Dispatch
R561

RNA Interference: Endogenous
siRNAs Derived from Transposable
Elements
The Piwi-interacting RNA interference pathway plays an important role in
suppressing transposable elements in the Drosophila germline. Now, deep
sequencing of short RNAs from somatic tissue and cell culture has identified
a novel class of endogenous siRNAs that may have a similar role in the soma.
Darren J. Obbard1
and David J. Finnegan2,*
Transposable elements (TEs) are
stretches of DNA that are able tomove around the genome bymechanisms that lead to duplicationof the element concerned, resulting inevolutionarily rapid changes in genome
size and a potentially high copy number
for TEs [1]. This will happen, andTEs will persist and spread within
a population, even if they are costly
to their host [2,3].
Although TE insertion canoccasionally be beneficial and may
indeed have been important in several
key evolutionary innovations [1], thevast majority of insertions are likely to
be deleterious or neutral [2,3], imposing
a fitness cost on the rest of the genome.
Costs arise directly when an insertion
disrupts functional sequences, orindirectly by increasing rates ofectopic recombination between distantsites. In either case, there is a conflict ofinterest between TEs, which need totranspose to survive, and the rest of the
genome, which would be better off ifthese elements did not transpose [2,3].
The situation is different in somatic
tissues. Here transposition cannot
benefit the TE by increasing its copy
number in subsequent generations
but may be harmful to the hostnevertheless. As a result, the interestsof the host and its TEs should bealigned, with both benefiting fromreduced transposition [3]. In principle,
this might even lead to the evolutionof different mechanisms for regulating
TEs in different tissue types, reflecting
conflict in the germline andcooperation in the soma.
Suppression of TE activity in the
germline is mediated, at least in part,by RNA interference (RNAi) and in the
animal germline depends on the Piwi
family of Argonaute proteins anda class of single-stranded 23–29
nucleotide (nt) Piwi-interacting RNAs

(piRNAs) [4]. Drosophila has two other
RNAi mechanisms (Figure 1) — themicro-RNA (miRNA) pathway, whichplays an essential role in controllinggene expression [5], and the siRNA
pathway, which is best understood as
an antiviral defence mechanism [6].
Although each of these pathways
utilizes short RNAs complexed withan Argonaute-family protein, they differ
in several ways. In summary, miRNAs
are about 22 nt long, are derived fromhost-expressed fold-back structures,
and associate primarily with Ago1 [5].
Conversely, antiviral siRNAs (viRNAs),
are about 21 nt, are derived fromdouble-stranded viral RNA, andassociate with Ago2 [6]. BothmiRNAs and viRNAs are produced bycleavage of double-stranded RNA bya Dicer-family protein: Dcr1 formiRNAs and Dcr2 for viRNAs. In
contrast, piRNAs appear to be
Dicer-independent and are generated
by Piwi-mediated cleavage ofsingle-stranded sense and antisense
TE transcripts, a process initiated in
the zygote by maternally derived
piRNAs [4]. There are also differencesin short-RNA structure between
pathways. In Drosophila, the 30 end ofboth piRNAs and siRNAs, but notmiRNAs, is 20 -O-methylated [7], while
piRNAs are preferentially antisenseand display a bias toward uridine at
the 50 end [4].
Plants [8] and nematodes [9] havea wider range of siRNAs than haveso far been found in Drosophila,
which, in this regard, has appeared
to be something of a ‘poor relation’.
Moreover, the different RNAi pathways
in Drosophila originally appeared quite
separate (e.g., [10]) in sharp contrast
to plants where there is appreciable
overlap between pathways and genefunctions [8]. These views are now
challenged by Chung et al. [11], ina recent issue of Current Biology, andby other groups [12–15] in studies that
identify a novel class of endogenous

Drosophila siRNAs derived from TEs
and host transcripts.
By deep sequencing the short-RNA
populations derived from adult flies
and tissue culture cells, these groups
have revealed a class of 30 -methylatedendogenous 21 nt RNAs derived from
TEs and several other loci, including
natural antisense transcripts and longfold-back structures [11–15]. These
endogenous siRNAs depend on Dcr2
and Ago2 of the antiviral siRNA
pathway but not Dcr1 or Ago1 of themiRNA pathway or on componentsof the piRNA pathway. Moreover, theendogenous siRNAs show a much
lower strand bias than is seen inpiRNAs and they do not have a strong U
bias in the 50 base. Although there had
been several previous reports hinting at
the involvement of the siRNA pathway
in TE suppression in Drosophila
somatic tissue (e.g., [16,17]), this is thefirst time that comprehensive studies
have quantified the number and originof these endogenous short RNAs. The
results identify TE-derived siRNAs asa distinct class of small RNAs and,because reduction in expression ofAgo2 or Dcr2 is associated bothwith decreased numbers of siRNAs
derived from TEs and with increased
levels of TE transcripts, it suggeststhat this siRNA pathway may actively
regulate TE transcript levels.
The discovery of TE-derived
endogenous siRNAs in many tissues,
including the germline, raises as
many questions as it answers. The
dependence of endogenous siRNAs
on Dcr2 and their reduced strand-biassuggests that they are derived froma long dsRNA precursor, but how is
the antisense transcript generated?
Is it through active host promoters,for example, the loci that give rise topiRNA-generating transcripts — Czech
et al. [13] suggest ‘yes’, Chung et al.
‘no’ [11]) — or are they the result of
incidental ‘background’ transcription?
Ghildiyal et al. [12] observe that there is
at least some overlap between piRNAand TE-siRNA generating, suggestingthat both may be involved.
Strikingly, both Czech et al. [13] andChung et al. [11] show that many
endogenous siRNAs depend on the
RNA-binding protein Loquacious(Loqs), the canonical partner of Dcr1 in
the miRNA pathway [18]. Loqs has notpreviously been found to associatewith Dcr2, which in the antiviral siRNA
pathway is thought to act with the Loqs

Current Biology Vol 18 No 13
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Known Drosophila RNAi pathways

Sense/antisenseTE dsRNA

Sense and antisense

Hairpin ssRNA

Viral dsRNANew?

TE ssRNA
Aub

Dcr2

References
Dcr2

Ago3

Dcr1

R2D2

Loqs

Aub

Loqs

Hen1
Hen1

Hen1

Hen1
Ago3

Aub
Ago2

Ago2

Ago1
Ago3
Hen1

RISC

RISC

Piwi

RISC ?

viRNA

miRNA

piRNA

TE-siRNA

Degradation ofviral transcripts
and genomes

Repression of mRNA Degradation of TE transcripts
translation and possible and TE-targeted formation ofmRNA degradation heterochromatin in the germline

Degradation of TE transcripts
and TE-targeted formationof heterochromatin?
Current Biology

Figure 1. The three well-studied RNAi pathways in Drosophila.
The viRNA pathway processes dsRNA derived from viruses and viral-replication intermediates
into w21 nt, 30 -methylated viRNAs that are used to recognise and bind viral targets for cleavage by the RNA-induced silencing complex (RISC). This processing is mediated by Dicer-2(Dcr2), Argonaute2 (Ago2) and R2D2, and viRNAs are methylated by Hen1. The miRNA pathway processes host-encoded fold-back structures (primary miRNAs) into short hairpins(pre-miRNAs) and then into w22 nt un-methylated miRNAs. This processing is mediated by
Dicer-1 (Dcr1), Argonaute-1 (Ago1) and Loquacious (Loqs). In Drosophila, miRNAs generally
include mismatches to their targets, which are usually 30 UTRs, and induce transcriptional repression. The piRNA pathway is functional only in the germline and is thought to alternately
cleave sense (red) and antisense (blue) TE transcripts, guided by a piRNA in complex with
one of the Piwi family of Argonaute proteins — Aubergine (Aub), Argonaute-3 (Ago3), or
Piwi. It has been proposed that cleavage not only reduces transcript levels but also generates
the 50 end of a new piRNA (the fragment is then bound by a partner Piwi-family member, shortened by an exonuclease, and methylated by Hen1). This feedback loop may be initiated by maternally generated piRNAs. The new pathway that has been identified in the recent studies
comprises components of both the viRNA and miRNA pathways and processes endogenous
dsRNA (as opposed to exogenous dsRNA, such as that of viral replication intermediates) into
21 nt methylated siRNAs. These derive from (and presumably target) transposable elements,
overlapping gene transcripts (especially 30 UTRs) and larger fold-back structures.

homologue R2D2 [6,19]. Does thishint at distinct mechanisms for thebiogenesis of TE siRNAs and viralsiRNAs? In a further twist, Kawamura
et al. [14] observe signs of RNA editingin their siRNAs, and this can also beseen in the data of Chung et al. [11],reinforcing the idea that RNA editing
and RNAi pathways may interact [20].
There are also questions of adaptive
significance. It seems clear that TEs do
indeed feed into an endogenous siRNA
pathway in both somatic and germline
tissue and that this affects theirtranscript levels [11–14], but whether
or not this is a ‘defence’ mechanism isfar from clear. As we point out above,
the interests of the TE and the rest

host genes, while the other — thepiRNA pathway — is solely directed
towards suppressing transposition?
We still have much to learn.

of the genome should be aligned insomatic tissues, with both benefitingfrom reduced transposition. It is notclear that the endogenous siRNA
pathway affects transposition per se,
only that it affects transcript levels,
and Kawamura et al. [14] suggest thatsiRNAs may be involved in the somatic
maintenance of heterochromatin:
perhaps both are affected. Why flies
should employ two different RNAi
pathways to suppress TE activity,
especially given that both are active in
the germline [13], is also not clear. Do
these pathways feed into each other, oris one — the TE-siRNA pathway — ofmutual benefit, perhaps being
derived from a system regulating

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Farley, G., Wang, P., and Zamore, P.D. (2007).
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small RNAs bind to Argonaute-2 in somatic
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19. Liu, Q., Rand, T.A., Kalidas, S., Du, F., Kim,
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1Institute of Evolutionary Biology, Universityof Edinburgh, Kings Buildings, West Mains
Road, Edinburgh EH9 3JT, UK. 2Institute

Phagocytosis: Dynamin’s Dual Rolein Phagosome Biogenesis
Dynamins have a well-established role in the fission of vesicles at sites of
endocytosis. In phagocytosis, however, a role for certain dynamin isoforms
has been reported in the full extension of pseudopods during phagosome
formation, not in fission of the phagocytic vacuole. Recent studies in
Caenorhabditis elegans have now uncovered a new function of dynamin inphagosome maturation.
Kassidy K. Huynh
and Sergio Grinstein
Phagocytosis is an integral part of ourinnate immunity, providing an earlyline of defense against invading
microorganisms. In addition,
phagocytosis plays a key role inwound healing, tissue morphogenesis
and development by removing
necrotic and apoptotic cells. Particle
intake is initiated by phagocytic
receptors that recognize ligands on thesurface of the target and unleash
a signaling cascade that culminates in
actin-driven extension of pseudopods
that ultimately encircle and engulf theparticle (Figure 1). However, additional
steps are required to complete theelimination of the phagocytic targets.
Full clearance requires the degradationof the internalized particles and thedisposal of their breakdown products,
which occur as the phagosomes
acquire degradative properties akin to
those of lysosomes by a process
known as phagosomal maturation.
Despite its critical importance to theoverall process, remarkably little is
known about phagosome maturation.
It has been studied much lessintensively than phagosome formation,
perhaps because the latter is mucheasier to visualize and quantify.
However, this gap is likely to narrowin the future through studies like therecent work by Kinchen et al. [1].
These authors took advantage of thefact that in Caenorhabditis elegans
phagosomes containing apoptotic
cells are identifiable under differential
interference microscopy by theirrefractile appearance and affinity for
nuclear dyes. Unusual persistence ofsuch refractile structures is indicativeof defective maturation and, whencombined with gene manipulation,
this analysis can provide valuable
information about proteins involvedin the maturation sequence. Indeed,because the genome sequence ofC. elegans is known and the worms are
genetically tractable, it should now be
possible to investigate systematicallythe role of individual proteins inphagosome maturation. This analysis
will be facilitated by the spontaneous
occurrence of multiple phagocytic
events because, during the course of
C. elegans development, over 600 cells
undergo programmed death and areingested by neighboring cells.
In the new work, Kinchen et al. [1]used a convenient and effectivemeans of silencing selected genes in
C. elegans to investigate their rolein the maturation of phagosomes
containing apoptotic cells. They fed to
the larvae bacteria transformed withdouble-stranded siRNA to manipulate
the expression levels of Rab5, Rab7,
EEA1, and the VpsC/HOPS complex.
These proteins were known to regulate
vesicular traffic in the endocytic andphagocytic pathways, controlling thetransition between early and latestages. By these means, the authors
were able to confirm the requirementfor Rab5 and Rab7 in the early stagesof the maturation process, asestablished in previous work inmammalian phagocytes [2]. They also
documented a role for the VpsC/HOPS
complex in phagosome maturation.

of Cell Biology, University of Edinburgh,Kings Buildings, Mayfield Road,
Edinburgh EH9 3JR, UK.
*E-mail: David.Finnegan@ed.ac.uk
DOI: 10.1016/j.cub.2008.05.035

This multi-subunit complex had been
proposed to bridge the transition fromRab5- to Rab7-positive vesicles
during endosome progression [3],but its involvement in phagosome
maturation had not been explored.
Kinchen et al. [1] systematicallysilenced the expression of individualcomponents of this complex and foundthat depletion of the Vps11, Vps16,
Vps18, Vps33, or Vps44 subunitsarrested phagosome maturation at
the Rab7-positive stage. These data
imply that, while the VpsC/HOPS
complex is essential forphagolysosome biogenesis, thecomplex is seemingly not needed forRab7 recruitment and may instead
regulate vesicle fusion and traffic, as
has been suggested for the endocytic
pathway [4–7]. A surprising observation
was that elimination of EEA1 had little
or no effect on the ability of C. elegans
to clear apoptotic corpses. This
contrasts with the results reported
using mammalian macrophages, whichwere unable to undergo maturation
following inactivation of EEA1 [8].
This discrepancy may indicate that
not all phagosomes are created orprocessed equally and that the natureof the particular phagocytic receptor
engaged may dictate the pathway
used for maturation.
The salient findings in the paper byKinchen et al. [1], however, refer tothe role of dynamin in the early stagesof phagosome maturation. The
involvement of dynamin in phagosome
formation had been exploredpreviously in professional phagocytes,
but its role in maturation was
unsuspected and therefore unexplored.
Unlike C. elegans, which has a single
form of dynamin, mammals express
three distinct isoforms: dynamin-1, -2,
and -3 [9]. Dynamin-2 is expressedubiquitously while dynamin-1 is present
exclusively in the brain and dynamin-3
in the testes, lungs and neurons [10].
It is noteworthy that dynamin-2 ispresent both in endomembranes
and in the plasma membrane,
while dynamin-1 is predominantly