4. Smith, S.A., Beaulieu, J.M., and
Donoghue, M.J. (2010). An uncorrelated
relaxed-clock analysis suggests an earlier
origin for flowering plants. Proc. Natl. Acad.
Sci. USA 107, 5897–5902.
5. Bell, C.D., Soltis, D.E., and Soltis, P.S. (2010).
The age and diversification of the angiosperms
re-revisited. Am. J. Bot. 97, 1296–1303.
6. Cardinal, S., and Danforth, B.N. (2013). Bees
diversified in the age of eudicots. Proc. R. Soc.
Biol. Sci. 280, 20122686.
7. Michez, D., Vanderplanck, M., and Engel, M.S.
(2012). Fossil bees and their plant associates.
In Evolution of Plant-Pollinator Relationships,
S. Patiny, ed. (Cambridge: Cambridge
University Press), pp. 103–164.
8. Graur, D., and Martin, W. (2004). Reading the
entrails of chickens: molecular timescales of
evolution and the illusion of precision. Trends
Genet. 20, 80–86.
9. Elliott, D.K., and Nations, J.D. (1998).
Bee burrows in the late Cretaceous (Late
Cenomanian) Dakota formation, northeastern
Arizona. Ichnos 5, 243–253.
10. Kay, K.M., and Sargent, R.D. (2009). The role
of animal pollination in plant speciation:
integrating ecology, geography, and genetics.
Annu. Rev. Ecol. Evol. Syst. 40, 637–656.
11. Grimaldi, D. (1999). The co-radiations of
pollinating insects and angiosperms in the
Cretaceous. Ann. Mo. Bot. Gard. 86, 373–406.
12. Ramı ´rez, S.R., Eltz, T., Fujiwara, M.K.,
Gerlach, G., Goldman-Huertas, B.,
Tsutsui, N.D., and Pierce, N.E. (2011).
Asynchronous diversification in a specialized
plant-pollinator mutualism. Science 333,
13. Neff, J.L., and Simpson, B.B. (1981).
Oil-collecting structures in the Anthophoridae
(Hymenoptera): morphology, function, and use
in systematics. J. Kansas Entomol. Soc. 54,
14. Renner, S.S., and Schaefer, H. (2010). The
evolution and loss of oil-offering flowers: new
insights from dated phylogenies for
angiosperms and bees. Philos. Trans. Roy.
Soc. B Biol. Sci. 365, 423–435.
15. Davis, C.C., Bell, C.D., Mathews, S., and
Donoghue, M.J. (2002). Laurasian migration
explains Gondwanan disjunctions: evidence
from Malpighiaceae. Proc. Natl. Acad. Sci. USA
16. Xi, Z., Ruhfel, B.R., Schaefer, H., Amorim, A.M.,
Sugumaran, M., Wurdack, K.J., Endress, P.K.,
Matthews, M.L., Stevens, P.F., and Mathews, S.
(2012). Phylogenomics and a posteriori data
partitioning resolve the Cretaceous angiosperm
radiation Malpighiales. Proc. Natl. Acad. Sci.
USA 109, 17519–17524.
17. Cosacov, A., Sersic, A.N., Sosa, V.,
De-Nova, J.A., Nylinder, S., and Cocucci, A.A.
(2009). New insights into the phylogenetic
relationships, character evolution, and
phytogeographic patterns of Calceolaria
(Calceolariaceae). Am. J. Bot. 96, 2240–2255.
18. Schaefer, H., and Renner, S.S. (2008). A
phylogeny of the oil bee tribe Ctenoplectrini
(Hymenoptera: Anthophila) based on
mitochondrial and nuclear data: evidence
for Early Eocene divergence and repeated
out-of-Africa dispersal. Mol. Phylogenet. Evol.
19. Davis, C.C., and Anderson, W.R. (2010).
A complete generic phylogeny of
Malpighiaceae inferred from nucleotide
sequence data and morphology. Am. J. Bot. 97,
20. Ascher, J. (2013). Discover Life Website. (http://
1Department of Organismic and Evolutionary
Biology, Harvard University Herbaria, 22
Divinity Avenue, Cambridge, MA 02138, USA.
2Biodiversitaet der Pflanzen, Technische
Universitaet Muenchen, D-85354 Freising,
Small RNA-Directed Silencing: The
Fly Finds Its Inner Fission Yeast?
Several recent studies demonstrate that piRNAs guide Piwi protein to repress
transposon transcription in fly ovaries, much as fission yeast use siRNAs to
silence repeat sequences. Still mysterious though is how Piwi targets
euchromatic transposons for silencing, but not the specialized
heterochromatic loci that produce piRNA precursors.
Daniel Tianfang Ge
and Phillip D. Zamore
Fungi, plants, and animals devote
considerable resources to thwart
transposable elements from increasing
their numbers or moving to new
genomic locations, particularly in germ
cells. In fungi and plants, small
interfering RNAs (siRNAs) act via the
RNA interference (RNAi) pathway
to silence transposons and other
types of repetitive DNA. In contrast,
animals use PIWI-interacting
RNAs (piRNAs), a class of small
silencing RNAs distinct from siRNAs,
to silence germline transposons
and ensure fertility. Like siRNAs
and the mRNA-regulating microRNAs
(miRNAs), piRNAs direct Argonaute
proteins to silence complementary
nucleic acid targets. Unlike siRNAs
and miRNAs, piRNAs guide a
specialized sub-class of Argonautes,
the PIWI proteins, which are found
exclusively in animals and nearly
always in the germline or
In Drosophila, piRNAs bind
three different PIWI proteins:
P-element-induced wimpy testes
(Piwi), Aubergine (Aub), and
Argonaute3 (Ago3). Aub and Ago3 act
strictly in the ovary and testis germline,
where they silence transposons by
destroying their RNA transcripts. In
contrast, Piwi resides in the nucleus,
where it represses transposons in
both germ cells and their supporting
somatic cells [1–3]. Now, four papers
demonstrate that Piwi silences
transposons, at least in part, by
repressing their transcription [4–7].
These genome-scale studies support
and extend earlier evidence that Piwi
directs transcriptional silencing in the
nucleus [3,8,9]. By depleting Piwi in
the ovarian germline [5,6], ovarian
somatic follicle cells , or cultured,
immortalized ovarian somatic cells
(OSCs) , or by inserting ectopic
piRNA target sites into the fly genome
, all four studies find that piRNAs
guide Piwi to its target loci, where
it recruits enzymes that establish
(Figure 1A). The papers generally
support the view that piRNAs
tether Piwi to nascent transcripts:
RNA is required for Piwi to
co-immunoprecipitate with chromatin
 and with proteins known to bind
nascent RNA . Piwi bound to
nascent RNA via its piRNA guide
appears to recruit Su(var)3-9 , a
histone methyltransferase that
methylates histone H3 on lysine 9.
These ‘H3K9me3’ marks bind
heterochromatin protein 1 (HP1,
officially named Su(var)205),
generating chromatin that is refractory
to transcription, as reflected by
reduced occupancy with RNA
polymerase II (pol II) . Supporting
this view, depletion of Piwi reduces
the amount of H3K9me3 [4–6,9] and
HP1  and increases the amount of
RNA pol II [4,5] and nascent transcripts
[4,6,8] at transposon sequences.
These findings call to mind the
silences repetitive sequences in the
fission yeast, Schizosaccharomyces
pombe. siRNAs bound to S. pombe
Ago1 guide the ‘RITS’ complex
to nascent transcripts from
transposon-like repeats near the
centromere, where it recruits
proteins that establish repressive
Current Biology Vol 23 No 8
heterochromatin  (Figure 1B).
Unlike fission yeast, whose siRNAs
derive from the silenced loci
themselves, transposon silencing in
flies requires transcription of both a
trigger locus — a ‘piRNA cluster’ —and
a transposon target locus. The model
that has emerged since 2006
maintains that piRNAs originate from
piRNA clusters, which record the fly’s
history of transposon invasion. The
new findings suggest that Piwi-bound
piRNAs bind the nuclear transcripts of
euchromatic transposons, initiating
their silencing. Does Piwi also ensure
the heterochromatic character of
piRNA clusters? How are the clusters
transcribed rather than silenced, and
why do their transcripts generate
piRNAs when RNA from other genes
In Piwi-depleted OSCs, the amount
of H3K9me3 is unaltered for
heterochromatic loci, where most
piRNA clusters reside . Similarly,
germline loss of Armitage — a protein
required for wild-type Piwi abundance
and localization — has no effect on
the transcription of at least two
heterochromatic elements . Yet
Piwi depletion in the ovarian germline
shows a general decrease of H3K9me3
in heterochromatic loci , although
a similar experiment by Hannon and
colleagues failed to detect such a
change in the chromatin of
germline-active piRNA clusters .
At present, it is not known if these
discrepant results reflect the different
tissues or cell populations studied, the
difficulty in accurately assigning highly
repetitive heterochromatic sequences
to specific genomic locations, or a
difference in the chromatin status of
piRNA clusters and the surrounding
Piwi helps maintain H3K9me3 marks
on euchromatic transposon copies
but may play a different role for
the transcriptionally active,
proteins that facilitate the processing
of piRNAprecursor transcripts, such as
Rhino , Cutoff  or UAP56 
(Figure 1A). Without Piwi, clusters may
still be transcribed, but the resulting
RNA may fail to be exported to the
perinuclear nuage for conversion into
piRNAs. Such a model helps explain
why Piwi depletion leads to nuclear
accumulation of transcripts
containing transposon sequences
, why steady-state level of
increases more than nascent
transcription output , and why
mature piRNA abundance, including
piRNAs presumably bound to Ago3,
declines when transposon-containing
transcripts accumulate .
In S. pombe, silencing can spread
beyond the region of heterochromatin
initially established by Ago1-bound
siRNAs. In Piwi-depleted OSCs,
protein-coding genes adjacent
to transposons show decreased
H3K9me3 and increased transcription
, suggesting that these regions are
normally silenced by the spreading of
heterochromatin beyond transposon
boundaries. Such spreading might
result from HP1 recruiting the histone
methyltransferase Su(var)3-9 or
Setdb1, which both deposit H3K9me3
marks on nearby nucleosomes, in turn
recruiting more HP1 . This process
would not require piRNAs
complementary to the sequences
flanking the transposons. While the
model is appealing, data from Toth and
colleagues suggest that expression of
genes adjacent to piRNA-silenced
loci is unaffected by loss of Piwi ,
and argue that much of the
observed increase in expression of
protein-coding genes reflects the
induction of stress responses triggered
by transposon activation and
associated DNA damage.
To date, all RNA silencing
pathways — that is, those mediated by
Argonaute proteins and directed by
small RNA guides — derive their
nucleic acid binding specificity from a
specialized guide region, the seed.
Argonaute proteins create the seed by
pre-organizing guidenucleotides 2–8in
a geometry resembling one strand of
an RNA helix. Given the remarkable
structural conservation of Argonaute
proteins from bacteria, fungi, and
animals, the field has assumed that all
Argonaute proteins derive their binding
specificity from complementarity
between the seed and target
sequences. Supporting the view that
Figure 1. Small RNA-guided transcriptional silencing.
(A) Current evidence suggests that only Piwi loaded with a mature piRNA is allowed to enter
the nucleus, where it binds to nascent transposon transcripts in euchromatin, recruiting
Su(var)3-9 or Setdb1 to methylate nearby histone H3 at lysine 9. HP1 binds to H3K9me3
and generates chromatin that is refractory to transcription. In Drosophila heterochromatin,
Piwi bound to nascent piRNA cluster transcripts may recruit Rhino, Cuff or UAP56 to facilitate
export of the transcripts to cytoplasmic sites of piRNA production. (B) In S. pombe,
siRNA-guided Ago1 binds nascent transcripts and Chp1 associates with chromatin at pericen-
tromeric repeats. Ago1 and Chp1 are components of the RITS complex. RITS recruits Clr4 to
methylate nearby histone H3 at lysine 9. Swi6 binds to H3K9me3 and generates transcription-
ally repressed chromatin. The nascent transcript may be cleaved by Ago1 and converted into
double-stranded RNA by Rdp1. Dcr1 generates siRNA duplexes and loads them into Ago1 to
target additional transcripts.
the general biochemical properties of
Argonaute proteins are conserved
among PIWI proteins, fly and mouse
PIWIs cleave their target RNAs at the
same phosphodiester bond as
Argonaute proteins acting in the
miRNA and siRNA pathways [16,17].
Moreover, mutation of the predicted
50-phosphate-binding pocket, which
anchors the small RNA guide to an
Argonaute protein, blocks small RNA
binding by Piwi in vivo . However,
Lin and colleagues find that target
mutations that disrupt pairing with
either the piRNA seed or regions
outside the seed have similar effects
in vivo . Given that the endonuclease
activity of Piwi is dispensable for Piwi
to silence transposons [2,4,18], their
data suggest that Piwi uses an
alternative, non-seed mechanism to
bind its targets. Clearly, rigorous
quantitative biochemical analysis will
be required to test this proposal.
The paradoxical requirement for
transcription to silence transcription
of a locus means that silencing is a
quantitative process: transcription of
repetitive sequences can be reduced
but not eliminated if the loci are
to remain silent. Thus, additional
mechanisms such as
post-transcriptional RNA destruction
are likely required to achieve complete
silencing. Indeed, although many
piRNAs appear to be bound by both
Piwi and Aub in the Drosophila
germline, transcriptional silencing
by Piwi-bound piRNAs is not
redundant with post-transcriptional
RNA destruction by piRNAs bound
to Aub. In fact, only some transposon
families recruit more RNA pol II  or
increase transcription  when Piwi is
depleted in the ovary using RNAi.
The contradictory result that in piwi
mutant flies all transposon families but
one recruit more RNA pol II  needs
to be interpreted with caution.
Traditionally, more weight has been
placed on observations made using
bona fide genetic mutations than RNAi.
However, piwi mutants have
rudimentary ovaries, making
comparisons even to the small ovaries
of newly eclosed females challenging.
Although adult flies have little if any
Piwi protein outside the gonads, the
use of whole flies instead of isolated
ovaries complicates the analysis of
chromatin immunoprecipitation (ChIP)
data for RNA pol II, HP1, and histone
methylation marks , because
deconvoluting the germline signal from
the broader somatic signal is not
Mapping the genomic locations of
Piwi protein is quite challenging [4,19],
particularly because Piwi appears tobe
tethered to the chromatin via nascent
RNA transcripts. To accomplish this,
Lin and colleagues employed an
nuclei were cross-linked with
formaldehyde, rather than the more
standard method of cross-linking
the cells or tissue prior to disrupting
the cell membrane. Moreover, their
protocol omits ionic detergents during
nuclear lysis, raising the theoretical
possibility that some Piwi binding
events may have occurred in the lysate
rather than in vivo.
Why do flies need two independent
yet seemingly overlapping
mechanisms to protect their germline?
Perhaps the requirement for nascent
RNA to recruit Piwi to euchromatic
transposon insertions limits the
effectiveness of the pathway, with Aub
subsequently mopping up the spillover
from Piwi-mediated silencing. If true,
this model suggests that all small RNA
silencing pathways that use nascent
RNA as staging for modifying nearby
chromatin will require a parallel
post-transcriptional mechanism to
ensure complete silencing of
transposons and repetitive RNAs.
It is tempting to speculate that the
reported post-transcriptional silencing
activity of yeast Ago1 may provide
such a backup function in
S. pombe .
1. Li, C., Vagin, V.V., Lee, S., Xu, J., Ma, S., Xi, H.,
Seitz, H., Horwich, M.D., Syrzycka, M.,
Honda, B.M., et al. (2009). Collapse of germline
piRNAs in the absence of Argonaute3 reveals
somatic piRNAs in flies. Cell 137, 509–521.
2. Saito, K., Ishizu, H., Komai, M., Kotani, H.,
Kawamura, Y., Nishida, K.M., Siomi, H., and
Siomi, M.C. (2010). Roles for the Yb body
components Armitage and Yb in primary piRNA
biogenesis in Drosophila. Genes Dev. 24,
3. Klenov, M.S., Sokolova, O.A., Yakushev, E.Y.,
Stolyarenko, A.D., Mikhaleva, E.A.,
Lavrov, S.A., and Gvozdev, V.A. (2011).
Separation of stem cell maintenance and
transposon silencing functions of Piwi protein.
Proc. Natl. Acad. Sci. USA 108, 18760–18765.
4. Sienski, G., Donertas, D., and Brennecke, J.
(2012). Transcriptional silencing of transposons
by Piwi and maelstrom and its impact on
chromatin state and gene expression. Cell 151,
5. Le Thomas, A., Rogers, A.K., Webster, A.,
Marinov, G.K., Liao, S.E., Perkins, E.M.,
Hur, J.K., Aravin, A.A., and Toth, K.F. (2013).
Piwi induces piRNA-guided transcriptional
silencing and establishment of a repressive
chromatin state. Genes Dev. 27, 390–399.
6. Rozhkov, N.V., Hammell, M., and Hannon, G.J.
(2013). Multiple roles for Piwi in silencing
Drosophila transposons. Genes Dev. 27,
7. Huang, X.A., Yin, H., Sweeney, S., Raha, D.,
Snyder, M., and Lin, H. (2013). A major
epigenetic programming mechanism guided
by piRNAs. Dev. Cell 24, 502–516.
8. Shpiz, S., Olovnikov, I., Sergeeva, A.,
Lavrov, S., Abramov, Y., Savitsky, M., and
Kalmykova, A. (2011). Mechanism of the
piRNA-mediated silencing of Drosophila
telomeric retrotransposons. Nucleic Acids Res.
9. Wang, S.H., and Elgin, S.C. (2011). Drosophila
Piwi functions downstream of piRNA
production mediating a chromatin-based
transposon silencing mechanism in female
germ line. Proc. Natl. Acad. Sci. USA 108,
10. Castel, S.E., and Martienssen, R.A. (2013). RNA
interference in the nucleus: roles for small
RNAs in transcription, epigenetics and beyond.
Nat. Rev. Genet. 14, 100–112.
11. Sigova, A., Vagin, V., and Zamore, P.D. (2006).
Measuring the rates of transcriptional
elongation in the female Drosophila
melanogaster germ line by nuclear run-on. Cold
Spring Harb. Symp. Quant. Biol. 71, 335–341.
12. Klattenhoff, C., Xi, H., Li, C., Lee, S., Xu, J.,
Khurana, J.S., Zhang, F., Schultz, N.,
Koppetsch, B.S., Nowosielska, A., et al. (2009).
The Drosophila HP1 homolog Rhino is
required for transposon silencing and piRNA
production by dual-strand clusters. Cell 138,
13. Pane, A., Jiang, P., Zhao, D.Y., Singh, M., and
Schupbach, T. (2011). The Cutoff protein
regulates piRNA cluster expression and piRNA
production in the Drosophila germline. EMBO J.
14. Zhang, F., Wang, J., Xu, J., Zhang, Z.,
Koppetsch, B., Schultz, N., Vreven, T.,
Meignin, C., Davis, I., Zamore, P., et al. (2012).
UAP56 couples piRNA clusters to the
perinuclear transposon silencing machinery.
Cell 151, 871–884.
15. Talbert, P.B., and Henikoff, S. (2006).
Spreading of silent chromatin: inaction at
a distance. Nat. Rev. Genet. 7, 793–803.
16. Gunawardane, L.S., Saito, K., Nishida, K.M.,
Miyoshi, K., Kawamura, Y., Nagami, T.,
Siomi, H., and Siomi, M.C. (2007). A
Slicer-mediated mechanism for
repeat-associated siRNA 50end formation
in Drosophila. Science 315, 1587–1590.
17. Reuter, M., Berninger, P., Chuma, S., Shah, H.,
Hosokawa, M., Funaya, C., Antony, C.,
Sachidanandam, R., and Pillai, R.S. (2011).
Miwi catalysis is required for piRNA
amplification-independent LINE1 transposon
silencing. Nature 480, 264–267.
18. Darricarrere, N., Liu, N., Watanabe, T., and
Lin, H. (2013). Function of Piwi, a nuclear Piwi/
Argonaute protein, is independent of its slicer
activity. Proc. Natl. Acad. Sci. USA 110,
19. Moshkovich, N., and Lei, E.P. (2010). HP1
recruitment in the absence of Argonaute
proteins in Drosophila. PLoS Genet. 6,
20. Sigova, A., Rhind, N., and Zamore, P.D. (2004).
A single Argonaute protein mediates both
transcriptional and posttranscriptional
silencing in Schizosaccharomyces pombe.
Genes Dev. 18, 2359–2367.
RNA Therapeutics Institute, Howard Hughes
Medical Institute, and Department of
Biochemistry and Molecular Pharmacology,
University of Massachusetts Medical School,
Worcester, MA 01605, USA.
Current Biology Vol 23 No 8