GW bodies: from RNA biology to clinical implications in autoimmunity.

Abstract

Evaluation of: Lian S, Fritzler M, Katz J et al. Small interfering RNA-mediated silencing induces target-dependent assembly of GW/P bodies. Mol. Biol. Cell 18, 3375-3387 (2007). GW bodies (GWBs) are also known as mammalian processing bodies and are involved in 5 -3 mRNA degradation. Conversely, siRNA is a powerful tool for silencing genes. Recently, components of RNAi have been associated with GWBs, but as more components of this complex pathway become known, such relationships remain to be clarified. This paper evaluates the induction of GWBs by siRNA transfection. The main results of these studies indicate that siRNA increased the GWBs, such an increase is also dependent on the endogenous expression of the target mRNA; siRNA increases require GW182 or Ago-2 proteins, but not rck/p54 or LSm1. Results of the present studies propose a regulatory function of RNAi in GWB assembly; therefore, cell biology implications of GWBs may open a new area in pathogenic mechanisms of autoimmunity.

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Key Paper Evaluation
10.1586/1744666X.4.1.21© 2008 Future Drugs LtdISSN 1744-666X
21
www.future-drugs.com
GW bodies: from RNA biology
to clinical implications
in autoimmunity
Expert Rev. Clin. Immunol. 4(1), 21–25 (2008)
Rafael Herrera-
Esparza†, Deyanira
Pacheco-Tovar and
Esperanza
Avalos-Diaz
†Author for correspondence
Universidad Autonoma de
Zacatecas, Department of
Immunology,
Chepinque 206, Col Lomas
de la Soledad, Zacatecas,
98040, Mexico
Tel.: +52 492 921 1640
Fax: +52 492 922 6070
rafael.herreraesparza@
gmail.com
Evaluation of: Lian S, Fritzler M, Katz J et al. Small interfering RNA-mediated silencing
induces target-dependent assembly of GW/P bodies. Mol. Biol. Cell 18, 3375–3387 (2007).
GW bodies (GWBs) are also known as mammalian processing bodies and are involved in
5’–3’ mRNA degradation. Conversely, siRNA is a powerful tool for silencing genes. Recently,
components of RNAi have been associated with GWBs, but as more components of this
complex pathway become known, such relationships remain to be clarified. This paper
evaluates the induction of GWBs by siRNA transfection. The main results of these studies
indicate that siRNA increased the GWBs, such an increase is also dependent on the
endogenous expression of the target mRNA; siRNA increases require GW182 or Ago-2
proteins, but not rck/p54 or LSm1. Results of the present studies propose a regulatory function
of RNAi in GWB assembly; therefore, cell biology implications of GWBs may open a new area
in pathogenic mechanisms of autoimmunity.
KEYWORDS: GW 182 • GW bodies • neuropathy • siRNA
The GW bodies (GWBs) are the equivalent to
mammalian processing (P) bodies, located in
cytoplasmic foci that have multiple decay factors
involved in the 5´–3´ mRNA degradation path-
way. Their name is derived from the GW182
marker protein, which contains repeats of glycine
(G) and tryptophan (W) and a RNA-binding
domain at the carboxyl end. Decay and process-
ing factors localized to GWBs include the deade-
nylase Ccr4, the
Dcp1a/1b/Dcp2, the
Ge-1/Hedls, rck/p54, RAP55 and exonuclease
Xrn1. GWBs colocalize and interact with the
stress granules (SGs), which process aggregates of
delayed translational complexes that accumulate
during cell stress. Therefore, SGs share some
components with GWBs. Interestingly, it was
found that GWBs are associated with compo-
nents of RNAi, which is a post-transcriptional
gene-silencing mechanism that uses specific
dsRNA molecules to silence genes in a sequence-
specific manner. These seminal observations gave
rise to the observations of intracellular RNAi
processing and that the mRNA degradation
mediated by RNAi requires the GWB compo-
nents. The fate of GWBs is dynamic, and the size
and number of GWBs depends on the cell cycle
phase, nutrient conditions and other variables, for
decapping
LSm1–7
complex
complex,
instance small GWBs, are observed in the early
S phase but are larger in late S and G2 phases,
GWBs are disassembled before mitosis and reas-
sembled in early G1. In addition, the size and
number of GWBs are affected by the amount of
mRNA and its blocking-decay intermediates, by
deadenylation, decapping, and 5´–3´ mRNA
degradation or translation. Finally, an important
observation of these authors is the demonstration
that if RNAi is blocked, the GWBs are disassem-
bled, whereas the introduction of siRNA into the
cells led to GWB reassembly.
Methods & results
The aim of the study was to investigate the rela-
tionship between RNAi activity and the forma-
tion of GWBs. The following antibodies were
used in the study: anti-GW182; anti-Ago2;
anti-Dcp1a; anti-LSm4; anti-lamin A/C 636;
anti-TIAR; anti-tubulin, anti-green fluorescent
protein (GFP); and anti-rck/p54 and anti-
LSm1. siRNA duplexes designed for hAgo2,
hrck/p54, luciferase GL2-duplex EGFP and
human lamin A/C as control were also used.
Construction of inducible GFP3 fibroblast
(TRE-GFP3T3) cells was required. pCAG20–1
and pUHD10–3Puro constructs were transfected
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Expert Rev. Clin. Immunol. 4(1), (2008)
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Herrera-Esparza, Pacheco-Tovar & Avalos-Diaz
into the 3T3 fibroblast cell line to express tTA under selection
with puromycin. The open reading frame of enhanced GFP
(EGFP) was amplified by PCR with specific primers and the
PCR fragment was ligated into the MluI–NotI restriction site of
the pTRE2hyg expression vector. HeLa, HSG, NIH3T3 and
GFP3T3 cells were cultured in Dulbecco’s modified Eagle’s
medium, transiently transfected with siRNA and, in some cases,
were cotransfected with two different siRNAs (24 h of difference
between transfections). The transfected cells were analyzed by
indirect immunofluorescence (IIF) and western blot. Statistical
analysis was performed with the PRISM 4.0 program.
The main results of the present investigation were:
• The size and number of GWBs increased in siRNA-trans-
fected cells. The induction of RNA silencing after transfec-
tion of siRNAs directed to endogenous targets indicated that
the increase in GWBs is target dependent;
• The increase of GWBs induced by siRNA started the first
day of transfection and was maintained over the following
4 days, peaking on day 3;
• The presence of GW182 and Ago2 is required to induce
siRNA activity and increase GWB; furthermore, Ago2
expression correlates with the siRNA activity. Additionally, a
decrease of GW182 inhibited GWB assembly;
• The decrease of LSm1 or rck/p54 did not inhibit the assembly
of GWBs induced by siRNA.
Discussion & significance
The GWB regulation under conditions of induced siRNA is
highly reproducible and GWBs may be considered a marker of
siRNA activity, since GWB formation implicates mRNA
silencing. The number of GWBs is dependent on mRNA frag-
ments. The pre-existing GWBs are the targets of siRNA, and
these structures can be observed under light microscopy.
Finally, rck/p54 contributes to the assembly of recently formed
GWBs [1].
Conclusion & expert commentary
GW bodies are cellular structures located in the cytoplasm that
play a key role as gene regulators. This control mechanism
switches off a number of genes and may modulate cell repro-
duction and development. The GWBs are conserved structures
in mammalian cells (Dcp-containing bodies or P mammalian
bodies), in yeast there are P bodies; however, there is no clear
evidence that all yeast have GWBs or equivalent GW182. In
human cells, gene regulation via RNAi is important in the
pathophysiology of cancer and autoimmunity.
RNA-silencing pathway
In the last decade, RNAi has become known as a natural mech-
anism for silencing of gene expression. This primordial cellular
response can inhibit the function of any chosen target gene,
and such mechanisms include the genes involved in cancer,
AIDS, hepatitis and other diseases. The knowledge in this area
has increased quickly, and is actually a useful tool in the func-
tional characterization of different genes and has rapidly
evolved to the development of technologies that are currently
applied as therapeutics [2].
Foundations of mechanisms involved in gene silencing arose
in the early 1980s when the group of Jorgensen attempted the
genetic modification of plants to get colorful petunias. Never-
theless, it was not until 1990 when two groups of investigators
led by Van der Krol and Napoli [3,4], manipulated the violet tone
of petunias by increasing the expression level of the chalcone
synthase gene, which is involved in violet pigmentation. These
experiments unpredictably resulted in white flowers; further-
more, the introduction of extra copies of the gene caused a
decrease rather than the expected increase in the pigment. Later,
similar observations were obtained in Neurospora crassa [5] dur-
ing attempts to introduce extra copies of the genes involved in
carotene production. Interestingly, the results of these experi-
ments confirmed the seminal observations in petunias and,
therefore, the transgenic fungus lost their orange color while
acquiring a whitish phenotype. Another group of investigators
from Cornell University (NY, USA), who inhibited the expres-
sion of genes involved in development by introducing antisense
sequences in Caenorhabditis elegans, also found that gene sup-
pression was more efficient if sense and antisense sequences were
introduced simultaneously [6].
In 1998, Fire and Mello demonstrated that gene-specific
post-transcriptional silencing (PTGS) was archived when a
dsRNA was processed into the cell, and was converted into an
siRNA, which in turn bound its single stranded complemen-
tary sequence [7]. In doing so, siRNA is capable of directing the
degradation of a target mRNA. It is believed that RNAi consti-
tutes a mechanism developed by cells to eliminate undesirable
genes present in high copy numbers, such as viral genes, trans-
posable elements, or experimentally transfected genes. Gene
silencing may take place during transcription; gene suppression
is chiefly archived post-transcriptionally.
Mechanism of siRNA
RNAi is initiated with the production of dsRNA that is com-
plimentary to a specific mRNA. The introduction of a dsRNA
triggers a cascade of events followed by the recognition and
processing to produce small RNA fragments of 21–25 nucleo-
tides called siRNAs. After processing, these siRNAs recognize
the cognate mRNA target by base pair-dependent binding, and
then a selective degradation of mRNA can occur. Events lead-
ing to this follows the cleavage of the dsRNA into 21–25
nucleotide double-stranded fragments by an ATP-dependent,
conserved nuclease known as Dicer, which is a member of the
RNase III family of double strand-specific ribonucleases [8].
The siRNA induce the formation of a multiprotein complex
designated as RNA-induced silencing complex (RISC), which
is involved in the recognition and silencing and, in some cases,

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GW bodies in autoimmunity
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degradation of the mRNA target. The RISC components
include different members of the Argonaut (Ago) family of pro-
teins eIF2C1 and eIF2C2, as well as Ge-1/Hedls and other
enzymes implicated in mRNA degradation and silencing. The
RISC also contains a helicase activity responsible for unwind-
ing both strands of RNA. The RISC then chooses a single anti-
sense strand; a homolog protein named Dicer 2 with nuclease
activity cleaves the strand of mRNA, which is complementary
to the siRNA sequence (FIGURE 1) [9].
There are other related mechanisms to suppress gene expres-
sion; among them are the noncoding RNAs, including tRNAs,
siRNAs and rRNA, and the micro-RNA (miRNA), which are
constituted by small fragments of approximately 22 nucle-
otides. Some miRNA are conserved from C. elegans through to
humans and are able to block the translation of specific
mRNAs. In contrast to siRNA, the mRNA targeted by the
miRNA is not necessarily destroyed during this process. Addi-
tionally, the nonsense-mediated mRNA decay, is a process that
probably participates in the quality-control mechanism to
remove nonsense transcripts, such as mRNA with premature
termination codons. PTGS and RNAi constitute the same phe-
nomenon, and are highly conserved along the phylogenic scale
from yeast to mammals, including humans.
Micro-RNAs
miRNAs are small noncoding RNAs of 21–23 nucleotides
that target specific mRNAs to prevent their translation via the
RNAi pathway. The genes encoding for miRNAs are tran-
scribed by RNA polymerase II. In animals, most miRNAs
regulate gene expression utilizing mechanisms of RNAi; how-
ever, this mechanism is accomplished in the absence of
siRNA-directed mRNA cleavage. As previously mentioned,
Ago proteins are a crucial part of the machinery required for
miRNAs function and their target mRNAs are localized in
the cytoplasm as scattered foci known as P bodies or GW
bodies. In this cellular compartment, the Ago proteins physi-
cally interact with GW182, and it has been demonstrated
that GW182 is critical for GWB formation. In addition,
silencing of GW182 dissociated the resident P-/GW-body
proteins and impaired the silencing of miRNA reporters
[10,11]. The multicomponent clustering
of Ago2 and GW182 is important for
the interaction as was demonstrated by
investigators from the University of
Florida (FL, USA) and Calgary (AB,
Canada), who showed that Ago2 and
transfected siRNAs were present within
the same cytoplasmic bodies where this
interaction appeared to take place. Fur-
ther, disruption of GWB interfered with
the silencing ability of a given siRNA, an
observation that supported the notion
that GW182 and/or the microenviron-
ment of the cytoplasmic GWBs contrib-
ute to the RNA-induced silencing com-
plex and to RNA silencing via miRNA.
Subsequent studies by the same group
substantiated this notion in that the
miRNA localization to GWBs was
important for the generation of these
structures as well as for silencing the
target gene [11].
GWB as targets in
autoimmune diseases
The GW182 protein was discovered
using an autoimmune serum from a
patient with motor and sensory neuro-
pathy to screen an expression cDNA
library. The identified gene possessed an
open reading frame with numerous GW
repeats and a single RNA recognition
motif. Both the patient’s serum and a rab-
bit serum raised against the recombinant
Figure 1. mRNA degradation by siRNA and formation of GW/P.
PARN: Deadenylation initiator; RISC: RNA-induced silencing complex.
Dicer RISC
mRNA
mRNA
mRNA
Cap
Cap
Cap
AAAAA
AAAAA
mRNA degradation
Deadenylation
Decapping
PARN2-PARN3
Xrn1
GW/P
mRNA decay factors
Exosome
dsRNA
siRNA
Small
fragments
Plasma membrane
Cytoplasm
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Expert Rev. Clin. Immunol. 4(1), (2008)
Key Paper Evaluation
Herrera-Esparza, Pacheco-Tovar & Avalos-Diaz
GW protein stained discrete cytoplasmic speckles named
GWBs, reactivity that did not overlap with the Golgi complex,
endosomes, lysosomes or peroxisomes.
Five-year view
The contribution of this group of investigators from Calgary and
Florida in the year of 2002 constitutes a tremendous advance in
biology, all of which started with the simple process of screening
an expression cDNA library with human autoantibodies [12]; this
initial observation was followed by a large and rapidly growing
body of information and all of which has resulted in a better
understanding of the biology of post-translational control of
gene expression via mRNA degradation and silencing. Studies by
this group into the clinical importance of anti-GW182 autoanti-
bodies indicate that approximately a third of patients possessing
this autoantibody have a motor and sensory neuropathy. In
another investigation of a cohort of autoimmune patients, they
found a link between anti-GW182 antibodies and Sjögren’s syn-
drome also accompanied by mixed motor/sensory neuropathy,
systemic lupus erythematosus and other conditions. It is remark-
able that this autoantibody distinguishes a subset of autoimmune
conditions with neurological pathology [13]. The pathogenic role
of anti-GW182 autoantibodies in autoimmune disease or with
neurological sequelae is still unknown. A study of cells from
neurological tumors may provide a clue because GWBs are
present in astrocytes and astrocytoma cells within cell bodies and
cytoplasmic projections [14]. Furthermore, the astrocytoma GWBs
exhibit a complex heterogeneity with combinations of LSm4 and
XRN1, as well as Ago2 and Dicer, key proteins involved in
mRNA degradation and RNAi, respectively. Also the GWBs
present in astrocytes contained the mRNA transport and stabiliza-
tion proteins SYNCRIP, hnRNPA1 and FMRP. In addition,
antiGW182 autoantibodies are also found in a particular subset of
patients with primary biliary cirrhosis. Taken together, these obser-
vations suggest the possible origin of anti-GWB production are
siRNA/miRNA complexes that may be presented to the immune
system during dysregulated apoptosis and the inefficient clearance
of apoptotic bodies described in lupus erythematosus and allied
conditions. In previous studies, apoptotic bodies have been shown
to contain other target autoantigens, such as small cytoplasmic
ribonucleoproteins, RNA-processing components and, perhaps,
even GWB elements. These authors have also demonstrated that
other components of the RNAi pathway that are targets of the
human autoimmune response include Ago2, Ge-1/Hedls (58%),
GW182 (40%) and Ago2 (16%) [15,16]. GWB autoantibodies
targeted epitopes of the N-terminus of Ago2 and the nuclear
localization signal-containing region of Ge-1/Hedls [16].
Finally, in spite of the aforementioned advances, the signifi-
cance of autoimmunity to GWBs and RNAi are still under inves-
tigation. The relationship between inflammation, innate immu-
nity and miRNA expression is just beginning to be explored.
Interesting results appeared recently indicating that murine mac-
rophages exposed to polyriboinosinic:polyribocytidylic acid or
the cytokine IFN-β upregulate a miRNA (miR-155) and such an
effect was induced by several Toll-like receptor ligands. In addi-
tion, upregulation by interferons was shown to involve TNF-α
autocrine signaling, the inhibition of the kinase c-Jun N-terminal
kinase (JNK) blocked induction of miR-155, suggesting that
miR-155-inducing signals use the JNK pathway. These findings
suggest that miR-155 is a common target of different inflamma-
tory mediators [17], and this result may constitute a possible link
between RNA silencing and the inflammatory response of
autoimmune diseases.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with
any organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the
manuscript. This includes employment, consultancies, honoraria, stock
ownership or options, expert testimony, grants or patents received or
pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
Key issues
• GW bodies (GWBs) are involved in 5’–3’ mRNA degradation.
• Components of RNAi are associated with GWBs.
• GW182 is critical for GWB formation.
• Disruption of GWBs interfered with the silencing ability of a
given siRNA.
• RNAi possesses a regulatory function in GWB assembly.
• The clinical importance of anti-GW182 autoantibodies
indicate that a third of patients possessing this autoantibody
have a motor and sensory neuropathy.
• Cell biology implications of GWBs may open a new area in
pathogenic mechanisms of autoimmunity.
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Affiliations
• Rafael Herrera-Esparza, MD, PhD
Universidad Autonoma de Zacatecas,
Department of Immunology,
Chepinque 206, Col Lomas de la Soledad,
Zacatecas, 98040, Mexico
Tel.: +52 492 921 1640
Fax: +52 492 922 6070
rafael.herreraesparza@gmail.com
Deyanira Pacheco-Tovar, BSc
Universidad Autonoma de Zacatecas,
Department of Immunology,
Chepinque 206, Col Lomas de la Soledad,
Zacatecas, 98040, Mexico
Tel.: +52 492 921 1640
Fax: +52 492 922 6070
qfbpato@gmail.com
Esperanza Avalos-Diaz, MD, PhD
Universidad Autonoma de Zacatecas,
Department of Immunology,
Chepinque 206, Col Lomas de la Soledad,
Zacatecas, 98040, Mexico
Tel.: +52 492 921 1640
Fax: +52 492 922 6070
avalosespera@gmail.com
•
•