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RISC hitches onto endosome trafficking
Haruhiko Siomi and Mikiko C Siomi
The RNA-induced silencing complex (RISC) downregulates expression of the genes targeted by RNA-silencing pathways.
But formation and turnover of the RISC complex itself is tightly regulated and requires endosomal membranes.
Over the past few years, we have seen remarkable progress in our understanding of how
small non-coding RNAs of 20–30 nucleotides,
including small interfering RNAs (siRNAs)and microRNAs (miRNAs)1, guide the downregulation of gene expression. These small
regulatory RNAs associate with Argonaute(AGO) proteins to form RISCs that direct
degradation or translational repression oftarget mRNAs. AGO proteins colocalize with
P‑body components, where enzymes involved
in mRNA decapping (Dcp1 and Dcp2) anddecay are concentrated, suggesting a physicalinteraction between components of RISCs andP‑bodies2,3. Targeting mRNAs away from thetranslational machinery into P‑bodies could
be an important mechanism in miRNA- andsiRNA-mediated silencing. However, where in
the cell RISC formation and/or turnover occurs
remains unclear. In two complementary papers
on pages 1143 and 1150 of this issue, Gibbings
et al.4 and Lee et al.5 show that RISC formationand turnover depends on the endosomal pathway. These results are of interest not just for
the insights they provide into the processes of
RISC formation and turnover, but also because
they provide a link between RNA silencing andmembrane trafficking6.
Many plasma-membrane proteins are internalized by endocytosis and are sorted to their
final destination by the endosomal pathway6.
Endosome maturation involves a gradualaccumulation of internal vesicles (Fig. 1a).
Late endosomes will have accumulated several hundred luminal vesicles that arise frominternal budding of the endosomal membrane.
These endosomes are often referred to as multivesicular bodies (MVBs). They can fuse withlysosomes, resulting in degradation of theircargo. MVBs can also fuse with the plasma
membrane, in an exocytic manner, to release
Haruhiko Siomi and Mikiko C Siomi are in the
Department of Molecular Biology, Keio University
School of Medicine, 35 Shinanomachi, Shinjuku-ku,
Tokyo 160‑8582, Japan.
e-mails: awa403@sc.itc.keio.ac.jp;
siomim@sc.itc.keio.ac.jp

internal vesicles (exosomes) into the extracellular milieu7 (these should not be confused withthe ribonuclease complex that is also known as
an exosome). The formation of inwardly budding vesicles in endosomes and the sorting of
selected cargo into these vesicles requires thesequential assembly of four ESCRT (endosomal
sorting complex required for transport) complexes on the endosomal membrane; these complexes are well conserved across the eukaryotic
lineage. Ubiquitylated cargoes engage the ubiquitin-binding domain of ESCRT components
for sorting into these vesicles.
Exosomes were recently shown to containmRNAs and miRNAs8,9 and can transfer these
nucleic acids into a recipient cell. Consistent
with these findings, Gibbings et al.4 purified
exosome-like vesicles from cultured monocytes
and found that they contained a large numberof miRNAs. They also contained low levels of
AGO2 as well as dramatically high levels of theP‑body component GW182, which is required
for miRNA function through its binding to
AGO proteins2. Knockdown of GW182 disrupts P‑bodies and significantly impairs theability of both miRNAs and siRNAs to repress
target gene expression2. However, the components of the P‑body decapping complex,
DCP1A and GE‑1, were barely detected inpurified exosome-like material.
Subsequent membrane fractionation analysis
showed that GW182 and AGO2 were enrichedin the endosomal/MVB fraction, suggestingthat crucial components of the RISC congregate in endosomes and MVBs4. These results
are consistent with the initial identification of
AGO2 as a membrane-associated protein10.
Gibbings et al. demonstrate that at least two
pools of AGO2-associated RNA-silencing components exist. One pool, defined as GW bodies,
is GW182-rich, DCP1A-poor and is associatedwith MVB components. The second pool does
not contain membrane-associated proteins, is
DCP1A-rich and GW182-poor and is therefore
defined as P‑bodies. These findings suggest that
the AGO2 and GW182 foci that colocalize with
MVBs constitute subcellular structures distinct

from P‑bodies (Fig. 1a). The findings also contradict previous results supporting a strong
association of GW182 with P‑bodies2,3. Gibbings
et al. propose that GW182 aggregates often,
albeit not always, correspond to endosomes/
MVBs. These results also raise the possibility
that MVBs constitute sites of RISC assembly
and/or function. Consistent with this proposal,
miRNA-repressible mRNAs accumulated in
fractions corresponding to MVBs. However,miRNA-repressible mRNAs are markedly
under-represented in exosome-like vesicles.
Thus, whereas miRNA-repressible mRNAs areenriched in GW182- and AGO2-associatedmembranous fractions, they seem selectively
excluded from exosome-like vesicles. These
results suggest that a pool of GW182 selectively
dissociates from membrane-bound, miRISC–
mRNA complexes to be sorted into MVBs andis subsequently secreted or degraded throughthe exosome–lysosome pathway (Fig. 1b).
Gibbings et al. found that blocking MVB
formation by knockdown of the ESCRT
component genes hrs and alix results in an
increase in cellular GW182, but not DCP1A,
levels. Furthermore, knockdown of hrs andalix inhibited miRNA activity, as monitored
by let-7a and its target reporter, and also
affected let-7a accumulation. Thus, blocking
the formation of MVBs generally compromises miRNA functions, probably by interfering with the sorting of GW182 into MVBs.
Gibbings et al. hypothesize that ESCRTdependent removal of GW182 molecules
into MVBs might constitute a limiting stepin miRNA loading onto AGO, or might allow
continuous assembly and disassembly of themembrane-associated RISC, required for its
maturation or function (Fig. 1).
Independently, Lee et al. carried out ascreen in Drosophila to identify mutations
that enhance siRNA- and miRNA-mediatedgene silencing. One of the mutated genes isorthologous to the gene responsible for a rareautosomal recessive disease — HermanskyPudlak Syndrome 4 (HPS4)11. Loss of HPS4
can also enhance RNAi in mammalian cells,

nature cell biology VOLUME 11 | NUMBER 9 | SEPTEMBER 2009
© 2009 Macmillan Publishers Limited. All rights reserved.

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a

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Exosom

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Pla

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*
*

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Early endosome*

Lysosome*

Late endosome/MVB
RISC
GW

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PB

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b

To next silencing cycle
miRNA

mRNA
AGO

AGO

GW182

GW

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GW

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Lysosome
ExosomeMVB
GW*
Figure 1 RISC assembly and turnover occurs at endosomes. (a) Membrane proteins (blue) are brought
inside the cell by incorporation into endocytic vesicles that fuse with an early endosome. Multivesicularbodies (MVBs) are late endosomal compartments, between early endosomes and lysosomes. MVBs
can also release internal vesicles, called exosomes, into their extracellular space. RISC formation and/or turnover appears to occur on MVBs. GW182 aggregates (GW) are often colocalized with MVBs anddefine subcellular structures distinct from P‑bodies (PB), the cytoplasmic aggregates that containtranslationally repressed ribonucleoprotein particles and are associated with the translation repression
and mRNA decay machinery. SG represents stress granules. (b) Blocking the fusion of MVBs withlysosomes or the plasma membrane stimulates RISC activity. In contrast, blocking the ESCRTdependent formation of MVBs from early endosomes inhibits RISC activity. Thus it is hypothesized that
ESCRT-dependent removal of GW182 into MVBs might be a limiting step in miRNA loading onto AGO,
or might allow the continuous assembly and disassembly of membrane-associated RISCs, required fortheir maturation or function. Asterisks represent cargoes in exosomes.

indicating that HPS4 attenuates RNAi acrossthe animal kingdom. The HPS4 gene is known
to be required for the fusion of MVBs withlysosomes11. Interestingly, Lee et al. found that
mutations in the ESCRT genes hrs and vps25
also impair miRNA-mediated silencing. In
contrast to the effect of mutations in the HPS4gene, the authors showed that blocking the formation of MVBs from early endosomes could
inhibit RISC activity (Fig. 1b).
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In Drosophila HPS4-mutant cells, AGO1, a
key component of miRNA–RISC (miRISC)1,
was dispersed throughout the cytoplasm. In
contrast, mutations in the ESCRT genes thatblock sorting of early endosomes into MVBs
and thereby generate enlarged early endosomes,
resulted in the concentration of AGO1 aroundenlarged early endosomes. These findings suggest that blocking MVB formation concentrates
miRISCs around early endosomes whereas

blocking MVB turnover disperses miRISCs
from lysosomes to the cytosol. Surprisingly,
the levels of miRNA associated with AGO1
were greater when AGO1 was immunopurified
from Drosophila HPS4-mutant cells, indicatingthat miRISC loading is enhanced when MVB
turnover is impaired.
Maturation of early endosomes into MVBs
depends upon the recognition of ubiquitylated
proteins in early endosomes by ESCRT proteins6. Lee et al. demonstrate that mutations
in a Drosophila ubiquitin ligase impair silencing mediated both by siRNAs and miRNAs.
A dominant-negative form of ubiquitin also
inhibits loading of AGO2 with exogenously
labelled siRNA. Together, these findings suggest that protein ubiquitylation stimulates RISC
activity through enhanced small RNA loading
onto RISCs, although effects through additional pathways cannot be excluded.
Gibbings et al. and Lee et al. thus provide
evidence that supports a model in which active
miRISCs associated with target mRNAs arerecruited into GW‑bodies that are physicallyassociated with MVBs. Alternatively, but notmutually exclusively, MVBs might regulatesilencing by increasing the ability of miRISCs
to load miRNAs, perhaps by promoting turnover of miRISCs (Fig. 1b). Reduced secretionor degradation would cause accumulation of
GW182; however, this would concomitantly
compromise the proposed assembly or turnover of RISCs. GW182 directly interacts with
AGO proteins. Thus, the GW182 association
with AGO may function as a temporal lock that
could compete with Dicer for loading empty
AGO with miRNA, or for reloading AGO withnew miRNA after an initial round of miRNAdependent repression. MVBs may be the sites
where miRNA biogenesis and RISC-assembly
pathways intersect, with continuous removalof GW182 being required for the efficientpassage of miRNA–miRNA antisense strand
duplex from DICER to AGO. Consistent withthis idea, AGO1 associates with Dicer‑1 and
GW182 in a mutually exclusive manner in
Drosophila12. How AGO is dissociated from
GW182 remains to be elucidated.
These new findings should help define themolecular pathways involved in RNA silencing.
They also underline the possibility that small
RNAs could be secreted from cells throughexosome pathways, for regulatory purposes.
Finally, the ESCRT machinery is used by enveloped retroviruses for outward virus budding at
the plasma membrane, a process topologicallynature cell biology VOLUME 11 | NUMBER 9 | SEPTEMBER 2009
© 2009 Macmillan Publishers Limited. All rights reserved.

news and views
identical to inward budding away from thecytoplasm to form MVBs6. These new results
raise the possibility that some retroviruses suchas HIV‑1 may contain GW182, miRNAs andsome AGO proteins within their virions, perhaps allowing them to use these RNA-silencing
components in the arms race with host cells.

1. Siomi, H. & Siomi, M. C. Nature 457, 396–404(2009).
2. Eulalio, A. et al. Nature Rev. Mol. Cell Biol. 8, 9–22
(2007).
3. Parker, R. & Sheth, U. Mol. Cell 25, 635–646 (2007).
4. Gibbings, D. J., Ciaudo, C., Erhardt, M, & Voinnet, O.
Nature Cell Biol. 11, 1143–1149 (2009).
5. Lee, Y. S. et al. Nature Cell Biol. 11, 1150–1156(2009).

6. Raiborg, C. & Stenmark, H. Nature 458, 445–452(2009).
7. Stoorvogel, W. et al. Traffic 3, 321–330 (2002).
8. Valadi, H. et al. Nature Cell Biol. 9, 654–659 (2007).
9. Skog J. et al. Nature Cell Biol. 10, 1470–1476 (2008).
10. Cikaluk, D. E. et al. Mol. Biol. Cell. 10, 3357–3372(1999).
11. Li, W. et al. Bioessays 26, 616–628 (2004).
12. Miyoshi, K. et al. RNA 15, 1282–1291 (2009).

Steroid hormone pulsing drives cyclic gene expression
Béatrice Desvergne and Christophe Héligon
Transcriptional cycling of activated glucocorticoid receptor (GR) and ultradian glucocorticoid secretion are well establishedprocesses. Ultradian hormone release is now shown to result in pulsatile gene transcription through dynamic exchange of GR withthe target-gene promoter and GR cycling through the chaperone machinery.
Endogenous glucocorticoid secretion by adrenal glands is controlled by the hypothalamicpituitary axis, resulting in a circadian (every
24 hours) and an ultradian (every hour) pattern of hormone release into the blood stream1.
On page 1093 of this issue, Stavreva et al. show
how nuclear GR translates the natural, ultradian
glucocorticoid pulses into a molecular responseof transcriptional pulses, an effect that is notobserved with synthetic glucocorticoids2.
Endogenous, active glucocorticoids, cortisol
in humans and corticosterone in rodents, are
key pleiotropic hormones regulating metabolism, stress response and the immune system
in adults3. Synthetic glucocorticoids are widelyused to treat chronic inflammatory diseases3,4.
GR, a steroid hormone nuclear receptor specificfor glucocorticoids, is expressed in virtually allcells, where it is required for glucocorticoiddependent gene expression. GR undergoes
rapid cycling on and off the promoters of its
targets in the presence of natural glucocorticoids5,6. In addition, the highly dynamic
association of GR and cortisol allows GR tosense rapid changes in cortisol concentration, which fluctuates, in vivo, in a circadianand an ultradian manner1. Stavreva et al. use
an engineered mouse cell line that expresses
fluorescent GR and contains an amplified
array of GR-responsive promoter structures,
including GR response elements, to monitor
GR association with the promoters in live cells.
Béatrice Desvergne and Christophe Héligon are at
the Centre for Integrative Genomics, University of
Lausanne, CH‑1015 Lausanne, Switzerland.
e-mail: beatrice.desvergne@unil.ch

The authors show that successive exposure tohormone drives dynamic cycles of association and dissociation of GR with the promoter
array. These cycles are mirrored by pulses oftranscriptional activity, which directly correlatewith the flux in hormone levels. In contrast,
synthetic glucocorticoids lead to GR association with the promoter array, but the receptor fails to dissociate after hormone washout(withdrawal), leading to sustained target gene
expression. Moreover, the authors found thatin vivo, when adrenalectomised rats were supplied with a natural glucocorticoid in an ultradian manner, both GR association with target
promoters and gene expression responded
dynamically to glucocorticoid levels, which was
similar to their observations in cell cultures.
These findings highlight fundamental mechanisms of transcriptional regulation in single
cells and demonstrate a rapid and dynamic
GR‑mediated transcriptional response to natural hormone pulses in vivo.
Oscillation in the transcriptional activity of various transcription factors is wellestablished and has been proposed to be acommon feature of gene regulation7. Nuclear
receptors, in particular, have been the focus
of detailed studies. In a simple model,
nuclear receptor-dependent transcriptional
cycling depends on hormone availability,
association and dissociation of the nuclear
receptor with and from its cognate response
element and removal of the nuclear receptor
and its cofactors through proteasomal degradation. These steps first generate transcriptional competence followed by productive

transcription and finally limit transcription
through promoter clearance8.
In such a scheme, the hormone itself is not acycling component. The following cycling events
have been described for GR: a slow, hormonedependent nucleo-cytoplasmic shuttling of GR;
continuous dissociation and re-association of
GR and hormone in the nucleus (occurringin minutes) and rapid exchange of GR on the
GR response element (occurring in seconds)5,9.
Chaperones have a role in each of these threecycles (Fig. 1). Ultradian rhythm of hormone
stimulation adds yet another time scale, in theorder of an hour, and must be integrated into
the model for GR‑driven transcription. When
independently analysed, all of the above-mentioned oscillations contribute to GR‑mediatedtranscriptional activity. However, the complexity generated by these additive layers of dynamic
patterns makes it difficult to predict transcriptional activity at a given time point.
So, what is the biological relevance of
GR‑driven gene pulsing? One proposal is that
nuclear receptor-driven cyclic transcription
may allow target gene transcription to rapidly
adjust to hormone fluctuations. This recent
study indeed demonstrates that ultradian oscillations of glucocorticoid are temporally followed by transcriptional oscillations. But why
the need for such reactivity? Ultradian andcircadian rhythms of glucocorticoid fluctuation are thought to allow synchronization andcoordination of diurnal and nocturnal events.
In addition, glucocorticoid response can be triggered by stress, at any time, to facilitate adaptation and contribute to restoring homeostasis.

nature cell biology VOLUME 11 | NUMBER 9 | SEPTEMBER 2009
© 2009 Macmillan Publishers Limited. All rights reserved.

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