Protoaurignacian underlies the Aurignacian (11, 12).
The best record of early symbolic artifacts
in Europe comes from the Swabian Aurignacian and from the Aurignacian of southwestern France and several other regions, andnot from the Protoaurignacian.
It is not clear, however, whether the term
Protoaurignacian has much meaning. For
example, the purported Protoaurignacianassemblage of Krems-Hundssteig has beenshown to be a mixed assemblage containing a strong component of artifacts fromthe Gravettian, an Upper Paleolithic culturalgroup that postdates the Protoaurignacianby ~10,000 years (13). Also, artifacts such as
split-based bone points and carinated scapers, often viewed as hallmarks of the Aurignacian, have been found at sites attributed
to the Protoaurignacian (see the figure). Thisis the case with the split-based bone points at
Trou de la Mère Clochette, Arbreda, and Fumane (14). Similarly, lithic assemblages withmixed signatures have been recovered from
Crimea, Romania, and the Basque Country(15). The concept of the Protoaurignacian isthus not as robust as the article by Benazzi
et al. implies.
While the research reported by Benazzi etal. is a welcome step forward in establishing the narrative of colonization of Europe
by modern humans, the archaeological andhuman fossil records will almost certainly
prove to be more complex and fascinating
than our current models suggest. It is only
through carefully excavating sites and establishing high-resolution regional signatures ofthe events and processes of the last glacial
cycle that a reliable picture of the spread ofmodern humans and the extinction of Neandertals will come into focus. ■Precursor piRNA(antisense)

Precursor piRNA(sense)

X

Zuc

Piwi
PrimarypiRNA

Aub

ResponderpiRNA

TertiarypiRNA
Trailer piRNA

Ago3 cleavage
site ofset
from Zuc Ago3

ZucTriggerpiRNA
Secondary piRNA
Ago3

scripts: Once a cleaved 3ⴕ transcript with1U is loaded onto Piwi, it follows repeatedrounds of cleavage with a ~27-nucleotide
interval. The interval may be determinedby a combination of Piwi’s footprint, Zuccleavage of Piwi-bound trail sequences, andthe preference of Piwi to bind 1U. Although
Zuc-dependent piRNAs are viewed as primary piRNAs, trailer piRNAs, which occupy
the vast majority of Piwi, may reasonably
be called “tertiary” piRNAs because theirproduction depends on secondary piRNAs.
Therefore, primary piRNAs make secondarypiRNAs, which make phased “tertiary piRNAs.” What initiates this sequential pathway
is Aub-bound antisense primary piRNAs,
which produce Ago3-bound sense “triggerpiRNAs” in the ping-pong cycle. This raises
the question of how such antisense primarypiRNAs are selectively produced.
Both Mohn et al. and Han et al. also observed phased piRNAs in ovarian somaticcells where only the primary piRNA production pathway operates, indicating that Zucdependent phasing can be initiated without
the ping-pong cycle. Because purified Zucexhibits no nucleotide preference for its
cleavage site (10, 11), ovarian somatic cells
may rely on factors replacing the functionof Ago3-trigger piRNA complexes to determine piRNA biogenesis initiation sites onparticular transcripts. This ping-pong–independent mechanism should also be available in germline cells to supply Aub-boundantisense primary piRNAs that produce
Ago3-bound trigger piRNAs.
Mohn et al. and Han et al. demonstratethat Zuc-mediated 3ⴕ end formation ofpiRNAs is also evident in mice, suggestingthat primary piRNA phasing is mechanistically conserved in mammals. Thus, important future challenges are to identify factorsthat endow specificity to Zuc both in germline and ovarian somatic cells to trigger
3ⴕ-directed and phased piRNA productionin the vast array of the transcriptome, andto elucidate the mechanisms that funnel
most Zuc-cleaved transcripts onto Piwi. ■ILLUSTRATION: C. SMITH AND V. ALTOUNIAN/SCIENCEREFERENCES

Expanding the piRNA arsenal. In Drosophila germ cells, Ago3-bound secondary piRNAs act as triggers to produce
responder piRNAs that bind to Aub. This initiates production of trailer piRNAs (through Zuc-dependent phased tertiary
RNA production) that mostly bind to Piwi. Protein X may determine piRNA cluster transcripts among cellular RNAs andinitiate phased piRNA production by cleaving the cluster transcripts. A similar ping-pong–independent mechanism
may also exist in ovarian somatic cells.

Thus, 5ⴕ- and 3ⴕ-end formation of piRNAs
is mechanistically coupled, precluding a3ⴕ-trimming event to form mature piRNAs.
Trailer piRNAs are selectively lost in ovaries
lacking Zuc, suggesting that Zuc determines
both the 3ⴕ and 5ⴕ ends of most germlinePiwi-bound piRNAs. By sequencing analysisof cleaved transcripts, Han et al. also con-cluded that production of Piwi- and Aubbound phased piRNAs depends on Zuc.
The findings of Mohn et al. and Han et al.
also suggest that cleavage in the ping-pongcycle by trigger piRNA (bound to Ago3) produces not only a corresponding ping-pongpartner piRNA but also piRNAs from theremaining 3´ portion of the cleaved tran-

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10.1126/science.aab3004
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Published by AAAS757

Phased piRNAs tackle transposons
Haruhiko Siomi and Mikiko C. Siomi
Science 348, 756 (2015);
DOI: 10.1126/science.aab3004

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