Erratum
The role of GW/P-bodies in RNA processing and silencing
Andrew Jakymiw, Kaleb M. Pauley, Songqing Li, Keigo Ikeda, Shangli Lian, Theophany Eystathioy, Minoru
Satoh, Marvin J. Fritzler and Edward K. L. Chan
Journal of Cell Science 120, 1702 (2007) doi:10.1242/jcs.03452
There was an error published in J. Cell Sci. 120, 1317-1323.
We apologise for two errors that occurred in the online and pdf versions of this article. The printed version is correct.
On p. 1317, in the Summary, the sentence ‘Formation of GW bodies appears to depend on both specific protein factors and RNA,
in particular, microRNA.’ appeared twice. The correct version of the summary is shown below.
Summary
GW bodies, also known as mammalian P-bodies, are cytoplasmic foci involved in the post-transcriptional regulation of
eukaryotic gene expression. Recently, GW bodies have been linked to RNA interference and demonstrated to be important
for short-interfering-RNA- and microRNA-mediated mRNA decay and translational repression. Evidence indicates that
both passenger and guide strands of short-interfering RNA duplexes can localize to GW bodies, thereby indicating that
RNA-induced silencing complexes may be activated within these cytoplasmic centers. Work over the past few years has
significantly increased our understanding of the biology of GW bodies, revealing that they are specialized cell components
that spatially regulate mRNA turnover in various biological processes. Formation of GW bodies appears to depend on
both specific protein factors and RNA, in particular, microRNA. Here, we propose a working model for GW body assembly
in terms of its relationship to RNA interference. In this process, one or more heteromeric protein complexes accumulate
in successive steps into larger ribonucleoprotein structures.
On p. 1319, right column, first paragraph, the word order of the penultimate sentence was incorrect and should read:
In particular, studies in Drosophila indicate that GW182 interacts with Ago1 and promotes miRNA-mediated degradation of a
subset of mRNA targets (Behm-Ansmant et al., 2006).
Commentary
1317
The role of GW/P-bodies in RNA processing and
silencing
Andrew Jakymiw1, Kaleb M. Pauley1, Songqing Li1, Keigo Ikeda1, Shangli Lian1, Theophany Eystathioy2,
Minoru Satoh3, Marvin J. Fritzler2 and Edward K. L. Chan1,*
1
Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
3
Division of Rheumatology and Clinical Immunology, Department of Medicine, and Department of Pathology, Immunology, and Laboratory
Medicine, University of Florida, Gainesville, FL 32610, USA
2
*Author for correspondence (e-mail: [email protected])
Accepted 21 February 2007
Journal of Cell Science 120, 1317-1323 Published by The Company of Biologists 2007
doi:10.1242/jcs.03429
Journal of Cell Science
Summary
GW bodies, also known as mammalian P-bodies, are
cytoplasmic foci involved in the post-transcriptional
regulation of eukaryotic gene expression. Recently, GW
bodies have been linked to RNA interference and
demonstrated to be important for short-interfering-RNAand microRNA-mediated mRNA decay and translational
repression. Evidence indicates that both passenger and
guide strands of short-interfering RNA duplexes can
localize to GW bodies, thereby indicating that RNAinduced silencing complexes may be activated within these
cytoplasmic centers. Formation of GW bodies appears to
depend on both specific protein factors and RNA, in
particular, microRNA. Work over the past few years has
significantly increased our understanding of the biology of
GW bodies, revealing that they are specialized cell
components that spatially regulate mRNA turnover in
various biological processes. The formation of GW bodies
appears to depend on both specific protein factors and
RNA, in particular, microRNA. Here, we propose a
working model for GW body assembly in terms of its
relationship to RNA interference. In this process, one or
more heteromeric protein complexes accumulate in
successive steps into larger ribonucleoprotein structures.
Introduction
The control of mRNA stability plays key roles in both the posttranscriptional regulation of eukaryotic gene expression
(Keene and Lager, 2005; Wilusz and Wilusz, 2004) and mRNA
quality control (Fasken and Corbett, 2005). The latter involves
the recognition and rapid degradation of aberrant mRNAs and
takes place when translation termination occurs too early
(nonsense-mediated decay) or fails to occur (non-stop decay)
(Fasken and Corbett, 2005) or when translation elongation
stalls (no-go decay) (Doma and Parker, 2006). In eukaryotes,
mRNA turnover is regulated by two major mechanisms. One
involves the multisubunit exosome, where transcripts are
degraded by 3⬘-to-5⬘ exonucleases (for a review, see van Hoof
and Parker, 1999). The second mechanism involves
cytoplasmic compartments termed GW bodies (GWBs), which
spatially control mRNA turnover by the 5⬘-to-3⬘ mRNA decay
machinery. These discrete cytoplasmic foci, also called Dcpcontaining bodies or processing (P)-bodies, constitute sites of
mRNA degradation, storage and translational repression
(Brengues et al., 2005; Coller and Parker, 2005; Cougot et al.,
2004; Eystathioy et al., 2002; Eystathioy et al., 2003; Sheth
and Parker, 2003; Van Dijk et al., 2002). Recently, they have
also been shown to function in RNA interference (RNAi)
(Jakymiw et al., 2005; Liu et al., 2005a; Liu et al., 2005b;
Meister et al., 2005; Pillai et al., 2005; Sen and Blau, 2005).
RNAi is a post-transcriptional silencing mechanism in which
small double-stranded RNA molecules induce sequence-specific
degradation and/or translational repression of homologous
mRNAs (for reviews, see Filipowicz et al., 2005; Meister and
Tuschl, 2004; Rana, 2007; Sen and Blau, 2006; ValenciaSanchez et al., 2006). The discovery that RNAi effector proteins
(Jakymiw et al., 2005; Liu et al., 2005a; Liu et al., 2005b; Sen
and Blau, 2005) and small RNAs localize to GWBs (Jakymiw et
al., 2005; Pauley et al., 2006; Pillai et al., 2005), and that GWB
assembly appears to be required for the proper functioning of the
RNAi pathway (Jakymiw et al., 2005; Liu et al., 2005a; Meister
et al., 2005), suggests that these foci are specifically involved in
short-interfering RNA (siRNA)- and microRNA (miRNA)mediated mRNA degradation and/or translational repression†.
Key words: GW bodies, P-bodies, RNA interference, mRNA
degradation, microRNA, short-interfering RNA
†
siRNAs are small RNAs of ~21 nucleotides in length and are derived from the progressive
cleavage of long, perfectly complementary double-stranded RNAs (dsRNAs) by an
RNase-III-type endonuclease, Dicer. They can originate from long dsRNAs transiently
introduced into cells by transfection or stably expressed hairpin-containing dsRNA
precursors derived from DNA constructs. They assemble into an RNA-protein complex
known as the RNA-induced silencing complex (RISC or siRISC), which includes
Argonaute 2 (Ago2), a key component of RNAi that possesses endonuclease activity.
RISC then targets and cleaves perfectly complementary mRNAs, generating 5⬘ and 3⬘
fragments, which are subsequently degraded. MiRNAs are similar in size to siRNAs but
originate from hairpin-containing precursors encoded by the genome. These non-coding
precursors of miRNAs have double-stranded regions with imperfect complementarity and
are sequentially processed by RNase-III-type enzymes Drosha and Dicer into mature
miRNAs. The mature miRNAs then assemble into an RNA-protein complex referred to
as the miRNA ribonucleoprotein complex (miRNP or miRISC), which is structurally
similar to RISC and contains at least one Argonaute protein. Multiple copies of miRNPs
are directed to the 3⬘-UTR of certain mRNAs that have imperfect complementarity to the
bound miRNAs. In plants, miRNAs cleave their regulated target mRNA, whereas in
mammals miRNAs promote translational repression of the targeted mRNA. Until recently,
this was considered the major distinction between siRISC and miRISC in mammalian
cells. However, new evidence suggests that miRNAs can also regulate mRNA degradation
similarly to siRNAs (Bagga et al., 2005; Jing et al., 2005; Yekta et al., 2004), thus blurring
the distinction between the two small-RNA-mediated silencing complexes.
Journal of Cell Science
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Journal of Cell Science 120 (8)
GWBs are enriched in mRNA decay factors and pools of
stored messenger ribonucleoproteins (mRNPs) (Bruno and
Wilkinson, 2006; Sheth and Parker, 2006). Moreover, they are
dynamic structures, whose size and number appear to depend
on specific intracellular processes. For instance, GWBs vary in
size and number throughout the cell cycle, the largest of which
are observed in late S and G2 phase (Yang et al., 2004).
Furthermore, stress (Kedersha et al., 2005; Teixeira et al.,
2005), cell proliferation (Yang et al., 2004), blocking mRNA
decay (Andrei et al., 2005; Cougot et al., 2004; Sheth and
Parker, 2003) and inhibition of translational initiation
(Brengues et al., 2005; Sheth and Parker, 2003; Teixeira et al.,
2005) all increase the size and number of GWBs. Conversely,
blocking transcription, deadenylation of mRNAs or
translational elongation decreases the size and number of
GWBs (Cougot et al., 2004; Sheth and Parker, 2003). Recent
studies have highlighted the importance of these structures in
the regulation of mRNA turnover and provided insights into
their involvement in RNA silencing. Here, we review the
current understanding of GWB function, focusing on its recent
links to RNAi, and discuss the role of these cytoplasmic foci
in RNA-induced silencing complex (RISC) activation. We also
examine the requirements for GWB formation and disassembly
and propose a working model for their genesis.
GWBs uncovered
GWBs were first identified and characterized in studies using
an autoimmune serum from a human patient with motor and
sensory neuropathy (Eystathioy et al., 2002). They were named
as such because they harbor the mRNA-binding protein
GW182. GW182 is characterized by glycine (G) and
tryptophan (W) repeat-rich domains and a canonical RNArecognition motif and has been demonstrated to associate with
a specific subset of transcripts in HeLa cells (Eystathioy et al.,
2002). The finding that a set of mammalian proteins involved
in mRNA degradation – Dcp1/2, Xrn1 and LSm1-7 – localize
to similar prominent cytoplasmic foci (Bashkirov et al., 1997;
Eystathioy et al., 2003; Ingelfinger et al., 2002; Van Dijk et al.,
2002), and that GWBs are active sites of mRNA decay in
human cells (Cougot et al., 2004), indicated that GWBs are
involved in the spatial regulation of mRNA turnover, and more
specifically, a selective 5⬘-to-3⬘ mRNA degradation pathway.
Concurrent studies identified GWB-related structures referred
to as P-bodies in budding yeast, and these were found to play
a role in mRNA decapping and 5⬘-to-3⬘ decay (Sheth and
Parker, 2003). In particular, Sheth and Parker demonstrated
that insertion of a poly(G) tract into mRNAs, which blocks 5⬘to-3⬘ exonuclease activity, causes accumulation of decay
intermediates within P-bodies, suggesting that the mRNA
decay process is associated with them (Sheth and Parker,
2003).
These bodies are thought to be conserved in budding yeast
and mammals because both the yeast P-bodies and mammalian
GWBs contain activators of decapping and decapping enzymes
(Cougot et al., 2004; Eystathioy et al., 2003; Ingelfinger et al.,
2002; Lykke-Andersen, 2002; Segal et al., 2006; Sheth and
Parker, 2003; Van Dijk et al., 2002). Note, however, that GWBs
differ from yeast P-bodies in that they contain translation
initiation factors and other proteins that have no yeast
counterparts, such as factors involved in RNAi [e.g. GW182
and Argonaute (Ago) proteins] (Anderson and Kedersha, 2006;
Segal et al., 2006). Furthermore, there are functional
differences between GWBs and yeast P-bodies in terms of their
responses to stress and cell growth (Schneider et al., 2006). For
example, P-bodies increase in size and number during growth
limitation, increased cell density, and stress (Teixeira et al.,
2005), whereas GWBs increase in size and number in
proliferating cells (Yang et al., 2004) and dynamically interact
with stress granules‡ in stressed mammalian cells (Kedersha et
al., 2005). Therefore, one needs to be cautious when
generalizing about these structures because differences do
exist, not only between species but even within a population of
GWBs in a single mammalian cell (Fig. 1). Interestingly, a
recent report similarly found that some GW/P-bodies that
contain Mex-3B, a newly described class of human RNAbinding protein that localizes to GW/P-bodies, are also devoid
of Dcp1 (Buchet-Poyau et al., 2007). The function of a
particular GW/P-body may therefore depend on its
organization or composition.
GW body function
mRNA degradation
The decay of mRNA in GW/P-bodies is thought to occur by
a 5⬘-to-3⬘ exonucleolytic process, which first requires the
removal of the 3⬘-poly(adenosine) [poly(A)] tail –
deadenylation. Subsequently, the 5⬘ mRNA cap is irreversibly
removed by a decapping complex and the body of the mRNA
is degraded by the 5⬘-to-3⬘ exonuclease Xrn1 (for a review,
see Coller and Parker, 2004). Evidence also suggests that
GW/P-bodies can regulate translation (Bhattacharyya et al.,
2006; Coller and Parker, 2005; Ferraiuolo et al., 2005; Pillai
et al., 2005) and store mRNAs (Bhattacharyya et al., 2006;
Brengues et al., 2005; Pillai et al., 2005). Moreover, GW/Pbodies also appear to be involved in other mRNA decay
pathways, such as nonsense mediated decay (NMD), AU-rich
element (ARE)-mediated decay (AMD) and stress-induced
decay (SID).
NMD is a surveillance mechanism that removes aberrant
mRNA transcripts containing premature termination codons.
Several factors involved in NMD, including UPF1-3, SMG5
and SMG7, as well as reporter mRNAs harboring nonsense
mutations have been found to localize to GW/P-bodies
(Fukuhara et al., 2005; Sheth and Parker, 2006; Unterholzner
and Izaurralde, 2004), which links these foci and the NMD
process. GWBs have also been linked to AMD, a decay process
involving messages containing AREs in the 3⬘-untranslated
region (UTR). In particular, depletion of three GWB proteins,
Xrn1, LSm1 or 4E-T, has been demonstrated to inhibit AMD
(Ferraiuolo et al., 2005; Stoecklin et al., 2006). Moreover,
evidence also indicates that the ARE-binding protein TTP,
which is known to destabilize ARE-containing mRNAs,
localizes to GWBs (Kedersha et al., 2005) and interacts with
and activates the decapping complex (Fenger-Gron et al., 2005;
Lykke-Andersen and Wagner, 2005). Finally, the observation
that the size and number of P-bodies increases during stress in
budding yeast cells (Teixeira et al., 2005) and that GWBs can
be induced to form and interact with stress granules by specific
stresses in mammalian cells demonstrates their importance in
SID (Kedersha et al., 2005). However, because budding yeast
‡
Stress granules are large RNP particles that store non-translating mRNAs when cells are
exposed to environmental stresses, but are absent in budding yeast cells.
GW/P-bodies in RNA processing and silencing
1319
Journal of Cell Science
Fig. 1. GW bodies (GWBs) are heterogeneous structures
with obvious differences in protein composition.
(Top) HEp-2 cells stained with human anti-GWB serum
(a prototype serum often used as a GWB marker and
known to contain antibodies to GW182, hAgo2 and Ge-1,
but not Dcp1; green) and rabbit anti-Dcp1 antibody (often
used as a marker for P-bodies; red) demonstrate that not
all GWBs contain Dcp1. (Bottom) HEp-2 cells stained
with mouse anti-hAgo2 monoclonal (green) and rabbit
anti-RCK/p54 (red) polyclonal antibodies demonstrate
that not all foci containing RCK/p54 contain hAgo2.
Conversely, not all foci that stain for hAgo2 contain
RCK/p54. Arrows, GWBs that do not contain both
protein factors. Arrowheads, GWBs that do contain both
protein factors. Nuclei (blue) were counterstained with
DAPI. Bar, 10 ␮m.
cells lack stress granules, the mechanism of SID probably
differs from that in higher eukaryotes.
In mammalian cells, GWBs and stress granules have been
reported to be compositionally and morphologically distinct
structures (Cougot et al., 2004; Kedersha et al., 2005);
however, evidence indicates that they are functionally and
spatially linked (Kedersha et al., 2005). Interestingly, both
entities share an assortment of proteins (e.g. CPEB1, Rck/p54,
FAST, Xrn1, eIF4 and TTP) and a single class of reporter
mRNA has been observed within both GWBs and stress
granules (Kedersha et al., 2005; Wilczynska et al., 2005).
Moreover, overexpression of TTP and CPEB1, a translational
regulator, induces the fusion of stress granules and GWBs,
which suggests that these proteins regulate the dynamic
interaction between these two structures (Kedersha et al., 2005;
Wilczynska et al., 2005). One model is that stress granules
serve as mRNA triage sites where transcripts are sorted for
storage, re-initiation of translation, or degradation, and that
mRNAs targeted for decay are exported from stress granules
to GWBs (Kedersha et al., 2005). Regardless of the
mechanistic differences between yeast and higher eukaryotes,
it is evident that GW/P-bodies have multiple functions, the
ultimate goal being mRNA decay and/or storage. It is therefore
not surprising that these foci comprise multiple factors and that
their specific structural composition and organization probably
determines their mode of action.
RNAi
Several groups recently demonstrated a link between GWBs
and RNAi (Jakymiw et al., 2005; Liu et al., 2005a; Liu et al.,
2005b; Pillai et al., 2005; Sen and Blau, 2005). In particular,
the Ago family of proteins, Ago1-4, which are components of
RISC, the key effector complex of RNAi, were found to be
concentrated in GWBs (Jakymiw et al., 2005; Liu et al., 2005a;
Liu et al., 2005b; Sen and Blau, 2005). Moreover, GWBs also
appeared to be sites involved in miRNA-mediated repression
of targeted mRNAs (Liu et al., 2005b).
In addition, we and others demonstrated that hAgo2, the
catalytic engine of RNA silencing, associates with several
components of GWBs, including GW182 (Jakymiw et al.,
2005; Liu et al., 2005a; Liu et al., 2005b). Moreover,
depletion of GW182, which disrupts GWBs (Yang et al.,
2004), perturbs both siRNA- and miRNA-mediated
repression (Jakymiw et al., 2005; Liu et al., 2005a). Dominant
interfering GW182 and hAgo2 mutants also disrupt GWB
formation and similarly inhibit RNA silencing (Jakymiw et
al., 2005). The impairment in RNAi appears to depend on
blocking the localization of hAgo2 to GWBs (Jakymiw et al.,
2005). Indeed, hAgo2 constructs containing point mutations
that prevent siRNA binding and localization to GWBs do not
repress target reporter mRNA, despite being tethered to the
target (Liu et al., 2005a). Additional evidence for the
involvement of GW182 and GWB in RNAi comes from
studies of the Drosophila melanogaster ortholog and
Caenorhabditis elegans functional analog (Ding et al., 2005;
Rehwinkel et al., 2005; Schneider et al., 2006). In particular,
studies in Drosophila indicate that GW182 interacts with
Ago1 and promotes degradation of a subset of miRNAmediated-decay mRNA targets (Behm-Ansmant et al., 2006).
GW182 may thus function as a molecular scaffold that
bridges components of the miRNA pathway with mRNA
decay enzymes (Behm-Ansmant et al., 2006).
A point of contention is whether GW182 and GWBs are
important for both slicer-dependent mechanisms (i.e. siRNAmediated RNAi) and miRNA-mediated repression or only the
latter. Work from our laboratory has demonstrated that
transfected fluorophore-labeled siRNAs associate with GW182
protein complexes and localize to GWBs and that GW182 and
GWBs play an important role in slicer-mediated functions
(Jakymiw et al., 2005). Moreover, Liu et al. found that
suppression of GW182 similarly impairs, albeit not as
effectively, the ability of an siRNA to silence its target by
mRNA cleavage (Liu et al., 2005a). In addition, silencing of
the GWB protein TNRC6B, a GW182 paralog, inhibits
cleavage of an mRNA reporter gene containing a target site
perfectly complementary to an endogenous miRNA (Meister
et al., 2005). TNRC6B thus appears to be important for slicerdependent mechanisms and subsequent mRNA degradation.
Other work has indicated a greater role for GW182 and
GWBs in miRNA-mediated translational repression by
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Journal of Cell Science 120 (8)
Table 1. Suppression of specific GWB factors demonstrates their interdependence on accumulation within and for
assembly of GWBs
Target*
Cell function
GWB foci†
GWB markers examined
References
GW182
RNAi
–
LSm4, hAgo2, hDcp1a, hDcp2
5⬘-to-3⬘ exonuclease
Deadenylation
Decapping
Decapping
Decapping
Decapping; translation
control; RNAi
Translation control
Translation control
Decapping
RNAi
Translation
+
–
–
–
+
–
hDcp2
LSm1, RCK/p54, eIF4E, eIF4E-T
Ccr4, RCK/p54, eIF4E, eIF4E-T
GW182, hDcp1a
LSm1, RCK/p54, eIF4E, eIF4E-T, Ge-1
hAgo2, LSm1, Ccr4, eIF4E, eIF4E-T
(Jakymiw et al., 2005; Liu et al., 2005a;
Yang et al., 2004)
(Andrei et al., 2005; Cougot et al., 2004)
(Andrei et al., 2005)
(Andrei et al., 2005; Chu and Rana, 2006)
(Kedersha et al., 2005)
(Andrei et al., 2005; Yu et al., 2005)
(Andrei et al., 2005; Chu and Rana, 2006)
–
–
–
–
–
Ccr4, RCK/p54, eIF4E, LSm1, hDcp1
hDcp1a
hDcp1a, hDcp2
GW182, hAgo2, hDcp1a
hDcp1a, LSm1, RCK/p54,
eIF4E, eIF4E-T
Xrn1
Ccr4
LSm1
LSm4
hDcp2
RCK/p54
eIF4E-T
RAP55
Ge-1/Hedls
miRNA‡
mRNA§
(Andrei et al., 2005; Ferraiuolo et al., 2005)
(Tanaka et al., 2006; Yang et al., 2006)
(Yu et al., 2005)
(Pauley et al., 2006)
(Andrei et al., 2005; Cougot et al., 2004)
Journal of Cell Science
GWB, GW body; miRNA, microRNA; RNAi, RNA interference; siRNA, short-interfering RNA; *proteins silenced through siRNA-mediated gene-specific
knockdown; †detected using fluorescence microscopy; ‡miRNA suppression was induced indirectly through siRNA-mediated knockdown of Drosha or DGCR8;
§
accumulation within GWBs inhibited using cycloheximide or actinomycin D.
demonstrating, using various reporter systems, that depletion
of GW182 impairs miRNA function (Chu and Rana, 2006; Liu
et al., 2005a; Rehwinkel et al., 2005). Furthermore, depletion
of GW182 has also been demonstrated to result in alterations
of mRNA expression profiles very similar to those seen in cells
depleted of the Drosophila miRNA effector Ago1 and not the
siRNA effector Ago2, which suggests that GW182 functions
in the miRNA pathway (Behm-Ansmant et al., 2006). Whether
the above disparities are because of the use of different reporter
systems (i.e. exogenously introduced versus endogenous
reporter systems), species variations or GW182 redundancy
(three paralogs have been identified in humans) will require
further study. Interestingly, in the study implicating GW182 in
slicer-mediated function, we used siRNAs with a fluorophore
conjugated to the 5⬘-end of the guide strand (Jakymiw et al.,
2005). The fluorophore might therefore have interfered with
the siRNA activity, making it behave more like an miRNA by
producing imperfect base-pairing between siRNA and target.
Regardless, the studies collectively demonstrate a role for
GW182 and GWBs in RNAi. More work will be needed to
determine whether GWBs represent converging sites for
siRISC and/or miRISC.
MiRNAs have similarly been identified within GWBs and
demonstrated to associate with GW182 protein complexes
(Pauley et al., 2006; Pillai et al., 2005). The identification of
siRNA and/or miRNA within GWBs suggests that RISC
activation, activity and/or recycling may occur within GWBs.
Are GWBs sites of RISC activation and activity? Evidence
supporting this possibility includes the observation that Ago2,
a known GWB component, is directly involved in passengerstrand cleavage of double-stranded siRNAs during RISC
assembly (Matranga et al., 2005; Rand et al., 2005). Also,
following transient transfection of HeLa cells with a
fluorophore-labeled passenger-strand siRNA duplex that has
no endogenous mRNA target, the siRNA localizes to GWBs –
similarly to a fluorophore-labeled-guide-strand siRNA duplex
that has an endogenous mRNA target (Jakymiw et al., 2005).
This suggests that the passenger strand and the guide strand
localize to GWBs independently of the mRNA target and that
passenger-strand cleavage and incorporation of the antisense-
strand into RISC may occur within GWBs. Docking of
siRNA/miRNA duplexes into RISC and its subsequent
activation could therefore be early events in GWB formation.
Interestingly, in fission yeast cells, a Dicer ortholog localizes
to structures resembling GWBs (Carmichael et al., 2006),
which suggests that GWB formation may take place even
before RISC activation.
GW body formation and structure
Immunogold electron microscopy of GWBs identifies
cytoplasmic electron-dense structures that are 100-300 nm in
diameter and which lack a membrane (Eystathioy et al., 2002;
Yang et al., 2004). Closer inspection reveals that they comprise
8-10 nm strands or fibrils (Yang et al., 2004). Currently, the
mechanism of GWB formation is not well understood. It
remains unclear whether GWBs form de novo or whether
mRNAs and their associated proteins are targeted to preexisting structures. Furthermore, we do not know whether
GWB components are targeted to GWBs independently or as
part of larger complexes that form higher-order structures that
can be visualized by conventional light microscopy. RNAimediated depletion of specific GWB factors in human cells has
demonstrated an interdependence of each of the proteins for
their accumulation in GWBs (see Table 1). GWBs may thus
form by the assembly of one or more heteromeric protein
complexes on mRNAs that can amass into larger mRNP
structures. The finding that specific enzymes involved in
decapping and subsequent 5⬘-to-3⬘ degradation are dispensable
for GWB formation (Andrei et al., 2005) – unlike factors
involved at earlier stages of mRNA decay (e.g. mRNA 3⬘-end
trimming) – indicates that earlier stages are more crucial for
GWB assembly.
Fig. 2 shows our working model, in which several factors,
including RISC, initially interact with the target mRNA to form
a specific RNP structure dependent on the type of decay or
storage process that will occur (e.g. siRNA-mediated decay
versus miRNA-mediated translational repression). This results
in the recruitment of other protein complexes, which depend
on the composition of the initial RNP; so their final
composition or structural organization promotes the proper
Journal of Cell Science
GW/P-bodies in RNA processing and silencing
1321
Fig. 2. A model linking RNAi and GW body (GWB) assembly and function. RNAi activity is triggered by siRNA/miRNA duplexes, which are
first processed from either double-stranded RNA (dsRNA) or precursor microRNA (pre-miRNA), respectively, by the RNase-III-type
endonuclease Dicer. These duplexes are then incorporated into RISC, where the passenger strand (black) is either cleaved and degraded or
removed by the bypass mechanism (Matranga et al., 2005). This activation process and assembly of the guide strand (red) into RISC is thought
to initiate early stages of GWB formation. Subsequent targeting by RISC results in further recruitment of one or more heteromeric protein
complexes (which could include GW182 and RCK/p54) on the mRNA, which forms a specific RNP structure that causes post-transcriptional
inhibition of gene expression (through siRNA-mediated cleavage or miRNA-mediated translational repression, depending on the degree of
complementarity between the guide-strand RNA and its target mRNA). The targeted mRNA is eventually degraded by further recruitment of 5⬘to-3⬘ mRNA decay factors, which include the deadenylase Ccr4, decapping factors (LSm1-7 ring, Dcp complex) and the 5⬘-to-3⬘ exonuclease
Xrn1. The accumulation of RNA decay factors during degradation could be responsible for the size increases in GWBs, which makes them
visible by conventional light microscopy. ORF, open reading frame.
decay mechanism and/or storage of the mRNA. Such a model
would explain why disruption of GWBs by depletion of other
GWB components, such as LSm1 or RCK/p54, does not
necessarily translate into impaired RISC activity (Chu and
Rana, 2006), whereas silencing of GW182 does (Jakymiw et
al., 2005). One possibility is that GW182 exists in a smaller
RNP complex with RISC components (e.g. hAgo2) that is
undetectable by fluorescence microscopy and supersedes the
requirement of LSm1 and RCK/p54 for initial target
recognition and endonucleolytic cleavage or translation
repression. Moreover, not all silencing of GWB factors impairs
formation of GWBs equivalently. Interestingly, Andrei et al.
have demonstrated that silencing of RCK/p54 has a less
pronounced effect on the disassembly of GWBs compared with
other factors (Andrei et al., 2005).
The integrity of GWBs also appears to depend on RNA.
Studies in yeast and mammals indicate that mRNA is an
integral component of GW/P-bodies (Andrei et al., 2005;
Cougot et al., 2004; Eystathioy et al., 2002; Teixeira et al.,
2005). Furthermore, silencing of Drosha or its required partner
protein, DGCR8, which together comprise the microprocessor
complex and are responsible for processing long nuclear
primary miRNA (pri-miRNA) transcripts to ~70-nucleotide
hairpin precursor miRNA (pre-miRNA), depletes cells of
mature miRNA and causes the disappearance of GWBs (Pauley
et al., 2006). This suggests that miRNAs are required for the
formation of GWBs. Although the possibility that the effect is
indirect cannot be ruled out, this seems unlikely because
siRNA transfected into Drosha-deficient cells can serve as a
surrogate for miRNA and drive the reappearance of GWBs.
The dependence of GWBs on miRNA means that many of
the functions attributed to GWBs similarly may depend on
miRNAs in mammalian cells. Processes such as translational
repression, mRNA decay and storage depend on miRNAs.
1322
Journal of Cell Science 120 (8)
Journal of Cell Science
Furthermore, miRNAs are involved in ARE-mediated
degradation events (Jing et al., 2005). All of these processes
are associated with GWBs. The primary function of GWBs
might therefore be to provide a microenvironment for miRNAmRNA interactions that lead to translational inhibition and/or
mRNA degradation. Whether NMD processes share this
requirement for miRNAs requires further study. Nevertheless,
if this hypothesis holds, a more appropriate name for these foci
in mammalian cells may be miRNA-induced bodies (miRBs).
Conclusions and perspectives
Recent cell biology and biochemical findings have
significantly enhanced our understanding of the spatial
regulation of mRNA decay and/or storage within eukaryotic
cells, and it is now evident that dynamic cytoplasmic foci,
GW/P-bodies, are crucial for these processes. The discovery of
a functional link between GWBs and RNAi has been
particularly instrumental in allowing us to decipher the
complexities of how small RNAs and their cognate proteins are
involved in post-transcriptional gene regulation (for reviews,
see Engels and Hutvagner, 2006; Eulalio et al., 2007; Jackson
and Standart, 2007; Pillai et al., 2007; Rana, 2007).
Our understanding of the cell biology of RNAi and its
relationship to GWBs is still limited, however. During the
course of writing this review, several articles were published
demonstrating that a large fraction of miRNAs and Ago
proteins reside in the cytoplasm (Leung et al., 2006; Maroney
et al., 2006; Nottrott et al., 2006), in particular on mRNAs
being actively translated by polyribosomes (Maroney et al.,
2006; Nottrott et al., 2006). Furthermore, inhibiting translation
initiation or inducing stress has been shown to result in
localization of miRNA and Ago to stress granules (Leung et
al., 2006). Data from these studies suggest that miRNPs first
associate with and suppress actively translating mRNAs in the
cytoplasm prior to completely dropping off ribosomes. Upon
miRNA-mediated repression, these mRNA are then believed to
be targeted to either stress granules for storage or sorted and
shuttled to GWBs for decay. Given the close relationship
between GWBs and stress granules, this would not be
surprising; however, one needs to be cautious in interpreting
the stress granule data because stress granules are generally
observed only during a stress response, whereas GWBs are
present continuously (Teixeira et al., 2005). Alternatively,
inhibition of actively translating polysomes on mRNAs by
miRISCs might directly trigger the recruitment of RNP
complexes involved in either early stages of GWB formation
and/or GWB targeting. Much more work will be needed if we
are to completely understand the interactions between GWBs
and other mRNP structures and the significance of their
heterogeneous composition.
Finally, although numerous studies have demonstrated that
RNAi occurs in the cytoplasm of a cell, a growing number of
reports suggest that it occurs within the nucleus as well. In
particular, recent studies in human cells have implicated the
RNAi effector proteins hAgo1 and hAgo2 in the posttranscriptional modulation of gene expression in the nucleus
(Robb et al., 2005) and transcriptional silencing (Janowski et
al., 2006; Kim et al., 2006). Furthermore, transfection of
siRNAs targeting small nuclear RNAs (e.g. 7SK and U6 RNA)
leads to their translocation into the nucleus and silencing of the
target genes (Berezhna et al., 2006). How the cytoplasmic and
nuclear RNAi pathways are interrelated and the relationship
between GWBs and nuclear RNAi are currently unclear.
Biochemical evidence suggests that these processes are linked
by a common requirement for Ago proteins; however,
immunofluorescence studies using a newly developed mouse
monoclonal antibody specific for hAgo2 show no evidence of
nuclear localization of this endogenous protein (Ikeda et al.,
2006), which is consistent with our earlier work using
polyclonal autoimmune sera (Jakymiw et al., 2006).
Regardless, the recent findings that GWBs appear to function
in siRNA/miRNA-mediated forms of post-transcriptional
regulation, the apparent requirement for GWBs in Drosophila
(Schneider et al., 2006) and C. elegans (Ding et al., 2005)
development, and the knowledge that small RNAs regulate
many cellular activities (Ambros, 2004), including
differentiation, stem cell division, and apoptosis, underline the
importance of GWBs for many biological processes.
We thank Jens Lykke-Andersen (University of Colorado) and Tom
Hobman (University of Alberta) for their generosity in providing
valuable antibody reagents. We apologize to colleagues whose
interesting work could not be cited owing to space limitations. This
work was supported in part by the National Institutes of Health Grant
AI47859 and the Canadian Institutes for Health Research Grant MOP38034 and the Canadian Breast Cancer Research Foundation Grant
16992.
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