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The Immune System’s Compact
Genomic Counterpart
Parasitic DNA has inﬁltrated our genome and do outside the cells of the germ line. “In
threatens our future. As in most other animals, mammals, the transposon-silencing function
much of the human genome derives from is just a small piece of what they do, but it’s
self-serving DNA strands known as transpo- the only piece we understand,” Zamore says.
sons. These genetic gypsies often jump to new One recent study, for instance, raises the poschromosome locations, sometimes disabling sibility that these molecules are important forgenes and even triggering cancer. In the germ learning. Researchers are also stumped as toline—sperm and eggs and the cells that spawn why mice generate hundreds of thousands,
them—a transposon hopping to a new posi- even millions, of piRNA varieties that have notion can lead to sterility, a disaster from a known transposon targets. “These are exciting
Darwinian point of view. “Failure to control times” in the ﬁeld, Pillai says.
transposons in most animals is the surest path
to extinction,” says biochemical geneticist Discovery of a new RNA
Phillip Zamore of the University of Massa- When researchers ﬁrst detected piRNAs inchusetts Medical School in Worcester.
2001, they were just beginning to grasp the
For that reason, a specialized group of importance of so-called small RNAs. These
RNA molecules known as piRNAs (pro- molecules, which are typically between 18 andnounced “pie-RNAs”) are the superheroes 40 nucleotides long and don’t code for proof animal genomes. Discovered in the past teins, were proving ubiquitous. “Small RNAs
decade, piRNAs team up with certain proteins have been harnessed by almost every single
to shackle transposons in animal germline life form we know,” Pillai says. Organisms
cells. Together, these protein-RNA combos deploy some small RNAs to turn down thecreate a molecular defense that scientists activity of their own genes, albeit indirectly.
liken to an immune system for the genome. Before a cell synthesizes the protein encoded
Like our immune system, piRNAs and their by a gene, it ﬁrst makes an RNA version ofpartners can tell friend from foe, mobilize a the gene, known as messenger RNA (mRNA).
response, and adapt to new invaders. Simi- The best-known types of small RNAs—
larly, our genome guardians have a memory, small interfering RNAs (siRNAs) anda record of past threats.
microRNAs—target these mRNAs, destroy“The complexity of this [piRNA] path- ing them or preventing the cell from translatway has exploded during evolution,” says ing them into proteins. Many organisms also
Julius Brennecke, a developmental geneticist enlist small RNAs to defend against pathoat the Institute of Molecular Biotechnology in gens. In plants and nematodes, for instance,
Vienna. The number of piRNA varieties that small RNAs help destroy viral RNA.
humans produce isn’t clear, but the total could
Eleven years ago, Alexei Aravin, then abe in the millions. “It’s not often that you dis- graduate student at Moscow State Univercover something that is so abuncontrol
piRNA
dant and that was missed for solong,” Zamore says. “It’s the perfect scientiﬁc problem.”Researchers intrigued by thisproblem have begun to sketch out
details of how these small RNAs
keep transposons in check. “We
are starting to learn what’s in the[piRNA] toolbox,” says molecular biologist Ramesh Pillai of
the European Molecular Biology
Laboratory in Grenoble, France. Shut down. Glowing egg cells stand out in worms whose piRNAs
But biologists still don’t know can’t silence a foreign DNA sequence (left panels). But when the
how cells manufacture this type piRNAs recognize and shut down the sequence, the egg cells areof RNA, or what piRNAs might dark (right panels).
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Guarding the germ line. DNA (blue) and two kindsof protective Piwi proteins (red, green) are visible inthis section of a mouse testis.

sity, and colleagues discovered several small
RNAs that shut down a transposonlike genein fruit ﬂies. At the time, the only hint thatthe molecules belonged to an unrecognizedgroup of RNAs was that they were slightly
longer than siRNAs, says Aravin, who is
now a molecular biologist at the California Institute of Technology in Pasadena. In afollow-up fruit ﬂy study 2 years later, however, he and colleagues identiﬁed more than
170 unique small RNAs that target transposons, suggesting that the insects have a
specialized type of RNA for this function.
Aravin would soon come across this newclass of small RNAs again, though by following a different research tack. He and otherbiologists were looking into the workings of
Piwi proteins, which studies had indicated arenecessary for fertility in several kinds of animals. Piwi proteins are part of the Argonautefamily. siRNAs and microRNAs work by consorting with non-Piwi Argonaute proteinsthat slice up RNA molecules. Some researchers speculated that Piwis also functioned bypartnering with RNAs. “It was only logicalto imagine that these similar family members
would also bind to small RNAs,” says molecular biologist Gregory Hannon of the Cold
Spring Harbor Laboratory in New York. In
2006, Aravin and colleagues, Hannon and coworkers, and two other groups independently
conﬁrmed this hypothesis, uncovering thousands of small RNAs that collaborate with
Piwi proteins in mice. Researchers realizedthat these small RNAs resembled the ones
Aravin and colleagues had initially identiﬁed
in fruit ﬂies and declared that all of them werean RNA family unto themselves, the Piwiinteracting RNAs, or piRNAs.
piRNAs differ from microRNAs andsiRNAs in several ways (see table, p. 27).
As Zamore’s team ﬁrst reported in 2006 in
Science (21 July 2006, p. 320), cells don’t
need the enzyme Dicer to make piRNAs.
However, Dicer is essential for the matu-

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CREDITS (TOP TO BOTTOM): A. A. ARAVIN ET AL., MOLECULAR CELL 31 (26 SEPTEMBER 2008) © 2008 ELSEVIER INC.; ADAPTED FROM M. P. BAGIJN ET AL., SCIENCE 337 (8 AUGUST 2012)

Small but powerful, piRNAs protect the genome and may have other functions as wellNEWSFOCUS

4 JANUARY 2013

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they key on transposons with complementary
sequences. But a study of nematodes indicatesthat the piRNA system might deploy a second
mechanism to prevent self-directed attacks,
suggests molecular geneticist Craig Melloof the University of Massachusetts Medical
School. He shared the 2006 Nobel Prize inphysiology or medicine for discovering RNAinterference: the ability of small RNAs to shut
down gene activity.
To move, transposons often make an RNA
copy, or transcript, of themselves that’s converted back to DNA in a new place. In the
6 July 2012 issue of Cell, Mello’s team proposed a novel way that piRNAs can avoid
mistaking this transposon RNA for a cell’svital RNA, such as messenger
RNAs. “People thought that piInitial
RNAs would target ‘aberrant
piRNAs
Detecting danger
RNA,’ ” that is, any sequence that
DNA with
Although the workings of thediffered from the animal’s own
piRNA system differ from thoseRNA sequences, Mello says. Heof our immune system, thesehas a different take: “Our ﬁndings
two defenses face many of thesuggest that a foreign sequence issame challenges. Their first job
recognized as foreign because it’sis detecting danger. piRNA clusnever been expressed”—used toters are crucial for this function.
make protein.
PIWI
PIWI
proteinin
They contain partial and complete
The researchers drew this contransposon sequences, and theyclusion after equipping nemaTTransposon
serve as the memory banks for thetodes with a fragment of wormtranscript
piRNA system. “It’s the way aniDNA that also included a foreignmals write down which transposequence—instructions for maksons have invaded their genome,”
ing the ﬂuorescent protein GFP.
Zamore says. Each piRNA targets
Mello and colleagues observed
transposons that contain a matcha curious pattern in the resulting
PIWI
ing sequence to its own RNA
mutant nematodes. In some ofproteineinisequence. By making piRNAs
the worms, piRNAs ignored thethat correspond to the transposon
inserted DNA, treating it as if it
piRNA cluster
sequences stored in the clusters,
were a normal gene. Those worms
transcript
animals can keep these selfish
made GFP and lit up.
strands in check.
But other worms remained
But what if an animal has todark because they reacted to thecontend with a transposon that it No game for transposons. In the ping-pong loop, one Piwi protein and a introduced DNA sequence as if
hasn’t encountered before? The piRNA slice up transposon RNA with a matching sequence. The transposon frag- it were a transposon and shut it
piRNA system relies on a nifty ments then join a different Piwi protein to produce more matching piRNAs.
down, preventing the productrick in these situations. “It makes
tion of GFP. These differences
use of the only thing that [selﬁsh] genetic ele- chromosomes. But as the flies grew older, remained steady from generation to generaments have in common—they move around they began to rein in the P element, crank- tion, Mello notes. “The ones that are on stay
the genome,” says molecular geneticist René ing out piRNAs that targeted it. Moreover, on, and the ones that are off stay off.”
Ketting of the Institute of Molecular Biology the researchers found that other transposons
According to Mello, why some inserted
in Mainz, Germany.
released by the P element began falling into DNA sequences are initially expressed andAs a new transposon migrates from loca- piRNA clusters, presumably allowing the ﬂies others are silenced is probably a matter oftion to location, it should eventually land in to make piRNAs to counter them as well. As chance. But if the DNA snippet is accepteda piRNA cluster. When that happens, the a result, the ﬂies regained some of their egg- and used to make proteins, the animal theretransposon becomes part of the memory producing capability.
after treats it as “self,” Mello suggests. He andbank, and the animal will begin producing
But how does a ﬂy or another animal tell a his colleagues hypothesize that worms have acomplementary, or matching, piRNAs to transposon from its own DNA? If the immune molecular pathway that keeps track of which
thwart the genomic interloper. Each piRNA system mistakes “self ” for microbial invad- DNA sequences have been active and prevents
cluster “is kind of a trap,” Pillai says. “Once ers, its responses can trigger autoimmune dis- piRNAs and Piwi proteins from interferinga transposon falls in, you have immunity.”eases. One way that piRNAs avoid triggering with them. The researchers haven’t pinpointed
Thanks to their genomic immune sys- genomic autoimmunity is their specificity; which molecules perform this job, Mello says,

26

tem, animals can recover from “infection” by
a new transposon, much as you get over theﬂu because your immune system defeats theinﬂuenza virus. For example, in a study published in the 23 December 2011 issue of Cell,
molecular geneticist William Theurkauf of the
University of Massachusetts Medical School,
Zamore, and colleagues followed what happened to young female ﬂies that inherited atransposon called the P element, which they
hadn’t tangled with before. At ﬁrst, the transposon got the jump on the insects. They wereinfertile and produced scant piRNAs that
had any ability to control the P element. The
genomic invader also unleashed other transposons that had been lurking in the flies’

CREDIT: C. BICKEL/SCIENCE

ration of microRNAs and siRNAs. Also,
unlike siRNAs and microRNAs, piRNAs
are exclusive to animals, occurring even in
ancient groups such as sponges.
The DNA sequences that code forpiRNAs are bunched in a few so-calledpiRNA clusters. One of the ﬁeld’s big mysteries is how these clusters give rise to piRNAs,
notes molecular geneticist Eric Miska of the
University of Cambridge in the United Kingdom. “piRNA biogenesis is still very enigmatic.” Cells likely make an RNA copy ofan entire cluster and then dissect it, hewing
the fragments into piRNAs. But the details of
this processing remain obscure. “More than
10 proteins are involved, but we know very
little about what steps they are
doing,” Aravin says.

NEWSFOCUS

Taking on transposons
Once immune cells meet an intruder, theycounterattack. piRNAs do the same, using
a variety of measures against transposons.
Some piRNAs dive into the fray. They trackdown transposon RNAs, and the Piwi proteins
they bring along slice up the rogue strands.
Some piRNAs let others do the dirty work.
In the 3 August 2012 issue of Science (p. 574),
Miska and colleagues described how piRNAs
boost their power by enlisting siRNAs to stiﬂe transposons. The reason, Miska suggests,
might be that although piRNAs come in manyvarieties—more than 16,000 in nematodes—
each germline cell harbors just a few copies ofeach one. “They can’t do much on their own,”
he says. In contrast, siRNAs are plentiful.
Other animals bolster their piRNAs
directly, relying on what’s called the pingpong ampliﬁcation loop. Hannon’s team anda group led by molecular biologist Mikiko
Siomi, now at Keio University School of
Medicine in Tokyo, independently described
this mechanism in ﬂies in 2007, but mice andzebraﬁsh also take advantage of a similar process. In the ping-pong loop, piRNAs and Piwi
proteins slice up transposon RNA. The resulting fragments undergo modiﬁcation and joinwith other Piwi proteins to cut up RNA transcripts of piRNA clusters, thus making newpiRNAs (see diagram, p. 26). The loop “onlyampliﬁes the useful piRNAs,” those with atarget available in the cell, says Brennecke, aco-author on one of the papers.
piRNAs can ﬁght back even if a transposon remains quiescent and hides as a stretchof genomic DNA. Researchers have foundthat in mice, piRNAs spur germline cells
to afﬁx methyl groups to transposon DNA,
preventing its transcription into RNA andthereby blocking the rogue strand’s movement to a new location in the genome.
Fruit fly piRNAs stymie transposon transcription using a similar mechanism thatinvolves molecular modiﬁcation of histones,
the protein spools around which DNA coils,
Brennecke and colleagues reported in the
21 November 2012 issue of Cell.
These strategies lead to long-term protection, and in that regard, piRNAs have ourimmune system beat. After you’ve recoveredfrom an infectious disease, you often will be
immune to the pathogen that caused it forlife—but your children and grandchildren

won’t be. Animals’ genomic guardians, bycontrast, can suppress some transposons for
multiple generations, Mello’s and Miska’steams revealed in the 6 July 2012 issue of
Cell. For example, this genomic resistancelasts for at least 20 generations in nematodes,
Miska and colleagues showed. Persistent
protection makes sense: Transposons in thegerm line can reactivate each generation, so
locking them down long-term is beneﬁcial.
More than defense?
piRNAs may do more than thwart transposons. Some scientists suspect that they, likesiRNAs and microRNAs, help adjust gene
expression. For example, Mello and col-germline cells seem to make Piwi proteins,piRNAs’ collaborators.
But a discovery from neuroscientist Eric
Kandel of Columbia University and colleagues suggests that piRNAs are active in thecentral nervous system, helping create memories. In the 27 April 2012 issue of Cell, theteam reported that they had identiﬁed piRNAs
in neurons from the sea slug Aplysia. The
piRNAs help block the production of a protein called CREB2, which inhibits memory
formation in these animals. Testing piRNAs’
role in learning in other creatures shouldn’t
be difﬁcult, Ketting says. If they do have arole, deleting Piwi proteins in animals such as
mice or ﬂies should cause memory lapses.

COMPARING DIFFERENT KINDS OF SMALL RNAS
LengthsiRNAmicroRNApiRNA

21–24 nucleotides

20–25 nucleotides

21–31 nucleotides

Organization

Double-stranded

Single-stranded

Single-stranded

Requires Dicer for maturation?

Yes

Yes

No

Found inAnimals, plants,
fungi, protists

Animals, plants,
protists

Only animals

FunctionControllinggene expression,
blocking transposons

Controllinggene expression

Blocking
transposons

leagues suggest that the targets of many nematode piRNAs are some of the worm’s own
genes, not transposons. They have shown thatabout 1000 of the roughly 20,000 nematodegenes are under piRNA control. Many of
the genes are normally turned off but switch
on in worms that lack one kind of Piwi protein, Mello and colleagues reported in the
6 July 2012 issue of Cell. One possibility,
Mello says, is that these genes perform functions that are useful in certain environmental conditions, such as when the worms areunder stress. When times are tough, a cell in
the germ line of a parent worm might rein in
piRNAs, allowing the genes to switch on andhelping the offspring cope with adversity.
The idea that piRNAs are tweaking
gene activity gets mixed reviews from otherresearchers. Some remain skeptical that piRNAs ever silence genes. Even if they accept
that possibility, other scientists question
whether animals other than nematodes avail
themselves of this gene-controlling mechanism. Few transposons trouble nematodes, so
the worms might have the freedom to divert
their piRNAs to new roles. “In most systems,
the evidence favors transposons being the targets” of piRNAs, and not genes, Ketting says.
Also unclear is whether piRNAs function
in nongermline cells. Most scientists havedismissed the possibility, as siRNAs quashtransposons in these cells. Moreover, only

www.sciencemag.org SCIENCE VOL 339
Published by AAASThe question that has researchers scratching their heads involves the pachytene
piRNAs, which are named for the stage ofmeiosis—the process that produces eggs
and sperm—in which they appear. Mammals
generate a huge number of different pachytene piRNAs—one recent study estimatedthe total for mice at more than 800,000,
but Zamore says that value is almost certainly too low. Yet the sequences of thepachytene piRNAs do not match those of
any transposons, suggesting that they aren’ttargeting the rogue strands. “What thesepachytene piRNAs are doing—nobody
knows,” Aravin says.
As piRNA researchers delve into such
mysteries, some also wonder if these genomic
superheroes sometimes take the day off tohelp a species adapt. We and other animals
are alive today because, over hundreds of millions of years, piRNAs helped our recent anddistant ancestors tamp down transposons. But
transposons aren’t necessarily all bad. They
also create genetic variation in the germ linethat is the raw material for natural selection.
A few researchers speculate that when conditions are rough, animals might inhibit their
piRNAs to unleash transposons and trigger
more mutations, speeding up their evolution.
That idea is “a very attractive hypothesis,”
Zamore says. “I’d like to think of a way to test
it experimentally.”–MITCH LESLIE

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but they suspect an Argonaute protein called
CSR-1 is the ringleader. Pillai describes thispotential recognition mechanism as “an interesting idea and plausible,” adding that the
Mello group’s paper is “the only one whichmight explain the available data.”